The beneficial properties of CBDA in diet-induced neuroinflammation

Karolina Konstantynowicz-Nowicka; Aleksandra Opęchowska; Maciej Domalewski; Magdalena Zabielska-Kaczorowska; Aleksandra Bogucka; Marcin Lipiński; Ewa Harasim-Symbor; Adrian Chabowski

doi:10.1007/s10787-026-02358-4

. 2026 Aug 21;34(9):6213–6242. doi: 10.1007/s10787-026-02358-4

The beneficial properties of CBDA in diet-induced neuroinflammation

Karolina Konstantynowicz-Nowicka ^1,^✉, Aleksandra Opęchowska ¹, Maciej Domalewski ¹, Magdalena Zabielska-Kaczorowska ², Aleksandra Bogucka ², Marcin Lipiński ³, Ewa Harasim-Symbor ¹, Adrian Chabowski ¹

PMCID: PMC13558366 PMID: 42627619

Abstract

Cannabidiolic acid (CBDA) is a phytocannabinoid found in the Cannabis plant. Understanding the effects of CBDA is essential to uncover its full potential and possible health benefits. The study was conducted on rats receiving standard rat chow (control) and a high-fat diet (HFD). Half of the animals in each group were administered CBDA intragastrically. The total lipid fractions and arachidonic acid (AA) contents were measured in the frontal and posterior cortex, hippocampus, and subcortical nuclei using gas-liquid chromatography. The expression of proteins involved in neurodegenerative diseases and insulin signaling pathway proteins in the frontal and posterior cortex was measured using Immunoblotting. RT-PCR was used to assess the expression of pro-inflammatory pathway proteins in the same regions. Additionally, untargeted and targeted metabolomic analyses were performed on cerebrospinal fluid (CSF). The results showed that a decrease in arachidonic acid levels and pro-inflammatory precursor proteins after CBDA treatment in high-fat-fed rats was simultaneous with improved insulin signaling, particularly in the posterior cortex. Inactivation of glycogen synthase kinase 3 (GSK-3β) in this region was concomitant with changes in neurodegenerative biomarkers in the cortex and CSF. Metabolomic studies revealed a significant diminishment in creatinine, phenylalanine, and sarcosine levels in the HFD+CBDA group, suggesting it plays an important role in neurological disorders. The results suggest that CBDA has anti-inflammatory properties by reducing the synthesis of lipid inflammatory mediators, which are concomitant with improved insulin signaling and probably reduced neurodegeneration. Thus, CBDA could be considered as a part of future clinical treatment for many inflammatory conditions.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s10787-026-02358-4.

Keywords: Neuroinflammation, Arachidonic acid, Phytocannabinoids, Cyclooxygenase, Brain cortex

Introduction

In today’s modern society, characterized by unlimited access to high-calorie diets, sedentary lifestyles, and chronic stress, there is an increased prevalence of obesity and its comorbidities(Celletti et al. 2026). One of them is inflammation, characterized by excessive production and release of pro-inflammatory cytokines, which in the central nervous system may lead to impairment in many signaling pathways (Van Greevenbroek et al. 2013; Arruda et al. 2011). Chronic, excessive availability of fatty acids in the diet leads to increased deposition of lipids not only in adipose tissue or the liver but also in the brain. As proven in studies on other tissues, not only is excessive accumulation associated with impairment in tissue function, but also the type of deposited lipids is vital, as some of them, like palmitic acid or arachidonic acid (AA), may be lipid precursors of inflammation development. Studies conducted in vitro on isolated rat astrocytes showed that incubation with saturated fatty acids, namely palmitic acid, induced increased release of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6)(Gupta et al. 2012) - two of the most important indicators of neuroinflammation occurrence. Moreover, studies on immortalized embryonic rat and mouse hypothalamic cell lines indicated that exposure to TNFα induced insulin resistance (IR) development through inhibited phosphorylation of protein kinase B (Akt), which has a similar effect to that observed in peripheral tissues (Clemenzi et al. 2019). Evidence has shown that changes in the signaling pathway of Akt that lead to glycogen synthase kinase 3 (GSK-3α/β) dysregulation and tau hyperphosphorylation have been associated with the development of Alzheimer’s disease (AD)(Lauretti et al. 2020; Razani et al. 2021). Thus, there is a strong link between obesity, inflammation, and the development of neurodegenerative diseases. Despite constant progress in pharmacology, new safe treatment methods targeting all elements in the etiopathogenesis of neurodegenerative diseases are still desired. The substance with great potential to become a new remedy for neurodegenerative diseases is cannabidiolic acid (CBDA). CBDA is a cannabinoid isolated from Cannabis sativa, as a precursor of cannabidiol (CBD). Apart from structural similarity to CBD, CBDA also shares similar beneficial effects, such as anti-inflammatory, anti-nociceptive, and anti-convulsant properties(Takeda et al. 2008; Vigli et al. 2021; Ben-Cnaan et al. 2022). The biological effects of CBDA are attributed to its pleiotropic action, characterized by the modulation of key enzymatic pathways involved in immune responses, among which the most important is the inhibition of cyclooxygenase-2 (COX-2) activity (Takeda et al. 2008, 2014; Singh et al. 2026a). Although CBDA is an acidic precursor to CBD, it does not interact with cannabinoid receptors (CB1 and CB2). However, CBDA exerts a remarkably high affinity to the transient receptor potential vanilloid 1 (TRPV1) and the 5-hydroxytryptamine receptor 1 A (HTR1A), alongside the peroxisome proliferator-activated receptors (PPARs) (Muller et al. 2019; D’Aniello et al. 2019; Rock et al. 2021; Hirao-Suzuki et al. 2022; Singh et al. 2026a). COX-2 emerges as of particular significance due to its pivotal role in inflammation development. It has been demonstrated that pharmacological inhibition of COX-2 diminishes the synthesis of prostaglandin E2 (PGE₂), which suppresses high-fat diet (HFD)-induced neuroinflammation and ameliorates neuronal insulin signaling in the rat brain (Zeng et al. 2025; Hassan et al. 2026).

Interestingly, CBDA has higher oral bioavailability in the bloodstream compared to CBD. Moreover, while CBD crosses the blood-brain barrier highly efficiently due to its neutral, highly lipophilic structure, CBDA exhibits lower brain penetration in mice (Anderson et al. 2019; Singh et al. 2026b). CBDA is still understudied, and its possible clinically relevant properties have become less significant due to the popularity of its derivative. A study by Formato et al. in 2020 showed that searching for “cannabidiol” in the PubMed library returned 2997 results, but only 104 matches were found when searching for information on CBDA (Formato et al. 2020). Today, a search for ‘cannabidiol’ returns 8639 results, compared to 338 outcomes for CBDA. Over the past 6 years, researchers have not shifted their focus to cannabidiolic acid, even though it has clearly exhibited potentially useful effects in rodent models. That is why, in our research, we focused on CBDA and deeply studied its effects.

Encouraged by literature data about CBDA’s properties, the present study aimed to evaluate the effect of short-term CBDA treatment on lipid deposition, inflammatory and insulin signaling pathways, as well as neurodegenerative markers in the frontal and posterior parts of the brain cortex of high-fat diet-induced obese rats. Moreover, to investigate the impact of CBDA on the metabolic state, an untargeted and targeted metabolomic analysis of cerebrospinal fluid (CSF) was performed. The main assumption of this study was that CBDA, through a decrease in lipid deposition, may diminish inflammation development, resulting in increased insulin sensitivity of the brain cortex and improved levels of neuroinflammatory markers assessed in brain tissue and CSF. If so, CBDA may in the future become a supportive drug in the treatment of comorbidities associated with obesity.

Materials and methods

Animals and experimental design

All the experiments were performed in accordance with the ARRIVE guidelines, and the Polish Animal Ethics Committee in Olsztyn under license number 35/2023 approved all procedures. Six-week-old male Wistar rats (initial body weight 70–100 g) obtained from the Centre for Experimental Medicine of the Medical University of Bialystok (Poland) were used in this study. The rats were given 7 days to acclimatize to their housing environment. Throughout the acclimatization period and the experimental period, the rats were maintained in a temperature- and humidity-controlled facility (22 ± 2 °C, 55 ± 5% humidity) on a reverse 12-hour light/dark cycle. Animals were housed in acrylic cages with two rats per cage, with ad libitum access to provided food and water.

Animals were selected and separated into four experimental groups using a random number generator’s randomization method:

Control group—was fed a basal rodent diet consisting of 12.4 kcal% fat, 57.1 kcal% carbohydrates, and 30.5 kcal% protein purchased from Animal Feed Manufacturer “Morawski” (Labofeed B, Kcynia, Poland) for 8 weeks. The nutritional and fatty acid formulations are available in the literature (Pastuszewska et al. 2000; Nowacki et al. 2017).
CBDA group—was fed the basal rodent diet (the same as the control group) for 8 weeks and during the last 14 days of the diet also treated once a day with freshly prepared just before use synthetic CBDA (0.1 mg/kg body mass, purity ≥ 99%; THC Pharm GmbH, Frankfurt, Germany) in a single intragastric dose at the same time of the day.
HFD group—was fed a rodent diet rich in fatty acids consisting of 60% fat, 20 kcal% carbohydrates, and 20 kcal% protein, purchased from Research Diets Inc. (cat. no.: D12492, New Brunswick, NJ, USA) for 8 weeks. The nutritional and fatty acid formulations are available in the literature (Research Diets 2026; Zalewska et al. 2019).
HFD+CBDA group—was fed a rodent diet rich in fatty acids (the same as the HFD group) for 8 weeks and during the last 14 days of the diet also treated intragastrically once a day with freshly prepared just before use synthetic CBDA (0.1 mg/kg body mass, purity ≥ 99%; THC Pharm GmbH, Frankfurt, Germany) in a single dose at the same time of the day.

Sesame oil was used as a vehicle for CBDA; it was given to animals from every group with or without CBDA in a volume of 1 ml/kg body mass through a gastric tube designed for adult rats. The CBDA dose was selected based on our preliminary studies and available literature as the most effective when administered intragastrically (Nadal et al. 2017; Rock et al. 2018). The total number of animals used in the study was 40, with 10 animals in each experimental group. Body weight and the amount of food consumed were measured every day until euthanasia. At the end of the experimental period, which was 24 h after the last dose of CBDA or its solvent, animals were anesthetized intraperitoneally with ketamine: xylazine (80 mg/kg:5 mg/kg body mass). From deeply anesthetized animals, large volumes of blood were collected, and the animals were killed by heart removal. The blood was collected in centrifuge tubes containing heparin as an anticoagulant and centrifuged for 15 min at 1000 x g at 4℃ within 30 min of collection. Next, the plasma was collected and aliquoted at -80℃ for further analysis. Immediately after sacrifice, samples of four brain regions, namely the frontal and posterior cerebral cortex, hippocampus, and subcortical nuclei (amygdala and nucleus basalis of Meynert), were anatomically identified with the use of a brain matrix and tissue punch needles (Palkovits punch technique), excised, frozen in liquid nitrogen with precooled aluminium tongs, and stored at -80℃ for further analysis (Herman and Watson 1987; Jaszczyk et al. 2022).

CSF collection

From dormant animals, samples of cerebrospinal fluid were collected and immediately frozen in liquid nitrogen according to the method described by Li et al. with slight modifications (Li et al. 2016). To collect high-quality CSF (without blood), a 1 ml insulin syringe with a 27 G needle (0.4 × 13 mm) was used. The dormant animal was placed on a brain stereotaxic platform with its head bent to 45°. After partial skin removal from the back of the neck, the area was disinfected with alcohol. Next, the depressed spot between the atlas and occipital protuberance was located, and the needle with the syringe was slowly inserted into the middle of the located spot, directed towards the apex of the nose until no resistance was felt. The lack of resistance indicated the moment when the end of the needle reached the cerebellomedullary cistern. Then, the plunger in the syringe was pulled back to create a negative pressure, drawing the CSF into the syringe. The amount of CSF collected from each rat varied between 100 and 150 µl.

Gas-liquid chromatography

Gas-liquid chromatography was used to determine the concentrations of individual fatty acid methyl esters extracted from samples of four brain regions (frontal and posterior cerebral cortex, hippocampus, and subcortical nuclei). Extraction was performed with a chloroform-methanol solution following the method described by Folch et al. (FOLCH et al. 1957). The internal standard, which was heptadecanoic acid (C17:0), was used at the start of the procedure. The extracted fatty acids were separated into four lipid fractions: phospholipids (PH), triacylglycerols (TAG), free fatty acids (FFA), and diacylglycerols (DAG) using thin-layer chromatography (TLC) as previously detailed. (Konstantynowicz-Nowicka et al. 2019, 2026) The individual fatty acid fractions were methylated in a solution containing 14% methanol with boron trifluoride and quantified based on the retention times of standards (Lanodan Research grade lipids, Stockholm, Sweden) using a Hewlett-Packard 5890 Series II Gas Chromatograph (Agilent Technologies, CA, USA) equipped with a flame ionization detector and a capillary column (HP-INNOWax 50mx0.25 mm inner diameter). The concentrations of PH, TAG, FFA, and DAG lipid fractions were calculated as the sum of the individual assessed fatty acid species in the selected fractions and expressed in nanomoles per gram of tissue. Additionally, the concentration of arachidonic acid (AA, 20:4) in specific lipid fractions was also expressed in nanomoles per gram of tissue.

Immunoblotting

The Western blot technique was used to assess the expression of specific proteins involved in the insulin signaling pathway and neurodegeneration. As previously described (Konstantynowicz-Nowicka et al. 2026), samples of the frontal and posterior cortex were homogenized in ice-cold lysis buffer (RIPA- radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors; Roche, Mannheim, Germany) and centrifuged. The obtained supernatant was used to assess total protein concentrations using the bicinchoninic acid (BCA) method and mixed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Next, 30 µg of total protein was separated by electrophoresis on Criterion TGX Stain-Free Precast Gels (Bio-Rad; Hercules, CA, USA). After electrophoresis, the gel was activated under UV light to form a cross-linked fluorescent product by binding the trihalo label (incorporated into the pre-cast gel) with tryptophan residues in the sample proteins. The separated proteins were transferred onto PVDF (semi-dry transfer) or nitrocellulose (wet transfer) membranes. The membranes containing fluorescently modified total proteins were then detected with a CCD imager and UV illumination. Subsequently, the membranes were blocked with either 5% non-fat dry milk or 5% BSA and incubated overnight with a suitable primary antibody against: beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) (1:1000), amyloid precursor protein (APP) (1:1000), tau (1:1000), ptau Ser202 (1:1000), ptau Ser396 (1:1000), ptau Ser404 (1:1000), protein kinase B (Akt) (1:1000) (Cell Signaling Technology Inc., Danvers, MA, USA), pAkt Ser473/474/472 (1:500; Abcam, Cambridge, UK), phosphorylated protein kinase B (pAkt Thr308/309/305) (1:500), pGSK-3α/β Tyr279/216 (1:500), lactate dehydrogenase (LDH) (1:500), TRPV1 (1:1000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GSK-3α/β (1:1000), pGSK-3α/β Ser21/9 (1:1000), insulin receptor substrate 1 (IRS-1) (1:1000), pIRS-1 Ser302 (1:1000) (Cell Signaling Technology Inc., Danvers, MA, USA), and HTR1A (1:3000) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). Next, horseradish peroxidase-conjugated (HRP) immunoglobulin G secondary antibody suitable for the primary antibody was used to incubate the membranes and visualize the protein bands with a chemiluminescence substrate (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA, USA). The ImageLab system attached to the ChemiDoc visualization system (Bio-Rad, Warsaw, Poland) was used to densitometrically analyze the immunoblotting signals of the obtained protein and total protein. To normalize the expression levels of the assessed protein, the image of the total protein and the protein of interest were overlapped in the ImageLab system. The control group was established at 100%.

Real-time polymerase chain reaction

The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the mRNA levels of genes from the inflammatory pathway. The TriReagent RNA isolation technique was used to extract total RNA from frontal and posterior cortex tissue according to the manufacturer’s protocol (Sigma Aldrich, Saint Louis, MO, USA; cat. No.: T9424) and the quality and concentration of obtained RNA were measured on a microplate Synergy H1 Hybrid Reader (BioTek Instruments, Winooski, VT, USA) at 260 and 280 nm wavelength. The synthesis of cDNA was performed from1 µg of mRNA using the EvoScript Universal cDNA Master kit (Roche Molecular Systems, Boston, MA, USA; cat. No.: 05893151001) according to the manufacturer’s instructions. The incubation conditions were as follows: 42 °C for 900s, 85 °C for 300s, 65 °C for 900s, and a cooling step at 4 °C for a minimum of 240s, which was performed on the LightCycler 96 Instrument with a real-time thermal cycler (Roche Diagnostics, Boston, MA, USA). The same instrument was used for the qRT-PCR with the use of the FastStart Essential DNA Green Master kit (Roche Molecular Systems, Boston, MA, USA; cat. No.: 04913850001). The reaction parameters were set as follows: preincubation (95 °C for 600s), three-step amplification (denaturation step at 95 °C for 15s, annealing step at 62 °C (COX-1, 5-LO, 15-LO), 55 °C (Il-10, Il-1β) 58 (TNFalpha, IL-6), 60 °C (glyceraldehyde-3-phosphate dehydrogenase - GAPDH, COX-2, cluster of differentiation 11b - CD11b, cluster of differentiation 68 - CD68) 64 °C (hexokinase 1 - HXK1), and 67 °C (ionized calcium-binding adapter molecule 1 - IBA1) for15s each, extension step at 72 °C for 15s, and a melting step at 95 °C for 10s, 70 °C for 15s, and 97 °C for 1s). The PCR product specificity was validated by the analysis of the melting curve obtained at the end of each reaction, and the mRNA levels of selected genes were normalized to the housekeeping gene GAPDH expression. According to the Pfaffl method (Pfaffl 2001; Dong et al. 2024) with slight modifications, the final gene expression levels were calculated, and the control group was set as 1. All the samples were analyzed in duplicate. The primer sequences used were:

GAPDH: Forward: 5′-TGCACCACCAACTGCTTA-3’,

Reverse: 5’-GGATGCAGGGATGATGTTC-3′.

COX-1: Forward: 5′-CCGGTACTGCTCACAGATGCT-3’,

Reverse: 5’-CCCGTGCGAGTACAGTCACAT-3’.

COX-2: Forward:5′- GCCCTTCCTCCTGTGGC-3’,

Reverse: 5’- TTGAATCAGGAAGTTCCTTATTTCCT-3’.

5-LO: Forward:5′- ACCCCCTTTTCAAGCTGCTGG-3’,

Reverse: 5’- CGGCAATCACGCTGACGATG-3’.

15-LO:Forward:5′-CGGGATCCGTGGGACTGGCTACTGGCC-3’,

Reverse:5’GGAATTCGCATGCTTGGCTGAGCAGG-3’

TNFalpha: Forward:5′-GCCAATGGCATGGATCTCAAAG-3’,

Reverse: 5’-CAGAGCAATGACTCCAAAGT-3’.

IL-6: Forward:5′- TCAACT CCATCTGCCCTTCAG-3’,

Reverse: 5’-AAGGCAGTGGCTAACAAC-3’.

IL-10: Forward:5′- GTTGCCAAGCCTTGTCAGAAA-3’,

Reverse: 5’-TTTCTGGGCCATGGTTCTCT-3’.

IL-1β: Forward:5′-CAGGAAGGCAGTGTCACTCA-3’,

Reverse: 5’-GGGATTTTGTCGTTGCTTGT-3’.

IBA1: Forward:5′-CTAAGGCCACCAGCGTCTGA-3’,

Reverse: 5’-AGCTTTTCCTCCCTGCAAATCC-3’.

CD11b: Forward:5′-ACAGAGACCAAAGTGGAGCC-3’,

Reverse: 5’-GCCACCGGCTTCATTCATCA-3’.

CD68: Forward:5′-TCCTTCACGGAGACACCT-3’,

Reverse: 5’-GGCTGGGAACCATTAGTC-3’.

HXK1: Forward:5′-CTGGGCTTCACCTTCTCATTT-3’,

Reverse: 5’-TCGCAAGTGGGTTCTTCATAC-3’.

Enzyme-linked immunosorbent assay

The quantitative determination of the concentration of BDNF in CSF and plasma was conducted using a commercially available colorimetric kit (Rat BDNF ELISA Kit, cat no.: NBP3-42309, Novus Biologicals, Centennial, CO, USA). The assessment was carried out according to the manufacturer’s instructions, and a 2-fold dilution of CSF and plasma was prepared. The absorbance of the obtained color product was measured immediately using Synergy H1TM (BioTek Instruments, Winooski, VT, USA) at a wavelength of 450 nm. The final concentration of BDNF was expressed in picograms per milliliter (pg/ml).

The same technique was used to determine the concentration of PGE2 (Rat Prostaglandin E2 ELISA Kit, cat no.: CSB-E07967r, Cusabio, Wuhan, China) and LTB4 (Rat Leukotriene B4 ELISA Kit, cat no.: CSB-E08035r, Cusabio, Wuhan, China) in the frontal and posterior parts of the cortex. Tissue homogenization and assessment were carried out according to the manufacturer’s instructions, and a 2-fold dilution of the obtained supernatant was prepared. The absorbance of the obtained color product was measured immediately using Synergy H1TM (BioTek Instruments, Winooski, VT, USA) at a wavelength of 450 nm with wavelength correction set to 540 nm for LTB4 measurement. The final concentration of LTB4 and PGE2 was expressed in picograms per milliliter (pg/ml).

Moreover, the quantitative determination of the concentration of beta-amyloid 42 (Aβ42) in frontal and posterior cortex was conducted using a commercially available colorimetric kit (Mouse Aβ42 ELISA Kit, cat no.: KMB3441, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). The frontal and posterior brain cortex homogenates were prepared according to the manufacturer’s instructions, and a final 2-fold dilution was prepared. The absorbance of the obtained color product was also measured immediately using Synergy H1TM (BioTek Instruments, Winooski, VT, USA) at a wavelength of 450 nm. The final concentration of Aβ42 was expressed in picograms per milliliter (pg/ml).

Metabolite extraction

A solution of 80% methanol in water was used to extract metabolites from cerebrospinal fluid (20 µl) as previously described (Lepoittevin et al. 2023; Chojnowski et al. 2025). Before extraction, equal quantities of the internal standard (IS; Cell Free Amino Acid Mixture − 13 C, 15 N, Sigma-Aldrich) were added to the samples. Samples were then vortexed and centrifuged twice to collect the supernatants (12 500 RPM, 15 min., 4 °C). Resulting extracts were evaporated to dryness using a SpeedVac centrifuge at 50 °C and stored at − 80 °C until analysis. Before LC-MS/MS analysis, dried extracts were reconstituted in 20 µl of 0.1% formic acid in water, vortexed, and centrifuged (12 500 RPM, 15 min., 4 °C). Quality control and calibration curve samples were prepared in the same matrix for absolute quantification (targeted analysis) and batch normalization (untargeted analysis).

Untargeted and targeted metabolomics analysis

The untargeted measurements were conducted on an Exploris 480 Orbitrap mass spectrometer coupled with a Vanquish UHPLC system (Thermo Fisher Scientific) separately in positive and negative ion mode as previously described (Chojnowski et al. 2025). Resulting measurement files were processed in Compound Discoverer 3.5 (Thermo Fisher Scientific). The resulting metabolite list was filtered using the following criteria: peak rating ≥ 4.5 in at least five samples, annotation mass tolerance within ± 2 ppm, annotation match in ≥ 3 sources, and exclusion of background ions. Statistical analysis was performed in the MetaboAnalyst 6.0 platform (Pang et al. 2024) on log2-transformed auto-centered values normalized by sum using Student’s t-tests to determine compounds with statistically significant changes between HFD+CBDA and HFD study groups (FDR-based p-adjusted value < 0.05, fold change ≥ 2). Identities of compounds with statistically significant changes between HFD+CBDA and HFD experimental groups were additionally confirmed with the FISh scoring algorithm applied to their registered fragment ion spectra.

The targeted analysis for amino acids and amino acid derivatives quantification was executed on a TSQ Vantage mass spectrometer coupled with a Surveyor HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode using a previously developed Multiple Reaction Monitoring assay (Olkowicz et al. 2017). Resulting files were processed with an automated method in the Xcalibur 4.7 SP1 software (Thermo Fisher Scientific) and manually inspected to confirm retention times to extract peak areas of 36 target compounds and corresponding internal standards. A linear regression analysis of the calibration curve samples was performed to establish absolute concentration in experimental samples. Statistical analysis was performed in the MetaboAnalyst 6.0 platform (Pang et al. 2024) on log2-transformed values using Student’s t-tests to determine compounds with statistically significant changes between HFD+CBDA and HFD and between HFD and control study groups (FDR-based p-adjusted value < 0.05).

Statistical analysis

All data were analyzed using GraphPad Prism for macOS Version 10.2.1 Software (San Diego, CA, USA) with the significance level set at p < 0.05. Data are given as the mean values and standard deviation (SD) for 6 animals per group (Western blot and PCR) or 10 animals per group (GLC, metabolomics, ELISA kits). The Shapiro‒Wilk and Bartlett’s tests were conducted to evaluate the normality of the data distribution and the homogeneity of the variance, respectively. Statistical analysis was conducted via one- or two-way ANOVA followed by the appropriate post hoc test. No correction for multiple comparisons was applied; if it were, the significance threshold would be a p-value less than 0.01250 when using the Bonferroni correction. It is important to mention that the large number of measured endpoints increases the possibility of type I error despite reporting exact p-values. The number of animals in each group was determined before the experiment using GraphPad Prism for macOS Version 10.2.1 Software (San Diego, CA, USA) to determine the size of the sample. The statistical analysis carried out included the following parameters: (1) the size of the expected effect between groups − 40% (n = 10) and 30% (n = 6), (2) standard deviation − 25 (n = 10) and 14 (n = 6), (3) the power of experience (the probability of a chance to detect statistical significance) − 0.9 (n = 10, n = 6), (4) statistical significance level − 0.05 (n = 10, n = 6).

Results

Effects of cannabidiolic acid treatment on total intracellular lipid fraction concentrations in four rat brain regions

In the group of rats fed with HFD and administered with CBDA, the concentration of DAG was significantly decreased in the frontal and posterior cortex (− 31.96%, p = 0.0079; − 34.48%, p = 0.0079; Fig. 1A, respectively) compared to the HFD alone group. Additionally, an increase in DAG was observed in the group of rats fed with HFD compared to the control group in the frontal cortex (67.24%, p = 0.0079; Fig. 1A).

The only change observed in the FFA fraction was an increase in FFA concentration in the posterior cortex (19.65%, p = 0.0232; Fig. 1B) compared to the HFD alone group.

A significant decrease in TAG concentration was observed in the HFD+CBDA group in the frontal and posterior cortex (− 30.79%, p = 0.0049; − 6.19%, p < 0.0001; Fig. 1D, respectively) compared to the HFD alone group. Additionally, there was a decrease in TAG concentration in the posterior cortex (− 30.86%, p = 0.0113; Fig. 1D) compared to the control group. Furthermore, in the group of rats fed with HFD, we observed a rise in TAG concentration in the posterior cortex (28.50%, p = 0.013; Fig. 1D) compared to the control group.

In the hippocampus and subcortical nuclei DAG concentration was significantly increased in the HFD group compared to the control group (129.39%, p = 0.0027; 197.85%, p = 0.001; Supplementary Fig. 1A and E, respectively) and decreased (− 31.85%, p = 0.0424; − 39.59%, p = 0.0288; Supplementary Fig. 1A and E, respectively) in the HFD+CBDA group compared to the HFD alone group. Additionally, an increase in DAG in the same group was observed in both regions (129.39%, p = 0.0027; 197.85%, p = 0.001; Supplementary Fig. 1A and E, respectively) when compared to the control group. A significant DAG elevation was noticed in the CBDA-only group (59.27%, p = 0.0023; Supplementary Fig. 1E) compared to the control group in subcortical nuclei. A significant decrease in FFA concentration was observed in the HFD+CBDA group in subcortical nuclei (− 24.92%, p = 0.0079; Supplementary Fig. 1F) compared to the HFD alone group. Conversely, an elevated concentration of FFA was measured in the group of rats only fed with HFD (48.74%, p = 0.0317; Supplementary Fig. 1F) compared to the control group in the same region. The concentration of PH notably increased only in subcortical nuclei in the group of rats administered with CBDA and fed with HFD (19.00%, p = 0.0165; Supplementary Fig. 1G) compared to the control group. A rise in TAG concentration in the HFD alone group was observed (54.84%, p = 0.0005; Supplementary Fig. 1D) in comparison to the control group in the hippocampal region. In the same brain region, a relevant drop in TAG concentration was measured in the HFD+CBDA group (− 54.27%, p < 0.0001; Supplementary Fig. 1D) when compared to the HFD alone group and to the control group (− 29.19%, p = 0.0038; Supplementary Fig. 1D).

Effects of cannabidiolic acid treatment on arachidonic acid concentration in lipid fractions in four rat brain regions

In the frontal and posterior cortex, the intracellular concentration of 20:4 in the DAG lipid fraction was decreased in the HFD group treated with CBDA (− 19.94%, p = 0.0317; − 25.38%, p = 0.0001; Fig. 2A, respectively) compared to the HFD alone group. Additionally, in the frontal cortex, it was reduced (− 30.25%, p = 0.0005; Fig. 2A) compared to the control group. Moreover, an elevation of 20:4 in DAG was observed in the posterior cortex of rats fed only a high-fat diet (25.52%, p = 0.0059; Fig. 2A) compared to the control group. The concentration of 20:4 in FFA was increased in both regions – frontal and posterior cortex – in the CBDA-only group (17.58%, p = 0.0435; 33.42%, p = 0.0334; Fig. 2B, respectively) compared to the control group. In the posterior cortex, we noticed a decrease in 20:4 in FFA concentration in the HFD+CBDA group (− 26.8%, p = 0.0079; Fig. 2B) compared to the HFD group, as well as an increase in the group of rats fed with a high-fat diet (63.93%, p = 0.0079; Fig. 2B) compared to the control group. The only significant change in 20:4 concentration in the PH was observed in the frontal cortex, where in the group of rats fed with a high-fat diet and administered with CBDA, a lowered concentration was noted (− 8.79%, p = 0.0294; Fig. 2C) compared to the HFD alone group. Moreover, in the HFD group, the 20:4 levels in the PH increased significantly (10.12%, p = 0.006; Fig. 2C) compared to the control group. In both the frontal and posterior cortex of the rats in the HFD+CBDA group, a significant decline in 20:4 TAG concentration was observed (− 46.94%, p < 0.0001; − 30.42%, p = 0.0094; Fig. 2D, respectively) compared to the HFD alone group. However, only in the frontal cortex was it significantly decreased (− 37.08%, p = 0.0002; Fig. 2D) compared to the control group. Furthermore, in the frontal and posterior cortex, 20:4 TAG levels were elevated in the HFD-only group (18.57%, p = 0.0074; 21.1%, p = 0.0369; Fig. 2D, respectively) compared to the control group.

Fig. 2 — The concentration of arachidonic acid (C20:4) in lipid fractions in the frontal and posterior cortex. The concentration of arachidonic acid in A diacylglycerols (DAG), B free fatty acids (FFA), C phospholipids (PH), and D triacylglycerols (TAG) in groups: fed a standard rat chow (CONTROL), fed a standard rat chow and treated with CBDA (CBDA), fed a high-fat diet (HFD), and fed a high-fat diet and treated with CBDA (HFD+CBDA). The data are presented as the mean values ± SD with n = 10 in each group. *–p < 0.05; **–p < 0.01; ***–p < 0.001; ****–p < 0.0001 significant change versus CONTROL group; #–p < 0.05; ##–p < 0.01; ###–p < 0.001; ####–p < 0.0001 significant change versus HFD group

In the hippocampus, the 20:4 concentration in the DAG fraction in the group of rats fed with HFD and administered with CBDA was significantly reduced (− 40.8%, p = 0.0061; Supplementary Fig. 2A) compared to the HFD alone group and (− 22.28%, p = 0.013; Supplementary Fig. 2A) compared to the control group. Furthermore, in the HFD alone group, 20:4 in the DAG was elevated (31.28%, p = 0.0496; Supplementary Fig. 2A) compared to the control group, along with a substantial drop in 20:4 in the DAG fraction in the CBDA-only group (− 37.63%, p = 0.0012; Supplementary Fig. 2A) relative to the control. On the other hand, in subcortical nuclei, the 20:4 concentration in DAG in the HFD+CBDA group was increased (43.69%, p = 0.0363; Supplementary Fig. 2E) compared to the control group. The only change in 20:4 concentration in FFA was observed in subcortical nuclei, where a significant increase was found in the rats from the HFD alone group (29.74%, p = 0.0187; Supplementary Fig. 2F) compared to the control group. In the PH lipid fraction, the 20:4 concentration was affected in the hippocampal region, where we observed a marked decrease in the HFD+CBDA group and CBDA-only group (− 17.39%, p = 0.0030; − 13.87%, p = 0.0178; Supplementary Fig. 2C, respectively) compared to the control group. Significant differences in TAG fraction in 20:4 concentration were notable in both brain regions. In the hippocampus and subcortical nuclei of the HFD+CBDA groups, a significant drop was observed (− 37.43%, p = 0.0001; − 46.99%, p < 0.0001; Supplementary Fig. 2D and Supplementary Fig. 2H, respectively) when compared to the HFD alone group. In the hippocampus, it was significantly lower (− 64.62%, p < 0.0001; Supplementary Fig. 2D) also in comparison with the control group. Furthermore, the concentration of 20:4 in TAG decreased significantly in the CBDA group (− 47.99%, p < 0.0001; Supplementary Fig. 2D) as well as in the HFD alone group (− 43.45%, p = 0.0001; Supplementary Fig. 2D) compared to the control in the hippocampal region. Additionally, changes were observed in the subcortical nuclei, where a significant decline was detected in the CBDA group (− 48.22%, p = 0.0079; Supplementary Fig. 2H) compared to the control group. On the other hand, a rise in 20:4 concentration in TAG was noted in the HFD alone group (53.72%, p = 0.0079) when compared to the control group in the same region.

Effects of cannabidiolic acid treatment on the expression of insulin signaling pathway proteins and glucose metabolism enzymes in the rat frontal and posterior cortex

Changes in the frontal cortex were as follows: in the group of rats fed a high-fat diet and administered with CBDA, the ratio of pAkt Ser473/474/472 to Akt protein was significantly decreased (− 41.91%, p = 0.0218; Fig. 3C) when compared to the control. Additionally, the pGSK-3α Tyr279 to GSK-3α ratio increased in the HFD-alone group and dropped in the CBDA-only group (44.27%, p = 0.0241; − 49.15%, p = 0.0048; Fig. 3E, respectively) in comparison to the control. The pGSK-3β Tyr216 to GSK-3β ratio went up in the HFD-only group (41.00%, p = 0.0087; Fig. 3G) when compared to the control, but on the other hand, it significantly declined in the HFD+CBDA group (− 30.20%, p = 0.0087; Fig. 3G) relative to the HFD-only group.

Fig. 3 — The expression ratio of phosphorylated proteins from the insulin signalling pathway to their non-phosphorylated forms in the frontal cortex. The ratio of A phosphorylated protein kinase B at Thr308 to protein kinase B (pAkt Thr308/Akt), B phosphorylated protein kinase B at Thr309 to protein kinase B (pAkt Thr309/Akt), C phosphorylated protein kinase B at Ser473/474/472 to protein kinase B (pAkt Ser473/474/472/Akt), D phosphorylated glycogen synthase kinase 3α at Ser21 to glycogen synthase kinase 3α (pGSK-3α Ser21/GSK-3α), E phosphorylated glycogen synthase kinase 3α at Tyr279 to glycogen synthase kinase 3α (pGSK-3α Tyr279/GSK-3α), F phosphorylated glycogen synthase kinase 3β at Ser9 to glycogen synthase kinase 3β (pGSK-3β Ser9/GSK-3β), G phosphorylated glycogen synthase kinase 3β at Tyr216 to glycogen synthase kinase 3β (pGSK-3β Tyr216/GSK-3β) in groups: fed a standard rat chow (CONTROL), fed a standard rat chow and treated with CBDA (CBDA), fed a high-fat diet (HFD) and fed a high-fat diet and treated with CBDA (HFD+CBDA). The data are expressed as the mean values ± SD with n = 6 in each group and presented as a percentage change relative to the Control set as 100% for Western blot. The displayed blots are representative examples, whereas the quantitative analyses were performed using all biological replicates. *–p < 0.05; **–p < 0.01; ***–p < 0.001; ****–p < 0.0001 significant change versus CONTROL group; #–p < 0.05; ##–p < 0.01; ###–p < 0.001; ####–p < 0.0001 significant change vs. HFD group

In the posterior cortex, the pAkt Thr309 to Akt ratio decreased in the HFD alone group (− 17.95%, p = 0.0431; Fig. 4B) when compared to the control. Furthermore, the pAkt Ser473/474/472 to Akt ratio declined in the HFD alone group (− 33.13%, p = 0.0427; Fig. 4C) in comparison to the control and rose in the HFD+CBDA group (67.54%, p = 0.0152; Fig. 4C) when compared to the HFD group. Significant changes also happened to the pGSK-3α Ser21/GSK-3α ratio, where in both CBDA alone and the HFD-only groups it decreased (− 36.81%, p = 0.0229; − 51.39%, p = 0.0087; Fig. 4D, respectively) in comparison to the control, whereas in the group of rats fed with HFD and administered with CBDA it went up (76.37%, p = 0.0087; Fig. 4D) when compared to HFD alone group. Additionally, the pGSK-3β Ser9 to GSK-3β ratio increased in the HFD+CBDA group (56.34%, p = 0.0214; Fig. 4F) relative to the HFD alone group.

In the frontal cortex, expression of LDH was elevated in both the CBDA-only and the HFD-alone groups (17.72%, p = 0.0152; 20.22%, p = 0.0152; Supplementary Fig. 3A, respectively) when compared to the control. On the contrary, it decreased in the HFD+CBDA group (− 10.84%, p = 0.0364; Supplementary Fig. 3A) in comparison to the HFD-only group. In the posterior cortex, LDH expression was increased in the HFD-only group (35.24%, p = 0.0053; Supplementary Fig. 3A) when compared to the control, whereas in the HFD+CBDA group it was decreased (− 28.52, p = 0.0029; Supplementary Fig. 3A) in comparison to the HFD-only group. The sole change in the ratio of pIRS-1 Ser302 to IRS-1 was observed in the posterior cortex in the HFD+CBDA group, where it was reduced (28.78, p = 0.0369; Supplementary Fig. 3B) relative to the control group. In posterior cortex, the HXK1 expression was decreased in both CBDA alone and HFD-only (− 6.08%, p = 0.0298; − 18.56%, p < 0.0001; Supplementary Fig. 3C, respectively) when compared to the control, whereas in frontal cortex it was elevated in CBDA-only and decreased in the HFD-alone groups (37.21%, p < 0.0001; − 50.88%, p < 0.0001; Supplementary Fig. 3C, respectively) in comparsion with the control group. Additionally, in the HFD+CBDA group, HXK1 expression was elevated (26.48%, p = 0.0023; 25.43%, p = 0.0022; Supplementary Fig. 3C, respectively) relative to the HFD alone group in both the frontal and posterior cortex, respectively. In addition to that, only in the frontal cortex in the HFD+CBDA group was the expression also decreased (− 37.87%, p < 0.0001; Supplementary Fig. 3C) compared to the control.

Effects of cannabidiolic acid treatment on the expression of proteins involved in the neurodegeneration process in the rat frontal and posterior cortex

In the frontal brain cortex, BACE1 expression was decreased in the HFD+CBDA group (− 32.48%, p = 0.0041; Fig. 5A) relative to the HFD group. Furthermore, it was increased in the HFD group (54.13%, p = 0.0029; Fig. 5A) compared to the control. The difference in APP expression was observed in the HFD-only group, where it was significantly increased (32.94%, p = 0.0032; Fig. 5B) compared to the control group. The sole change in tau protein was an increase in the ptau Ser202 to tau protein ratio in the HFD-only group (63.86%, p = 0.0018; Fig. 5D) when compared to the control.

In the posterior part of the brain cortex, the expression of the BACE1 protein was significantly decreased in the HFD+CBDA group (− 24.62%, p = 0.0021; Fig. 6A) compared to the HFD group. Furthermore, in the rats only fed with HFD, the expression of the above-mentioned protein was increased (15.17%; p = 0.0001; Fig. 6A) in comparison to the control group. Changes in APP expression were noticed in the HFD+CBDA group, where it was significantly decreased (− 24.50%, p = 0.0015; Fig. 6B) in comparison with the HFD-only group.

Effects of cannabidiolic acid on the expression of proteins associated with the inflammatory process in the rat frontal and posterior cortex

The expression of COX-1 was significantly decreased in both the frontal and posterior cortex in the HFD+CBDA group (− 9.02%, p = 0.0271; − 9.4%, p = 0.0304; Fig. 7A, respectively) in comparison with the HFD group. Additionally, in both regions, the expression of COX-1 was elevated in the HFD group (14.08%, p = 0.0028; 14.61%, p = 0.0007; Fig. 7A, respectively, in the frontal and posterior cortex) when compared to the control. Significant alterations in COX-2 expression were noticed in the HFD+CBDA group in the frontal and posterior cortex (− 9,74%, p < 0.0001; − 7.6%, p = 0.0108; Fig. 7B, respectively) when compared to the HFD alone group. An increase was detected in each region in the HFD-only group (10.37%, p < 0.0001; 7.44%, p = 0.0153; Fig. 7B, respectively, in the frontal and posterior cortex) relative to the control. In the HFD+CBDA group, changes in 5-LO expression were measured in the frontal cortex, where it was decreased (− 16.74%, p = 0.0043; Fig. 7C) in comparison to the HFD-only group, whereas in the posterior cortex it dropped (− 25.12%, p = 0.0036; Fig. 7C) compared to the control. In both regions, 5-LO expression was elevated in the HFD group (27.96%, p = 0.0022; 36.82%, p = 0.0008; Fig. 7C, respectively, in the frontal and posterior cortex) compared to the control group. An enhancement in 15-LO expression was observed in the HFD+CBDA group in the frontal and posterior cortex (20.92%, p = 0.0459; 32.25%, p = 0.0004; Fig. 7D, respectively) compared to the HFD alone group. Additionally, in the posterior cortex, there was a significant elevation (32.44%, p = 0.0022; Fig. 7D) compared to the control.

The expression of IL-1β was decreased in the CBDA group (− 13.69%, p = 0.0008; Fig. 8A) only in the posterior cortex, whereas in both the frontal and posterior cortex it was elevated in the HFD alone group (24.07%, p = 0.0007; 10.78%, p = 0.0208; Fig. 8A, respectively) when compared to the control. In addition to that, in the HFD+CBDA group the expression was decreased (− 23.16%, p < 0.0001; Fig. 8A) in comparsion with the HFD alone group in frontal cortex, Similarly, in posterior cortex it was lowered relative to the control and to the HFD-only group (− 18.82%, p < 0.0001; − 26.72%, p < 0.0001; Fig. 8A, respectively). IL-6 expression was increased in both brain regions in the HFD-alone group (27.14%, p = 0.0018; 38.31%, p < 0.0001; Fig. 8B, respectively) when compared to the control. Moreover, it was increased in the HFD+CBDA group (16.26%, p = 0.003; Fig. 8B) in comparison to the control solely in the posterior cortex. In the same group, a decrease in expression (− 16.17%, p = 0.0021; − 15.94%, p < 0.0001; Fig. 8B, respectively) was observed in both regions relative to the HFD-only group. Decreased expression of IL-10 was observed in the HFD-only group (− 27.49%, p = 0.0022; − 22.21%, p < 0.0001; Fig. 8C, respectively) in both the frontal and posterior cortex when compared to the control. Furthermore, in the group of rats fed with a high-fat diet and administered with CBDA, in both brain regions the expression of IL-10 was elevated (81.29%, p = 0.0022; 26.95%, p = 0.0004; Fig. 8C, respectively) relative to the HFD-only group, as well as it was increased (31.46%, p < 0.0001; Fig. 8C) when compared to the control only in the frontal cortex. The expression of TNFα in the HFD+CBDA group was reduced in both regions (− 14.52%, p = 0.0029; − 2.04%, p = 0.0152; Fig. 8D, respectively, in the frontal and posterior cortex) compared to the HFD alone group. It was also significantly decreased in the posterior cortex (− 11.99%, p = 0.0043; Fig. 8D) in comparison with the control. In the frontal cortex, TNFα expression was higher in the HFD group (19.16%, p = 0.0029; Fig. 8D) compared to the control. Conversely, within the posterior cortex, a reduction was observed in the CBDA-only group (− 9.01%, p = 0.0045; Fig. 8D) relative to the control. The concentration of PGE2 was increased in both the frontal and posterior cortex in the HFD alone group (29.45%, p = 0.001; 98.70%, p < 0.0001; Fig. 8E, respectively) when compared to the control. Additionally, the concentration of the above-mentioned compound was decreased in the HFD+CBDA group (− 30.67%, p < 0.0001; − 27.36%, p = 0.0002; Fig. 8E, respectively) in comparison with the HFD-only group in both regions, and it was elevated in the same group (44.33%, p = 0.0003; Fig. 8E) relative to the control solely in the posterior cortex. In the posterior cortex, the concentration of LTB4 was increased in the CBDA-only group (9.88%, p = 0.0327; Fig. 8F) relative to the control. In both regions, the concentration of LTB4 was increased in the HFD-alone group (109.52%, p < 0.0001; 32.04%, p < 0.0001; Fig. 8F, respectively) when compared to the control. Furthermore, a decrease was observed in the HFD+CBDA group (− 40.52%, p < 0.0001; − 8.48%, p < 0.0001; Fig. 8F, respectively) in comparison with the HFD-only group in both the frontal and posterior cortex. Only in the frontal cortex, the concentration was increased in frontal cortex in the HFD+CBDA group (24.63%, p = 0.0243; Fig. 8F) relative to the control group.

Effects of cannabidiolic acid treatment on β-amyloid concentration in the frontal and posterior cortex and BDNF concentration in cerebrospinal fluid and plasma

Changes in β-amyloid 42 concentration were observed only in the frontal cortex, where in both the HFD-only and HFD+CBDA group it was elevated (55.35%, p < 0.0001; 27.22%, p = 0.0003; Fig. 9A, respectively) compared to the control. Additionally, in the HFD+CBDA group, the concentration was lower (− 18.11%, p = 0.0116; Fig. 9A) in comparison to the HFD alone group. BDNF concentration in the CSF was significantly increased in the group of rats fed with HFD and administered with CBDA (34.81%, p = 0.017; Fig. 9B) compared to the HFD alone group. On the other hand, there was a significant drop in BDNF cerebrospinal fluid concentration in the HFD and CBDA alone groups (− 24.48%, p = 0.0088; − 27.71%, p = 0.026; Fig. 9B, respectively) compared to the control group. The only change in BDNF plasma concentration was observed in the group of rats fed with HFD, where there was a significant decrease (− 25.94%, p = 0.0202; Fig. 9C) compared to the control group.

Fig. 9 — The concentration of β-amyloid 42 in the frontal and posterior cortex and the concentration of BDNF in CSF and plasma. The A concentration of β-amyloid 42 (Aβ42) and the concentration of brain-derived neurotrophic factor (BDNF) in B cerebrospinal fluid (CSF) and C plasma in groups: fed a standard rat chow (CONTROL), fed a standard rat chow and treated with CBDA (CBDA), fed a high-fat diet (HFD), and fed a high-fat diet and treated with CBDA (HFD+CBDA). The data are presented as the mean values ± SD with n = 10 in each group. *–p < 0.05; **–p < 0.01; ***–p < 0.001; ****–p < 0.0001 significant change versus CONTROL group; #–p < 0.05; ##–p < 0.01; ###–p < 0.001; ####–p < 0.0001 significant change versus HFD group

Effects of cannabidiolic acid treatment on microglial activation markers in the frontal and posterior cortex.

In both regions, the expression of IBA1 was decreased in the CBDA group (− 29.44%, p = 0.0022; − 7.63%, p = 0.0411; Supplementary Fig. 4A, respectively), whereas it was increased in the HFD alone group (24.40%, p = 0.0004; 24.03%, p = 0.0001; Supplementary Fig. 4A, respectively) when compared to the control. Moreover, in the HFD+CBDA group, the expression was reduced in the frontal cortex (− 5.65%, p = 0.0316; − 24.15, p < 0.0001; Supplementary Fig. 4A, respectively) and in the posterior cortex (− 20.30%, p = 0.0002; − 35.74%, p < 0.0001; Supplementary Fig. 4A, respectively) when compared to both the control and HFD-only groups. In the frontal and posterior cortex, the CD11b expression was increased in the HFD group (27.10%, p < 0.0001; 15.90%, p = 0.0052; Supplementary Fig. 4B, respectively) in comparison with the control. Furthermore, in the HFD+CBDA group, it was lowered (− 9.23%, p = 0.0006; − 19.65%, p = 0.0022; Supplementary Fig. 4B, respectively) when compared to the HFD-only group in both regions. In the same group, the expression of CD11b was increased in the frontal cortex, whereas in the posterior cortex it was decreased (15.37%, p = 0.0032; − 6.87%, p = 0.026; Supplementary Fig. 4B, respectively) in comparison with the control. In the frontal cortex, the CD68 expression was decreased in the CBDA group (− 20.29%, p = 0.0002; Supplementary Fig. 4C) when compared to the control. In the HFD alone group, the expression was elevated (17.39%, p = 0.0007; Supplementary Fig. 4C) in comparison to the control. Furthermore, in the HFD+CBDA group, it was decreased (− 28.87%, p < 0.0001; − 39.41%, p < 0.0001; Supplementary Fig. 4C, respectively) relative to both the control and HFD-only group. In the posterior cortex, the rise in CD68 expression was observed in the HFD-only group (13.64%, p = 0.0006; Supplementary Fig. 4C) when compared to the control. On the contrary, in the HFD+CBDA group, the expression was decreased (− 12.36%, p < 0.0001; Supplementary Fig. 4C) relative to the HFD-only group.

Effects of cannabidiolic acid treatment on amino acids and amino acid-related metabolites concentrations in the cerebrospinal fluid.

The targeted metabolomic analysis was performed to evaluate the changes exerted by CBDA treatment on amino acids and amino acid-related metabolites concentrations in cerebrospinal fluid. To visualize the metabolic variations, a heatmap with hierarchical clustering was generated for the 36 targeted metabolites (Fig. 10A). The analysis revealed a lack of distinct metabolic signatures for the control and experimental groups. A Principal Component Analysis (PCA) plot (Fig. 10B) showed no distinct separation between the experimental and control groups, indicating similar profiles of amino acids and amino acid-related metabolites, suggesting that the high-fat diet as well as CBDA treatment had no significant impact on the overall profile. In the volcano plot, significant differences between the HFD and control groups were observed in the concentration of only two compounds. The concentration of L-NMMA (N^G-monomethyl-l-arginine) was down-regulated (log2FC= 2.1005, p-adjusted value = 0.00000026153; Fig. 10C) while the concentration of lysine was up-regulated (log2FC = 0.56419, p-adjusted value = 0.015977; Fig. 10C). Between the HFD+CBDA and HFD alone groups a significant down-regulation was observed in the concentration of creatinine (log2FC= − 0.45906, p-adjusted value = 0.0033586; Fig. 10D), phenylalanine (log2FC= − 0.4223, p-adjusted value = 0.046182; Fig. 10D), and sarcosine (log2FC= − 0.36211, p-adjusted value = 0.046182; Fig. 10D) in the volcano plot.

Fig. 10 — A targeted metabolomics. A targeted analysis of 36 amino acids and related compounds in CSF across groups: fed a standard rat chow (CONTROL), fed a standard rat chow and treated with CBDA (CBDA), fed a high-fat diet (HFD), and fed a high-fat diet and treated with CBDA (HFD+CBDA), n = 10. A Hierarchical clustering of ln(x + 1)-transformed compound concentrations generated using ClustVis. (Metsalu and Vilo 2015) Rows are centered; unit variance scaling is applied to rows. Rows are clustered using correlation distance and average linkage. B A Principal Component Analysis (PCA) score plot on ln(x + 1)-transformed compound concentrations generated using ClustVis. (Metsalu and Vilo 2015) X and Y axis show principal components 1 and 2 that explain 42.8 and 13.2% of the total variance, respectively. C Volcano plot of differential compounds between the group fed a high-fat diet (HFD) and the group fed a standard rat chow (CONTROL) (threshold: p-adjusted value < 0.05). D Volcano plot of differential compounds between the group fed a high-fat diet and treated with CBDA (HFD+CBDA) and the group fed a high-fat diet (HFD) (threshold: p-adjusted value < 0.05)

Effects of cannabidiolic acid treatment on the metabolome in cerebrospinal fluid

To visualize the variations in the metabolome, a heatmap with hierarchical clustering was generated (Fig. 11A). The preliminary analysis revealed distinct metabolic signatures for the control and HFD groups. A Principal Component Analysis (PCA) plot (Fig. 11B) showed distinct separation between the control and CBDA groups and the HFD and HFD+CBDA groups, indicating distinct metabolic profiles, suggesting that the HFD significantly affected the metabolome. In the volcano plot, significant differences between the HFD + CBD and HFD groups were observed in the concentration of only two compounds. The concentration of Thr-Pro (threonine and proline dipeptide) was down-regulated (log2FC = − 1.5519, p-adjusted value = 0.023011; Fig. 11C) between the HFD+CBDA and HFD alone groups. Moreover, the concentration of 2,4-Undecadiene-8,10-diynoic acid 2,3-dehydropiperidide was down-regulated (log2FC = − 1.388, p-adjusted value = 0.033458; Fig. 11C).

Effects of cannabidiolic acid treatment on the expression of CBDA’s receptor proteins in the frontal and posterior cortex

Changes in expression were observed only in the posterior cortex, where the expression of HTR1A was increased in the CBDA-only group and the HFD-alone group (34.76%, p = 0.0155; 43.57%, p = 0.0119; Supplementary Fig. 5B, respectively) in comparison to the control. Additionally, in the HFD+CBDA group, the expression was elevated (32.99%, p = 0.0119; Supplementary Fig. 5B) when compared to the control.

Discussion

Peripheral insulin resistance, typically accompanying the course of type 2 diabetes mellitus (T2DM), often correlates with the development of cerebral IR, recently referred to as type 3 diabetes mellitus (T3DM) (Chapple et al. 2025). Adherence to a diet rich in saturated fats and refined carbohydrates, apart from evoking metabolic diseases such as the previously mentioned T2DM or obesity (Clemente-Suárez et al. 2023), is associated with systemic chronic low-grade inflammation affecting the long-term functioning of body organs, including the brain (Komleva et al. 2021; Clemente-Suárez et al. 2023). In previously conducted studies, it was broadly demonstrated that a HFD could induce diet-dependent low-grade neuroinflammation in rodents, even prior to substantial weight gain (Thaler et al. 2012; Hersey et al. 2021; Costa et al. 2026). This was somewhat confirmed in our study regarding the contents of AA, the precursor of proinflammatory lipid mediators, namely the eicosanoids (Kursun et al. 2022), when comparing control rats and HFD-fed rats. The significant increase in its content in the frontal cortex in the fraction of phospholipids (PHs) and triacylglycerols (TAGs), and its increase in the posterior parts of the cortex in free fatty acids (FFAs), diacylglycerols (DAGs), and TAGs, suggest a region-specific proinflammatory shift after the 8-week maintenance of the fat-enriched diet.

The observed reduction in the contents of AA after the implementation of CBDA subsequently supports the anti-inflammatory and probably neuroprotective role of this compound. Interestingly, the most pronounced effect of CBDA was noted in the TAG class lipids in all assessed brain regions. While CBDA remains one of the lesser-studied cannabinoids, studies conducted using CBD (a CBDA derivative) and other cannabinoids have shown that these agents may also exert anti-inflammatory effects in brain tissue, based on both in vivo and in vitro studies(Gugliandolo et al. 2018; Rodrigues et al. 2023; Sabbag et al. 2025). Importantly, according to recent reports, although necessary in certain amounts, an excess of AA derivatives can be associated with the development of neuroinflammation and neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD)(Lin et al. 2024). Therefore, since eicosanoids are generally considered pro-inflammatory agents that enhance the immune response, regulating the excessive amount of AA and intervening in AA metabolic pathways can be beneficial (Zhang et al. 2023). On the other hand, as AA is important in many physiological processes, such as maintaining cell membrane fluidity and structure or acting as a second messenger (Kursun et al. 2022), excessive decrease in its content may disrupt synaptic plasticity or lead to psychiatric disorders such as schizophrenia (Gao et al. 2026). Thus, maintaining the proper homeostasis in AA content and its proper functioning metabolism in the brain is vital. In our study, the expression of cyclooxygenase 1 and 2 (COX-1 and COX-2), implicated in the synthesis of proinflammatory lipid modulators derived from AA, was increased in both the frontal and posterior parts of the cortex in rats fed an HFD. This increase was subsequently reduced by the application of a cannabinoid. The dependency of elevated levels of COX proteins in response to lipid overload is consistent with the results of a previous study conducted by Zhang et al., which demonstrated that a high intake of fat leads to a significant increase in the expression of COX-1 and COX-2 enzymes in the rat cerebral cortex (Zhang et al. 2005). In general, the COX-1 isoform is considered constitutive, present in most tissues, while COX-2 is inducible in cases of inflammation (Minghetti 2004; Kursun et al. 2022). COX-2 protein is overexpressed in the brain tissue of AD patients (Minghetti 2004), possibly linking the proinflammatory shift with the occurrence of neurodegenerative processes in AD. Moreover, emerging evidence suggests that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX enzymes, is associated with a reduced risk of dementia (Andersen et al. 1995; vom Hofe et al. 2025). Therefore, the search for substances that can inhibit COX induction and thus diminish the development of neuroinflammation is necessary. Our study demonstrated that the application of CBDA hinders the expression of both cyclooxygenases in HFD rats. There are no direct reports in the literature determining the effect of CBDA on COX-1 and COX-2 expression in in vivo models. However, in an in vitro model, Takeda et al. noted that CBDA is a much more potent inhibitor of COX-2 than Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and can also downregulate the expression of COX-2 in human breast cancer cells (Takeda et al. 2008, 2014). Taking into account the investigation of the properties of other cannabinoids, corresponding conclusions were drawn from both in vitro studies(Navarrete et al. 2018; Wang et al. 2023; Cosentino et al. 2023) and our previous animal studies (Opęchowska et al. 2024). Interestingly, Bartkowiak-Wieczorek et al. discovered that in rats with neuropathic pain, the implementation of Cannabis sativa extract B (containing CBDA: 1.2 mg/g; CBD: 220.2 mg/g; Δ9-THC-A: 0.1 mg/g; Δ9-THC: 15.5 mg/g) increased the level of COX-1 mRNA, while COX-2 mRNA was significantly decreased in the cortex tissue. Additionally, extract D (CBDA: 1.2 mg/g; CBD: 215.2 mg/g; Δ9-THC-A: 0.15 mg/g; Δ9-THC: 13.3 mg/g) dose-dependently increased COX-1 mRNA and protein expressions in the rat hippocampus and cortex. These data provide somewhat contradictory conclusions, indicating that Cannabis extract simultaneously increases and decreases the expression of COX proteins. This could even suggest that the Cannabis extract exerts proinflammatory properties, particularly in the rat hippocampus, which contradicts the results of our study. However, researchers also reported an extract-dependent reduction in TNFα protein levels, a proinflammatory cytokine, in the rat cerebral cortex (Bartkowiak-Wieczorek et al. 2025). The explanation for various effects described in the literature may be that the amount of active compounds differs in each extract. In the review described by Russo et al., studies were presented where different combinations of tetrahydrocannabinol and cannabidiol were used in the treatment of many diseases, such as spasticity, pain, or rheumatoid arthritis. The obtained results showed that, knowing the properties of each compound, a combination of two phytocannabinoids was more effective (Russo and Guy 2006). Thus, the present study is the first to show the possible treatment effects of CBDA alone, as decreased COX-1 and − 2 expressions occur simultaneously with diminished expression of pro-inflammatory cytokines, namely interleukin 6 and interleukin 1β (IL-6 and IL-1β), as well as decreased PGE2 and leukotriene B4 (LTB4) contents in the HFD+CBDA group in both parts of the cortex. This finding seems to be of prime importance because decreased neuroinflammation is essential in decreasing microglial activation markers, namely ionized calcium-binding adaptor molecule 1 (IBA1), cluster of differentiation 11b (CD11b), and cluster of differentiation 68 (CD68), observed in both the frontal and posterior cortex of HFD-fed rats after CBDA treatment.

5-lipoxygenase (5-LO) activity is associated with the progression and promotion of inflammatory diseases such as nonalcoholic fatty liver disease (NAFLD). Additionally, its expression was upregulated in Alzheimer’s disease patients (Martínez-Clemente et al. 2010; Ma et al. 2017; Kumar et al. 2020). The reduction in its expression in HFD-fed animals after the CBDA application, significant in the frontal cortex, suggests the achieved diminishment of the process of pro-inflammatory lipid mediator formation in this brain region (Rad̊mark and Samuelsson 2009). The promising inflammation-resolving properties of the CBDA derivative, CBD, were already noted in the metabololipidomics analysis of human monocyte-derived macrophages, where CBD could hinder the 5-LO-mediated leukotriene production (Peltner et al. 2023). This finding is confirmed in our study by the observed reduction in the content of leukotriene B4 in both tissues in the HFD+CBDA group. Other cannabinoids can also inhibit the activity of 5-LO as per the results of the study using the molecular docking technique (Bouchentouf et al. 2017), which consistently suggests a beneficial role of cannabinoids in inhibiting pro-inflammatory processes, restoring the oxidative balance in the tissue necessary to slow down aging and neurodegenerative processes. The biological roles of 15-lipoxygenase (15-LO) are characterized by significant functional pleiotropy, often exerting physiological effects distinct from other lipoxygenase isoforms. Instead of the pro-inflammatory activity, achieved through the production of leukotrienes, this enzyme generates a range of pro-resolving mediators (SPM), facilitating the resolution of inflammation (Snodgrass and Brüne 2019; Zamora et al. 2024). The use of CBD led to the activation of the 15-LO enzyme, which has an effect similar to our results, where CBDA increased 15-LO expression in the HFD-fed rats. However, in the cited work, they used only the in vitro model, incorporating the innate immune cells (Peltner et al. 2023).

The observed reduction in the contents of AA is consistent with the fact that CBDA implementation reduces the amount of lipids in the entire TAG and DAG classes in both the frontal cortex and posterior parts of the cortex, along with the lipids of the FFA class in the posterior cortex of HFD-fed animals. Differences in the observed changes in the contents of AA and the entire lipid classes deposition could be related to the functions that these lipids perform in mammalian cells. Although PHs are mainly the building components of cell membranes (Akutsu 2020), DAGs act as signaling molecules (Wang and Kazanietz 2006; Cooke and Kazanietz 2022), and TAGs are the storage material of the cell (Li et al. 2017b). The high content of AA in the TAG fraction in both tissues and in the rest of the lipid fractions of frontal or posterior parts of the cortex after an HFD shows a pro-inflammatory potential of deposited lipids. Therefore, considering the proven greatest influence of CBDA on the TAG and DAG classes, it can be concluded that these classes are the first targets for the action of this cannabinoid.

To further characterize the neuroinflammatory profile, we quantified the expression of the pro-inflammatory cytokine TNFα. Consistent with our hypothesis, HFD-induced metabolic stress significantly augmented the expression of TNFα in the frontal cortex compared to control rats. This remains in accordance with other studies demonstrating that consuming an HFD or a diet rich in saturated fatty acids significantly increases TNFα expression in the hypothalamus (De Souza et al. 2005; Wang et al. 2012; Valdearcos et al. 2014), hippocampus (Jeon et al. 2012; Spagnuolo et al. 2015; de Paula et al. 2021; González-Castillo et al. 2026), cortex (Pistell et al. 2009; Cavaliere et al. 2019), and other brain regions. The application of CBDA appeared to diminish TNFα levels in both the frontal and posterior parts of the cortex. TNFα is regarded as a potent pro-inflammatory agent, actively involved in the potentiation of immune activation (Gonzalez Caldito 2023); therefore, reducing its level may help in the protection of the brain tissue from inflammation-related comorbidities. It has been proven that TNFα is able to interact with the insulin signaling cascade, leading to insulin resistance development associated with obesity (Nieto-Vazquez et al. 2008; Mohallem and Aryal 2020). Moreover, it may also cause neurodegeneration affecting tau protein hyperphosphorylation (Probert et al. 1997; Frankola et al. 2011). Comparable pharmacological observations regarding other phytocannabinoid analogs and TNFα have been reported in the literature. In a study by Sabbag et al., using a similar model with a shorter exposure to HFD (2 weeks vs. 8 weeks in our experiment), CBD was shown to significantly decrease TNFα expression in the ventromedial prefrontal cortex (Sabbag et al. 2025). Additionally, in a study by Rodrigues et al. involving offspring of obese Wistar rat females, treatment with CBD oil attenuated the overexpression of TNFα and IL-6 in the hypothalamus (Rodrigues et al. 2023). Bartkowiacz-Wieczorek et al. demonstrated that TNFα expression in the rat brain cortex in neuropathic pain was reduced after treatment with Cannabis sativa extracts, consistent with our findings where decreased expression of both TNFα and IL-6 was observed after CBDA treatment in both cortex parts in HFD-fed animals (Bartkowiak-Wieczorek et al. 2025). These data support the idea that cannabinoid sensitivity is a function of the metabolic and physiological heterogeneity inherent across distinct neuroanatomical regions. CBDA treatment in HFD-fed animals significantly diminished the expression of IL-6 and IL-1β, whereas the expression of anti-inflammatory interleukin 10 (IL-10) was significantly increased in both cortex regions. Although under physiological conditions, IL-1β is expressed at low levels in the brain tissue and behaves as a neuromodulator involved in memory processing, during neurodegeneration, its production by activated microglia is significantly increased. High levels of IL-1β promote the release of itself and also TNFα, producing a vicious cycle of pro-inflammatory mediator deposition (Shaftel et al. 2008). Therefore, decreased expression of both cytokines, IL-1β and TNFα, together with decreased CD11b, CD68, and IBA1 microglial activation markers, after CBDA treatment in the frontal and posterior cortex of HFD rats, confirms this phytocannabinoid potential as a neuroinflammation-decreasing agent.

Following a high-fat diet that mimics the Western dietary pattern, induces not only neuroinflammation but also a state of brain IR, ultimately contributing to cognitive decline, as demonstrated in both animal and human studies (Greenwood and Winocur 1996; Li et al. 2017a; Tang et al. 2026). Additionally, insulin pathway effector proteins are involved in the pathogenesis of Alzheimer’s disease, characterized by beta-amyloid (Aβ) protein accumulation and neurofibrillary deposits composed of hyperphosphorylated tau protein (Hampel et al. 2021; Limantoro et al. 2023).

The activation of GSK-3α/β, a downstream enzyme of the insulin signaling pathway, particularly its β isoform, is a central hub linking this pathway with tau phosphorylation. Literature data indicate that this activation may be achieved by Aβ, inactivating the phosphatidylinositol 3-kinase/Akt signaling pathway. Subsequently, active GSK induces tau protein hyperphosphorylation, leading to the activation of neurofibrillary tangles and neuroinflammation in AD (Vallée et al. 2017). The second route for GSK activation is the interaction of TNFα with the phosphatidylinositol 3-kinase/Akt signaling pathway and subsequently with GSK (Clemenzi et al. 2019). We suspect that both mechanisms may be present in this study, especially in the frontal cortex, as increased TNFα expression and beta-amyloid 42 (Aβ42) content were observed in the HFD group. However, mechanistic studies are still needed to confirm this association. Regardless of the mechanism of activation, GSK-3β activity is inhibited by Ser9 phosphorylation, while phosphorylation at the Tyr216 site is required for maximal catalytic potency.

In the case of the GSK-3α isoform, phosphorylation at the Ser21 site serves as a critical inhibitory phosphorylation, effectively suppressing its catalytic activity, with Tyr279 as an activating phosphorylation (Ahmed et al. 2014; Pandey and DeGrado 2016). In our study, CBDA was able to reduce the phosphorylation rate of one enzyme isoform, calculated as the pGSK-3β Tyr216 to GSK-3β ratio in the frontal part of the brain cortex, which indicates that activating phosphorylation was diminished in this tissue. In the posterior cortex, an increase in the inactivating phosphorylation at Ser21 and Ser9 of GSK-3α/β was achieved, calculated also as the ratio of phosphorylated form to total, supporting diminished activity of both subunits in the posterior parts of the brain cortex. As the obtained changes in the phosphorylation of insulin signaling proteins after CBDA treatment are only partially a sign of improved insulin sensitivity, the expressions of hexokinase (HXK1) and lactate dehydrogenase (LDH) were evaluated. HXK1 is an enzyme predominantly expressed in the brain that plays a pivotal role in catalyzing the first irreversible step of glucose metabolism. Literature data showed that the expression of this enzyme was significantly decreased in the brains of streptozotocin-induced diabetic rats (Muthuraman and Srikumar 2010) and in the brains of 6-week Western diet-fed rats (Nowacka-Chmielewska et al. 2021). Our results are in line with cited findings, as we observed a diminishment in HXK1 expression in the HFD group in both cortex parts, which indicated that glucose metabolism was affected. CBDA treatment increased the expression of this enzyme in the HFD group in both tissues, and this upregulation occurs alongside improved insulin signaling. Studies showed that the significant decline in HXK1 expression is associated with neurodegeneration development observed in mouse models of AD (Farooq et al. 2026). Thus, enhanced HXK1 expression may also be associated with neurodegeneration improvement in our study; however, it is only speculation without mechanistic confirmation. Additionally, in neurodegeneration and during the aging process, LDH expression is upregulated, leading to an abnormal accumulation of lactate in the brain tissue (Frame et al. 2024). Similar impairment is also observed in an insulin-resistant brain, where this overexpression is a hallmark of the glycolytic shift that occurs when the brain is not able to utilize glucose (Meng et al. 2025). Therefore, decreased expression of LDH in HFD-fed animals after CBDA treatment in both tissues seems to be advantageous not only in the aspect of glucose metabolism but also in the development of possible neurodegenerative disorders. Thus, changes in insulin signaling proteins and glucose metabolism enzymes occurring simultaneously with reduced neuroinflammation markers in both tissues, increased BDNF levels in CSF, and decreased Aβ42 in the frontal cortex suggest that CBDA may have anti-neurodegenerative potential at least in the frontal cortex (Ahmed et al. 2014). However, these findings still should be confirmed with mechanistic, behavioral, memory, or cognitive assessments because the analysis of tau protein phosphorylation levels did not change. In contrast, studies conducted on PC12 neuronal cells stimulated with Aβ showed that CBD treatment inhibited hyperphosphorylation of the tau protein. Unfortunately, in the cited studies, there is no information about the phosphorylation sites affected by CBD. However, a similar aspect to our studies is that the decrease in phosphorylation of GSK-3β (in our study observed only in the frontal cortex) was responsible for changes in tau phosphorylation (Esposito et al. 2006).

Finally, to resolve the possible role of CBDA in neurodegeneration, we examined the expression of the amyloid-cleaving enzyme, beta-secretase 1 (BACE1), the amyloid precursor protein (APP), and Aβ42. CBDA had significant effects in both cortical regions, reducing the expression of BACE-1. This was further supported by the reduction in APP expression in the posterior parts of the cortex, consistent with other studies involving endocannabinoids (Brar 2013). Although APP is the precursor molecule to Aβ42, it is an integral membrane protein that plays an important role in many physiological processes in the brain (Orobets and Karamyshev 2023). Thus, changes in its expression do not precisely reflect the neurodegenerative process. Therefore, we also performed the assessment of Aβ42 content in the frontal and posterior parts of the cortex. Importantly, according to some reports, the medial prefrontal cortex and medial parietal cortex are the locations where the formation of early Aβ plaques begins (Hampel et al. 2021). In our study, we observed a significant decrease in the Aβ42 content only in the frontal cortex, and with concomitant decreased expression of microglial activity markers, we may suspect that the frontal cortex may be the main site affected by CBDA.

Moreover, as plasma CSF is a valuable source of biomarkers and often reflects neurological biomarkers, we performed an assessment of the levels of the BDNF peptide, a critical mediator of synaptic plasticity and neuronal survival in both CSF and plasma. Given its role as a key indicator of neurobiological homeostasis, a reduction in BDNF expression is characteristic of impaired neurogenesis, disruption in brain tissue function, or an early stage of neural tissue damage (Colucci-D’amato et al. 2020). Moreover, a decrease in its content can serve as a precursor to diet-induced neurodegeneration. In an animal study by Cavaliere et al., it was demonstrated that after following the HFD for 18 weeks, the content of BDNF in the mice’s brain cortex homogenates was significantly lower than in mice receiving a standard diet (Cavaliere et al. 2019). Interestingly, in our study, we detected a reduction in BDNF content after only 8 weeks of HFD-feeding, both in the CSF and in the plasma of the animals. This suggests that already at a very early stage of using an HFD, significant disturbances in the functioning of brain tissue occur, which is particularly pronounced in our study, since the reduction in BDNF level was noted not in the brain tissue homogenates themselves, but in BDNF secreted into the CSF and plasma. Additionally, a significant increase in microglial activation markers and pro-inflammatory cytokine expressions confirmed that HFD disrupted brain homeostasis. More importantly, the cannabinoid could raise its concentration in the CSF, supporting the ability of CBDA to restore this disrupted homeostasis. Although CBDA treatment exerted a similar increasing trend in BDNF levels in the plasma, it did not reach the level of significance. Similar to our results, a meta-analysis revealed that the level of BDNF in the blood serum is not significantly different between cannabis users and non-users (Shafiee et al. 2023). This may indicate that BDNF content is a specific biomarker only present in CSF, and more time is probably needed to reflect CSF BDNF levels in plasma. Comparable findings may also be drawn from changes in Aβ42, whose content was only decreased in the frontal cortex, although beneficial anti-neuroinflammatory effects were observed in both tissues.

Another possible biomarker of neurodegeneration present in plasma and CSF may be arginine and its metabolites. Studies conducted by Fleszar et al. indicated that in the plasma of patients with dementia, significant alterations in arginine, citrulline, asymmetric (ADMA) and symmetric (SDMA) dimethylarginine, and dimethylamine (DMA) were associated with the degree of brain tissue loss and severity of cognitive impairment (Fleszar et al. 2019). Thus, in the present study, targeted metabolomics was used to assess possible changes in the concentrations of amino acids and amino acid-related metabolites in high-fat diet-fed animals after CBDA treatment. Surprisingly, the concentration of arginine and its metabolites was not affected by HFD, which was in accordance with neurodegeneration results, suggesting very preliminary neurological disorders. In the HFD+CBDA group compared to HFD, a significant decrease was observed in the concentration of creatinine. As creatinine is the by-product of creatine breakdown, which is a non-enzymatic, irreversible process, the decrease in one compound results in the decrease of another (Clarke et al. 2020). Taking into account this relationship, and the fact that, unlike skeletal muscles, the brain can produce creatine endogenously (Vietor et al. 2025), as observed in our study, a decrease in this by-product may indicate a diminishment in the production of creatine. Although some scientists indicated that brain creatine production may be inhibited in response to this product supplementation (Roschel et al. 2021), in our experiment, this was not the case. Therefore, a possible decrease in creatine may be disadvantageous since patients suffering from genetic creatine deficiency disorders, wherein creatine metabolism or transport is disrupted, have been shown to have intellectual disabilities (Schulze 2003). Moreover, it was proven that creatine supplementation exerts beneficial effects in neurodegenerative conditions and brain injury (Genius et al. 2012). On the other hand, high serum levels of creatinine were associated with an elevated risk of incident dementia development (Zammit et al. 2016). Keeping in mind these contradictory findings, more research is needed to evaluate the association between CBDA and creatinine, not only in the brain, but also in skeletal muscles, where creatine is the most important in the energy production process (Walker 1979). Another compound that was notably decreased in the HFD+CBDA group was phenylalanine. Studies conducted on CSF obtained from patients suffering from phenylketonuria with high levels of phenylalanine in plasma indicated a strong correlation between high levels of phenylalanine and elevated tau, ptau, and Aβ proteins. Furthermore, high levels of phenylalanine were also associated with neuropsychiatric symptoms, motor-evoked potential latency, or parietal lobe atrophy (Pilotto et al. 2021). Although phenylalanine is an essential amino acid, important in the synthesis of proteins and neurotransmitters, its elevated levels act as a direct neurotoxin that inhibits enzymes producing components for the myelination process, as it was observed in in vitro and in vivo models of phenylketonuria. Moreover, increased levels of phenylalanine may also interfere with other cerebral enzyme systems, such as pyruvate kinase, resulting in diminished energy metabolism in the brain (van Spronsen et al. 2009). The strong connection between high phenylalanine levels and neurodegeneration suggests that lowering phenylalanine levels with CBDA could be crucial in treating neurological disorders in phenylketonuria. However, this conclusion is only preliminary, and further studies on phenylketonuria models are needed. Another important aspect of altered phenylalanine levels is the fact that it plays a significant role as an amino acid precursor. Phenylalanine is converted to tyrosine, which may be further converted into catecholamines (Pascual 2017). Therefore, excessive amounts of phenylalanine inhibit tyrosine hydroxylase, an enzyme responsible for converting tyrosine to 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine, which results in a disruption of this enzymatic pathway leading to catecholaminergic deficit present in Parkinson’s and Alzheimer’s diseases (Tabrez et al. 2012; Herman et al. 2019). Observations from phenylketonuria patients indicated that mental dysfunction may also be caused by disruption in the transport of large neutral amino acids through the blood-brain barrier. Because phenylalanine shares the same transporter as other essential amino acids when passing the blood-brain barrier, its high concentrations in the blood cause phenylalanine to be effectively transported ahead of other vital amino acids, and as a result, the production of neurotransmitters like serotonin or dopamine is disrupted (van Spronsen et al. 2009). Another compound decreased in CSF in the HFD+CBDA group was sarcosine. The observed change may be interpreted as bidirectional. Although it can improve cognitive and negative symptoms in schizophrenia (Socała et al. 2010), its levels in the CSF of sarcosinemia patients are increased, which is also associated with the development of neurological disorders such as Parkinson’s disease and multiple sclerosis (Leung et al. 2009). Thus, the observed decrease in sarcosine levels induced by CBDA in the present study may be beneficial among patients suffering from sarcosinemia. However, schizophrenic patients treated with sarcosine may experience a significant reduction in the therapeutic effect when combined with CBDA. On the other hand, studies conducted on in vitro and in vivo AD models treated with sarcosine indicated that this glycine derivative exerted a neuroprotective effect, alongside decreased oxidative stress and neuroinflammation (Tanas et al. 2022). Moreover, in the rat model of schizophrenia, sarcosine not only reversed behavioral impairments, but also decreased oxidative and nitrosative stress, improved mitochondrial function, and diminished neuroinflammation (Kumar et al. 2023). Therefore, when considering beneficial anti-inflammatory effects exerted by sarcosine supplementation, the observed decrease in its levels in the HFD+CBDA group seems to be disadvantageous. Moreover, it is contradictory to the results obtained from cortex tissues, where CBDA in the HFD group decreased proteins associated with neuroinflammation and microglial activation markers. This indicates that CBDA may affect various metabolic pathways in different ways, because lipid metabolism, neuroinflammation, insulin pathway signaling, and the initiation of neurodegeneration are closely related, in a chain-linked manner. Excessive amounts of consumed fats, especially saturated fats, lead to changes in the content of lipid fractions in brain tissue and increase the production of lipid-derived inflammatory mediators. This entails the development of chronic neuroinflammation, slowly and successively damaging the primarily healthy tissue. Although the HFD used in our study derives most of its fat from lard, which is a main source of saturated fats, it also contains small amounts of soybean oil. Studies conducted on mice indicated that animals fed soybean oil developed more severe neuroinflammation than animals fed solely lard (Liu et al. 2024). However, there are also studies showing that using soybean oil instead of saturated fat improved serum lipid profile (Baer et al. 2021). Thus, the use of this particular type of diet resulted in a specific fatty acid profile that may also influence the lipid and inflammatory alterations observed in our study.

Simultaneously, a caloric surplus and weight gain promote the development of insulin resistance not only in the skeletal muscles or liver, but also in the brain. When occurring together, neuroinflammation and impaired insulin signaling may eventually initiate neurodegenerative processes that snowball over time, resulting in the development of the clinically observed dementia.

Limitations

Unfortunately, the established experiment exhibits significant limitations that may compromise its translational relevance. Primarily, the experiment was conducted on an animal model, which cannot be directly translated to humans. Moreover, the short duration of the treatment protocol might not reveal the long-term therapeutic efficacy or potential delayed adverse effects of the intervention, despite CBDA being described as safe. The study scope was also narrowed by the exclusive use of male animals, which precludes the assessment of sex-based differences in response to the analyzed cannabinoid (Craft et al. 2013). Based on previous studies evaluating non-psychoactive cannabis compounds, scientists indicated that oral CBD administration resulted in higher relative exposure to CBD metabolites over time in females than in males, which may be caused by present sex differences in this phytocannabinoid metabolism or elimination (MacNair et al. 2024). Moreover, as CBD acts on cannabinoid receptors, their hypothalamic expression varies between males and females (Riebe et al. 2010), and their expression undergoes estrous cycle-dependent changes in females (González et al. 2000). Studies conducted on an animal model of orofacial neuropathic pain indicated that CBD and cannabigerol administered to male and female rats alleviated neuropathic pain in females, whereas males required significantly higher doses to achieve the same therapeutic outcome (Vivanco-Estela et al. 2025). Thus, taking sex differences into account in future studies is vital. Although the most effective dose of CBDA was selected based on existing literature, the study is devoid of dose-dependent response evaluation, which restricts our understanding of the optimal therapeutic window. Moreover, the implemented doses might not be sufficient for humans. Furthermore, the lack of behavioral testing and mechanistic studies prevents us from correlating biochemical improvements in the tissue with functional outcomes in animal activity. More dose-dependent experiments, combining two or more phytocannabinoids, conducted on larger groups of male and female animals, are needed before testing CBDA in patients suffering from inflammatory or neurological diseases. Before introducing CBDA into human studies, it is also required to develop a safety and toxicity profile for long-term use of CBDA. To better reflect the mechanisms occurring in brain tissue under conditions of more severe induced insulin resistance, a diet enriched in simple, refined sugars should be implemented. Additionally, these findings only characterize a prodromal neuroinflammatory state, representing the preliminary shift towards chronic neurodegeneration. More studies using inhibitors, indicating the precise molecular mechanism of CBDA action under high availability of fatty acids in the diet, are still in high demand. Concluding the use of CBDA as a future treatment method, based on the current stage of research in the rat model used, should be approached with caution (Fig. 12).

Fig. 12 — Schematic summary of the proposed mechanism of action of CBDA in the HFD-fed animal model

Conclusion

Our study demonstrated a preliminary analysis of the impact of CBDA on the inflammatory profile of the brain under conditions of excess calories from fat. The results suggest an anti-inflammatory role for this cannabinoid, particularly in inhibiting the synthesis of lipid inflammatory mediators. Changes in the total content of deposited TAG and DAG lipid fractions and decreased arachidonic acid content in them were concomitant with diminished cyclooxygenase expression and PGE2 and LTB4 content in both frontal and posterior parts of the brain cortex. CBDA also significantly decreased the expression of pro-inflammatory cytokines, which also affected the attenuation of microglial activation markers. Thus, it indicates that CBDA under high availability of fatty acids is able to exert anti-neuroinflammatory properties. Moreover, in addition to changes in lipid deposition and attenuated inflammation, CBDA improved the phosphorylation profile of insulin signaling proteins, especially in the posterior brain cortex, and enhanced expression of enzymes responsible for glucose metabolism in both tissues. The present study also showed decreased BACE1 expression in both brain regions and increased BDNF levels in CSF after CBDA usage. Although the decrease in Aβ42 deposition was observed in the frontal cortex, the CBDA effect on neurodegeneration in our study is only partial and needs further investigation with memory, cognitive, or behavioral testing. Although creatinine, phenylalanine, and sarcosine levels were not affected by HFD, the diminishment in its deposition exerted by CBDA treatment may be both advantageous and disadvantageous in the treatment of some neurological disorders, suggesting a new direction for future studies. However, in our study, the precise mechanism through which CBDA is able to exert the described changes was revealed only partially. We suspect that the observed changes may result from COX-2 inhibition. In future studies, it is necessary to conduct research with specific COX-2 inhibitors to confirm our findings. Moreover, identifying more specific receptors responsible for the observed results is important. The present study sheds light on the need to deepen the knowledge about CBDA as a new therapeutic option.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(4.4MB, pdf)}

Supplementary Material 2^{(6.1MB, docx)}

Acknowledgements

This work was funded by the Minister of Education and Science of Poland from the state budget under the program “Student science clubs create innovations” (grant no. SKN/SP/630507/2025) and the Medical University of Białystok (grant no. B.SUB.26.297).

Author contributions

Conceptualization, K.K.N., A. O.; methodology, K.K.N., E.H.S., M.Z.K., A.B., M.L.; software, M.D., M.Z.K., A.B., M.L.; validation, K.K.N.; formal analysis, K.K.N., M.Z.K., A.B., M.L.; investigation, K.K.N., E.H.S.; resources, K.K.N.; data curation, K.K.N., M.Z.K., A.B., M. L.; writing, K.K.N, A.O., M.D.; review and editing, K.K.N.; visualization, M.D., A.B.; supervision K.K.N.; project administration, K.K.N., A.O.; funding acquisition, K.K.N., A.C. All authors have read and agreed to the published version of the manuscript.

Funding

Funded by the Minister of Education and Science of Poland from the state budget under the program “Student science clubs create innovations” (grant no. SKN/SP/630507/2025) and the Medical University of Białystok (grant no. B.SUB.26.297).

Data availability

The data presented in this study are available upon request from the corresponding author.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

The study was conducted according to the EU Directive 2010/63/EU for animal experiments, and approved by Ethical Committee for Animal Testing in Olsztyn, Poland (permission no. 35/2023, date of approval: 19 April 2023).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(4.4MB, pdf)}

Supplementary Material 2^{(6.1MB, docx)}

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

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PERMALINK

The beneficial properties of CBDA in diet-induced neuroinflammation

Karolina Konstantynowicz-Nowicka

Aleksandra Opęchowska

Maciej Domalewski

Magdalena Zabielska-Kaczorowska

Aleksandra Bogucka

Marcin Lipiński

Ewa Harasim-Symbor

Adrian Chabowski

Abstract

Supplementary Information

Introduction

Materials and methods

Animals and experimental design

CSF collection

Gas-liquid chromatography

Immunoblotting

Real-time polymerase chain reaction

Enzyme-linked immunosorbent assay

Metabolite extraction

Untargeted and targeted metabolomics analysis

Statistical analysis

Results

Effects of cannabidiolic acid treatment on total intracellular lipid fraction concentrations in four rat brain regions

Fig. 1.

Effects of cannabidiolic acid treatment on arachidonic acid concentration in lipid fractions in four rat brain regions

Fig. 2.

Effects of cannabidiolic acid treatment on the expression of insulin signaling pathway proteins and glucose metabolism enzymes in the rat frontal and posterior cortex

Fig. 3.

Fig. 4.

Effects of cannabidiolic acid treatment on the expression of proteins involved in the neurodegeneration process in the rat frontal and posterior cortex

Fig. 5.

Fig. 6.

Effects of cannabidiolic acid on the expression of proteins associated with the inflammatory process in the rat frontal and posterior cortex

Fig. 7.

Fig. 8.

Effects of cannabidiolic acid treatment on β-amyloid concentration in the frontal and posterior cortex and BDNF concentration in cerebrospinal fluid and plasma

Fig. 9.

Effects of cannabidiolic acid treatment on microglial activation markers in the frontal and posterior cortex.

Effects of cannabidiolic acid treatment on amino acids and amino acid-related metabolites concentrations in the cerebrospinal fluid.

Fig. 10.

Effects of cannabidiolic acid treatment on the metabolome in cerebrospinal fluid

Fig. 11.

Effects of cannabidiolic acid treatment on the expression of CBDA’s receptor proteins in the frontal and posterior cortex

Discussion

Limitations

Fig. 12.

Conclusion

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Conflict of interest

Ethical approval

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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