Pioglitazone protects against trimethyltin hippocampal injury by reducing pyroptosis, mitochondrial dysregulation and ER stress

Abstract

Hippocampus-specific neurotoxic trimethyltin (TMT) is routinely used to mimic a reliable murine phenotype of neurodegeneration as well as cognitive loss and is accordingly appropriate to analyze pathogenesis of the prevalent neurodegenerative disorders, i.e. Alzheimer’s disease (AD), and to examine the effectiveness of novel therapeutics. Antidiabetic medication pioglitazone has exhibited neuroprotective effects with promising clinical indications for neurodegeneration-based illnesses. This study was accomplished for studying the neuroprotective effect of pioglitazone against TMT-initiated cognitive decline and allied hippocampal neurodegeneration. For this purpose, rats received intraperitoneal TMT (8 mg/kg) to generate a model of AD-like neurodegeneration and subsequently had oral daily administration of pioglitazone for 3 weeks (20 mg/kg). The acetylcholinesterase inhibitor and certified anti-AD drug donepezil (4 mg/kg) was similarly used as a positive control medicine. Pioglitazone treatment was accompanied by lower cognitive deficits in novel object recognition test and Barnes maze paradigm in addition to mitigation of astrogliosis severity with glial fibrillary acidic protein (GFAP) as its specific indicator and lower CA1 neuronal loss. Furthermore, pioglitazone partially normalized hippocampal factors of oxidative stress and neuroinflammation together with downregulation of pyroptotic parameters comprising caspase 1 and NLR family pyrin domain containing 3 (NLRP3). Moreover, less activity of acetylcholinesterase (AChE) and greater quantity of mitochondrial health-allied factors comprising peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial membrane potential (MMP), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ (PPARγ) were likewise detected after pioglitazone treatment. These advantageous properties of pioglitazone were accompanied by inferior quantity of specific AD-allied markers comprising presenilin1 (PSEN1) and hyperphosphorylated tau (p-tau) as well as downregulation of endoplasmic reticulum (ER) stress, as observed by lower levels of PKR-like ER kinase (PERK), C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), and inositol-requiring enzyme 1α (IRE1α). While anti-AD donepezil treatment was associated with improvement of cognitive function, however, it was not capable to significantly yield most advantageous effects of anti-diabetic PPARg agonist pioglitazone. This study disclosed the underlying pathways for neuroprotective effect of pioglitazone in TMT neurodegeneration and AD-like phenotype.

Introduction

Alzheimer’s disease (AD) is the leading cause of dementia in people older than 65 years worldwide, characterized by progressive neurodegeneration that results in profound cognitive and behavioral impairments¹. The vast majority of AD cases are sporadic and late-onset, influenced by intricate interactions between genetic and environmental factors. Pathologically, AD is hallmarked by the accumulation of hyperphosphorylated tau (p tau) proteins in the neurofibrillary tangles besides amyloid beta (Aß) plaques, which contributes to widespread loss of synapses, neuronal dysfunction, and cognitive decline². These pathological features are compounded by neuroinflammation, a critical driver of disease progression. Neuroinflammation is characterized by glial cell activation and the release of pro-inflammatory mediators, including cytokines and chemokines, which exacerbate neuronal damage³. Unfolded protein response (UPR) and associated endoplasmic reticulum (ER) stress as a result of the accumulation of unfolded and/or misfolded proteins perturbs ER and cellular homeostasis which contributes to the onset and development of AD and this may help to explore new therapeutic approaches in the management of AD⁴. Mitochondrial dysfunction irrespective of it being as an initiating factor or as its sequel is strongly involved in AD pathogenesis⁵. There is also compelling evidence that NLR family pyrin domain containing 3 (NLRP3) inflammasome-linked pyroptosis is strictly associated with the AD pathogenesis⁶.

The neurotoxic agent trimethyltin (TMT) in rodents specifically injures neuronal cells in the limbic system, particularly in the hippocampal region^7,8. Specific neurodegeneration induced by the TMT is accompanied by marked cognitive deterioration, aggressive behavior, and even seizure bouts^7,9. Mitochondrial dysregulation, nitrosative and oxidative stress, inflammation, intracellular calcium burden, and ER stress, higher deposition of Aβ, and higher production of hyperphosphorylated tau (p-tau) have been documented subsequent to the TMT challenge^7,10–12. TMT-provoked ER stress can lead to UPR with ensuing apoptosis and development of various dysfunctions¹³. All of these changes are also indicated during the development of AD^14,15. Thus, TMT-initiated cognitive deficit in rodents is valuable for preclinical assessment of prospective therapeutics in AD-related setting^16,17.

Recent studies have highlighted the role of peroxisome proliferator-activated receptor-γ (PPAR-γ), a nuclear receptor involved in glucose metabolism, lipid regulation, and anti-inflammatory pathways in addition to PPAR-γ coactivator-1 alpha (PGC1α) as potential therapeutic targets for AD^18,19. The thiazolidinedione pioglitazone as a PPAR-γ agonist is clinically administered for controlling diabetes type II and with emerging as a hopeful candidate for AD therapy due to its ability to inhibit neuroinflammation and oxidative stress in the brain tissue^20,21. In this respect, PPARγ activation is involved in regulation of insulin-degrading enzyme (IDE) transcription, leading to amyloid β (Aβ) degradation, a principal pathological hallmark of AD²². PPARγ activation is also associated with inhibition of the transcription of Aß degrading enzyme 1, i.e. BACE1²³. Importantly, pioglitazone can penetrate the blood-brain barrier, enhancing cerebral glucose metabolism, improving mitochondrial function, and reducing inflammatory responses in the central nervous system^20,21. In the context of AD, PPAR-γ agonists like pioglitazone exhibit neuroprotective effects by suppressing the expression of pro-inflammatory genes and upregulating anti-inflammatory pathways mediated by microglia and macrophages^24,25. Furthermore, pioglitazone attenuates oxidative stress and mitochondrial dysfunction²⁶, two critical contributors to AD pathogenesis. Beyond its effects on neuroinflammation, pioglitazone also demonstrates anti-apoptotic properties and can mitigate Aβ-induced toxicity, highlighting its multifaceted therapeutic potential²³. In experimental models of neurodegeneration, pioglitazone has shown significant efficacy in reversing neurocognitive deficits, reducing inflammation, and restoring mitochondrial integrity. For instance, pioglitazone alleviates hippocampal neurodegeneration caused by toxic insults^27,28, which mimic key aspects of AD pathology. Additionally, preclinical studies demonstrate its potential to protect the blood-brain barrier (BBB) from chronic peripheral inflammation²⁹, a hallmark of aging and neurodegenerative disorders. Thereby, we explored the therapeutic potential of pioglitazone in TMT-induced neurodegeneration and AD-like phenotype, focusing on its mechanisms of action, including modulation of neuroinflammation and oxidative stress, pyroptosis, mitochondrial dysregulation, and ER stress.

Materials and methods

Animals

In this study, a total of 32 adult male Wistar rats weighing 200–235 g were obtained from the Laboratory Animal Breeding and Research Center at the University of Tehran. All animals were housed under standard laboratory conditions, including a controlled temperature environment, a 12-hour light/dark cycle, and ad libitum access to food and water. All procedures involving animals were conducted in accordance with our institutional guidelines for the care and use of laboratory animals, which were approved by Shahed University Ethics subcommittee in 2024 (approval no. IR.Shahed.REC.1403.041). In addition, this study is reported in compliance with ARRIVE guidelines.

The animals (n = 32) were randomly assigned to four testing groups (n = 8/group) as follows: Control group, animals received the vehicle (Cremophor 10%, SigmaAldrich, USA) of the pioglitazone orally via gavage daily for 3 weeks. Additionally, a single intraperitoneal injection of the trimethyltin (TMT) vehicle (distilled water) was administered; TMT group, animals received a single i.p. injection of the neurotoxin TMT (8 mg/kg), as previously described³⁰, they were also administered the pioglitazone vehicle orally via gavage daily for 3 weeks; TMT + Pioglitazone group, animals received a single i.p. injection of TMT (8 mg/kg) and were then treated with pioglitazone at a dose of 20 mg/kg/day orally via gavage for 3 weeks; TMT+ Donepezil as the positive control anti-AD drug, animals received a single i.p. injection of TMT (8 mg/kg) and were then treated with donepezil at a dose of 4 mg/kg/day orally via gavage for 3 weeks. Administration of therapeutics including pioglitazone and/or donepezil started one hour following TMT injection. Dose of pioglitazone, i.e. 20 mg/kg, was in accordance to its beneficial effect against high fat diet-instigated metabolic disorder and associated depression and hippocampal astrocytic alterations³¹ and its effect against lipopolysaccharide (LPS)-prompted amyloidogenesis besides cognitive deficit³². Dose of donepezil as the positive control drug of this study, i.e. 4 mg/kg, p.o., was derived from an earlier investigation on its protective effect against memory deficit in streptozotocin murine phenotype of type-2 diabetes³³. The experimental protocol is summarized in Fig. 1.

Fig. 1.

Diagrammatic experimental plan of the study. TMT was injected (i.p. route, 8 mg/kg, once) to provoke neurotoxicity. One hour after TMT, pioglitazone (p.o., 20 mg/kg, daily for 3 weeks) or donepezil (p.o., 4 mg/kg, daily for 3 weeks) were administered.

Behavioral assessment

Barnes maze test

Barnes maze is a dry-based behavioral test that has been developed to study spatial memory in rodents^30,34. It is obviously one of the optimized apparatuses for hippocampal-dependent memory task and rats have to find a relation between surrounding cues and a fixed escape area. The apparatus consisted of an elevated circular platform with 20 holes on the edge, evenly spaced and with a diameter of 120 cm. Since rodents show aversive behavior for light and open area, there is a strong motivation behavior for animals to find a dark and closed space. During the training phase, animals used random, serial and spatial strategies to find the hidden safe space during 2-min sessions. Following the acquisition phase, the escape box was removed and spatial memory was assessed. Memory retention was tested on day 18 after TMT injection. Surface of the apparatus was cleaned with 70% alcoholic solution to remove olfactory cues. The number of errors and the time latency in finding the correct hole were recorded for each rat.

Novel object recognition (NOR) test

This test is used to evaluate some aspects of learning and memory in rodents, i.e. animal ability to recognize a novel object in the environment^30,35. It consisted of three phases, i.e. habituation, training, and testing. Rats were placed in the apparatus for being habituated. On the following day, rats were allowed to get familiar with the two identical objects (training day). One of the objects was replaced with a new one on the following day. Since rodents have an innate preference for novel objects, they spend more time on the new replaced one. The novel object recognition index was considered as the time each rodent spent on the new object and expressed it in percentage by the formula (novel object exploration time/total exploration time for both objects) * 100.

Biochemical analyses

Tissue lysate preparation

At the end of the behavioral assessments, for euthanasia, animals were deeply anesthetized in a CO2 chamber connected to 100% CO2 cylinder and with a filling rate of 40–55% displacement/min and brains were immediately separated. Left-side hippocampal blocks were homogenized in 50 mM Tris lysis buffer with pH at 7.4 to yield a 5% lysate (w/v). Homogenized samples were centrifuged (5,000 rpm at 4 °C) and the supernatants were gathered for the following assays.

Total protein of the samples was assessed by the bicinchoninic acid (BCA) assay reagent (Kiazist, Iran). In this experiment, proteins reduce bivalent copper to monovalent copper at 55 °C in an alkaline condition in the presence of bicinchoninic acid. After 30 min of the reaction, absorbance readings were done at 560 nm and with albumin protein as its standard.

Quantitation of oxidative stress markers

Griess reagent was used for quantitation of nitrite as a byproduct of nitric oxide (NO) metabolism (Cat no. G7921, Thermo Fisher Sceintific, USA). In this reaction, sulfanilic acid is converted to a diazonium salt by its reaction with nitrite under acidic condition with subsequent coupling with N-(1-naphthyl)ethylenediamine and formation of an azo dye that can be quantitated with its absorbance reading at 548 nm.

Hippocampal quantity of malondialdehyde (MDA), which is regarded a valid chemical indicator of lipid oxidation³⁶, was determined with its specific kit (Kiazist, Iran). Briefly, 2-thiobarbituric acid dissolved in glacial acetic acid was used with boiling for 35 min and absorbance reading was done at 530 nm.

For analysis of superoxide dismutase (SOD) activity, its specific kit from Kiazist (Iran) was used with its reaction in the presence of xanthine oxidase (XO) and SOD reagent and finally taking its absorbance at 565 nm.

To assess catalase activity, its commercial kit obtained from KiaZist (Iran) was employed with its reagent comprising specific buffer, methanol, hydrogen peroxide as its substrate, purpald, potassium periodate, and potassium hydroxide solution and finally absorbance reading at 545 nm.

Measurement of ER stress-, mitochondrial dysregulation-, neuroinflammation- and neurodegeneration-allied factors by the ELISA method

The quantity of related crucial markers for these biochemical events were quantitated in the hippocampal lysed samples by ELISA routine procedure and specific antibodies raised against NLR family pyrin domain containing 3 (NLRP3) (Cat # ab277086, Abcam, USA), tumor necrosis factor α (TNFα) (Cat # sc-52746, Santa Cruz Biotech, USA), interleukin 10 (IL-10) (Cat # sc-365858, Santa Cruz Biotech, USA), phosphorylated tau (P-tau) (Cat # sc-32275, Santa Cruz Biotech, USA), presenilin-1 (Cat # sc-365495, Santa Cruz Biotech, USA), mitochondrial transcription factor A (mtTFA, TFAM) (Cat # sc-166965, Santa Cruz Biotech, USA), ER stress-related factors including C/EBP homologous protein (CHOP; Cat # MBS1607940, MyBioSource, USA), glucose-regulated protein 78 (GRP78; Cat # MBS1600255, MyBioSource, USA), protein kinase RNA-like ER kinase (PERK; Cat # LS-F21585, LSBio, USA), inositol-requiring enzyme 1-alpha (IRE1α; Cat # sc-390960, Santa Cruz Biotech, USA), peroxisome proliferator-activated receptor γ (PPARg; Cat # MBS160956, MyBioSource, USA), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α; Cat # sc-518025, Santa Cruz Biotech, USA). Pertinent horseradish peroxidase (HRP)-coupled secondary antibody was obtained from Santa Cruz Biotech, USA (m-IgGκ BP-HRP, Cat # sc-516102). Finally, absorbance readings were done at 450 nm using a convenient microplate reader (BioTek, USA).

Determination of mitochondrial membrane potential (MMP)

MMP index is a consistent factor denoting functional integrity of the mitochondrial compartment and its preservation is essential to establish mitochondrial homeostasis³⁷. This index was assessed as mentioned in a previous research report³⁸. The mitochondrial part was isolated via differential centrifugation of the supernatant samples at 10,000 rpm for 10 min. Then, obtained mitochondria fraction was reacted with rhodamine 123 (Cat # R8004, SigmaAldrich, USA) and kept at 37 °C for a period of 5 min. Quantitative estimation of MMP was done at prearranged wavelengths (λex at 488 nm and λem at 525 nm). The fluorescence intensity was then shown in arbitrary fluorescence unit (AFU).

Quantitation of acetylcholinesterase (AChE) activity

Activity of the enzyme AChE was quantitated by a modified Ellman’s protocol, as mentioned before³⁹. In this assay, activity was measured by assessing the generated product upon reaction of Ellman reagent with thiocholine due to breakdown of the acetylthiocholine. The absorbance at 412 nm was finally taken with reporting in nmol of the substrate/minute/g of protein.

Quantitation of myeloperoxidase (MPO) activity

The enzyme MPO is regarded as a specific marker of neutrophil invasion and microglial activation with significance during neurodegeneration⁴⁰. Activity of this enzyme was determined in accordance to an earlier study⁴¹ and with its reagent comprising K2HPO4 buffer, hexadecyltrimethylammonium bromide (CTAB), O-dianisidine dihydrochloride, and H2O2. Absorbance was finally obtained at 457 nm and results were shown in Unit/g tissue.

Determination of caspase 1 activity

The enzyme caspase 1 is regarded a key player during inflammation owing to its involvement in activation of various pro-inflammatory cytokines, i.e. IL-1β and IL-18⁴². A specific colorimetric assay reagent (Cat. # ab39470, Abcam, USA) containing assay buffer, dithiothreitol (DTT), and YVAD-p-NA as the substrate was employed to determine its activity at 405 nm.

Histopathological analyses

Three weeks post-TMT, right-side hippocampal blocks were prepared, fixed in formalin (10%), embedded in paraffin, and 5 μm coronal sections were taken for subsequent assessment with Nissl staining by 0.1% Cresyl violet and immunohistochemical (IHC) evaluation of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes.

For IHC, a series of alternate sections underwent deparaffinization, antigen unmasking, and reaction with primary GFAP antibody (Cat. # sc-33673, Santa Cruz Biotech, USA; dilution = 1:70) for 13 h at 4 °C. Immunodetection was then conducted using secondary HRP-coupled antibody (Cat. # sc-516102, Santa Cruz Biotech, USA; dilution = 1:90). Visualization was made by diaminobenzidine-tetrahydrochloride (DAB; Cat. # sc-209686, Santa Cruz Biotech, USA) in the presence of hydrogen peroxide. For GFAP quantification, an image analysis software was used (ImageJ 1.54, NIH). GFAP immunoreactivity (IRA) was reported in integrated optical density (IOD).

To assess neuronal density and neurodegeneration, healthy pyramidal neurons in the hippocampal CA1 area with distinct boundary and discernable nucleolus were counted in at least four sections (at least 150 μm apart), equivalent to stereotaxic planes 3.2–4.3 behind the bregma reference point. All analyses were performed on coded slides to avoid biased judgement.

Statistical tests

Statistically, data analysis was made in GraphPad Prism Package (10.4.1) environment. Data normality was verified using Shapiro-Wilk test. All data were shown as means ± standard error. Existing differences between the tested groups were found out by one-way ANOVA and successive Tukey test with significance level at p < 0.05.

Results

Pioglitazone diminished behavioral neurocognition deficits in NOR and barnes maze tasks

Novel object recognition (NOR) task was used to assess neutral context-dependent memory⁴³. Our Tukey analysis showed that TMT group has a significant fall of NOR index when compared to the control data (p < 0.01) and pioglitazone administration to the TMT group significantly improved this index relative to the TMT group (p < 0.05) (Fig. 2a).

Fig. 2.

Neurocognitive performance of rats in novel object recognition (NOR) test (a) and Barnes maze task (b and c). One-way ANOVA revealed significant and marked inter-group differences for NOR index (F3,28 = 5.96, p < 0.01) and Barnes maze indices including errors (F3,28 = 11.19, p < 0.001) and latency (F3,28 = 10.73, p < 0.001). Donepezil given to the TMT group was also associated with significant improvement of NOR index and fall of errors relative to the TMT group. Data are shown as mean ± SEM (n = 8/testing group).

Barnes maze test as a hippocampal-dependent task was used to assess spatial learning and memory³⁴. Our analysis showed that TMT group has a significant increase of errors (p < 0.001) and latency (226.5%, p < 0.001) as compared to the control data and pioglitazone administration to the TMT group significantly lowered errors (p < 0.05) and latency (p < 0.05) relative to the TMT group (Fig. 2b and c).

Pioglitazone alleviated hippocampal oxidative stress

Data analysis demonstrated that hippocampal levels of MDA (Fig. 3a) (p < 0.001) and nitrite (Fig. 3b) (p < 0.05) was significantly greater and activity of both SOD (Fig. 3c) (p < 0.05) and catalase (Fig. 3d) (p < 0.05) was significantly less in the TMT group when compared to the relevant data of the control group. By contrast, pioglitazone administration to the TMT group partly reversed back these abnormal alterations for MDA (p < 0.05), SOD (p < 0.05) and catalase (p < 0.01) and with no significant change of nitrite (p > 0.05), when compared to the TMT group values.

Fig. 3.

Hippocampus oxidative stress-associated factors consisting of malondialdehyde (MDA) quantity as an indicator of lipid peroxidation (a), nitrite as a by-product of gaseous NO metabolism (b), and activity of superoxide dismutase (SOD) enzyme (c) and catalase enzyme (d) as determinant antioxidant elements. Data analysis for these factors exhibited significant inter-group differences with respect to MDA (F3, 24 = 7.34, p < 0.01), nitrite (F3, 24 = 4.29, p < 0.05), SOD activity (F3, 24 = 4.57, p < 0.05), and catalase activity (F3, 24 = 7.29, p < 0.01). Donepezil fed to the TMT group was associated with significant reduction of MDA (p < 0.05) and improvement of catalase activity (p < 0.05) with no significant effect on nitrite and SOD activity (p > 0.05). Data are shown as mean ± SEM (n = 7/testing group).

Pioglitazone prevented alteration of inflammatory and AD-associated factors

Tukey analysis indicated that levels of TNFα (Fig. 4b) (p < 0.001), presenilin-1 (Fig. 4c) (p < 0.01), and p-tau (Fig. 4d) (p < 0.001) are significantly elevated and IL-10 (Fig. 4a) quantity was significantly less (p < 0.01), when compared to comparable data of the TMT group. Contrariwise, pioglitazone treatment of the TMT group was associated with significant reduction of TNFα (p < 0.05), p-tau (p < 0.05), and presenilin-1 (p < 0.05) and significant improvement of IL-10 (p < 0.01), as compared to the TMT group.

Fig. 4.

Hippocampal quantities of inflammation-allied factors including IL-10 (a) and TNFα (b) in addition to Alzheimer’s neurodegeneration-specific factors including presenilin-1 (c) and hyperphosphorylated tau (p-Tau) (d). One-way ANOVA showed significant inter-group variations for IL-10 (F3, 24 = 5.68, p < 0.01), TNFα (F3, 24 = 11.49, p < 0.001) and AD-associated factors including presenilin-1 (F3, 24 = 6.20, p < 0.01) and hyperphosphorylated Tau (p-Tau) (F3, 24 = 7.29, p < 0.01). Meanwhile, donepezil treatment of the TMT group did not significantly alter hippocampal levels of these variables. Data are shown as mean ± SEM (n = 7/testing group).

Pioglitazone decreased hippocampal cholinergic (AChE) and mitochondrial malfunction besides pyroptosis-associated factors

Tukey analysis showed elevated AChE activity (Fig. 5c) (p < 0.01), higher MPO activity (Fig. 5d) (p < 0.01), greater activity of caspase 1 (Fig. 5a) (p < 0.001) and higher level of NLRP3 (Fig. 5b) (p < 0.001) along with lower levels of MMP index (Fig. 5e) (p < 0.001), TFAM (Fig. 5f) (p < 0.001), PGC1α (Fig. 5g) (p < 0.001), and PPARg (Fig. 5h) (p < 0.001), when compared with relevant values of the control group animals. Contrariwise, pioglitazone treatment of the TMT group was associated with significant fall of AChE activity (p < 0.05), MPO activity (p < 0.05), caspase 1 activity (p < 0.05), NLRP3 (p < 0.05) and significantly higher levels of MMP (p < 0.01), PGC1α (p < 0.01), PPARg (p < 0.05), and TFAM (p < 0.01) relative to the comparable values of the TMT group.

Fig. 5.

Hippocampal activity of pyroptotic factors consisting of caspase 1 activity (a) and NLRP3 level as a specific factor for inflammasome-associated pyroptosis (b), activity of the ACh degrading enzyme acetylcholinesterase (AChE) (c), myeloperoxidase (MPO) activity as an index of neutrophilic invasion (d), mitochondrial membrane potential (MMP) index (e), and levels of mitochondrial transcription factor A (TFAM) (f), the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (g), and peroxisome proliferator-activated receptor gamma (PPARg) (h) as indicators of mitochondrial function. One-way data analyses indicated significant inter-group variations for pyroptosis-related indices comprising caspase 1 activity (F3, 24 = 9.49, p < 0.001) and NLRP3 level (F3, 24 = 16.63, p < 0.001), AChE activity (F3, 24 = 6.29, p < 0.01), MPO activity (F3, 24 = 5.21, p < 0.01), and mitochondrial integrity-related indices comprising MMP index (F3, 24 = 13.20, p < 0.001) and TFAM level (24 = 13.58, p < 0.001) besides PGC1α (F3, 24 = 18.07, p < 0.001) and PPARg (F3, 24 = 14.58, p < 0.001). Donepezil treatment of the TMT group was associated with reduction of NLRP3 (p < 0.05) and AChE activity (p < 0.05) and with no reversal of other factors. Data are shown as mean ± SEM (n = 7/testing group).

Pioglitazone lowered hippocampal ER stress-allied factors

Tukey analysis showed greater levels of CHOP (Fig. 6a) (p < 0.001), PERK (Fig. 6b) (p < 0.001), GRP78 (Fig. 6c) (p < 0.001), and IRE1α (Fig. 6d) (p < 0.001), as compared to comparable data of the control group. In contrast, pioglitazone treatment of the TMT group led to significant attenuation of CHOP (p < 0.05), PERK (p < 0.05), and GRP78 (p < 0.05) in comparison with the relevant data of the TMT group.

Fig. 6.

Hippocampal quantity of endoplasmic reticulum (ER) stress-associated factors consisting of C/EBP homologous protein (CHOP) (a), PKR-like ER kinase (PERK) (b), glucose-regulated protein 78 (GRP78) (c), and inositol-requiring enzyme 1 alpha (IRE1α) (d). One-way ANOVA for ER stress-allied factors showed significant inter-group variations regarding CHOP (F3, 24 = 11.29, p < 0.001), PERK (F3, 24 = 8.21, p < 0.001), GRP78 (F3, 24 = 12.88, p < 0.001) and IRE1α (F3, 24 = 12.63, p < 0.001). Donepezil treatment of the TMT group was associated with significant fall of CHOP (p < 0.05), GRP78 (p < 0.05), and IRE1α (p < 0.05). Data are shown as mean ± SEM (n = 7/testing group).

Pioglitazone treatment lowered CA1 neurodegeneration along with mitigation of astrogliosis

Tukey post-test revealed significantly elevated degeneration in the TMT-injured rats relative to the control (Fig. 7) (p < 0.001) and pioglitazone treatment of the TMT group attenuated intensity of this degeneration in comparison with the TMT group (p < 0.05). In addition, Tukey analysis disclosed significantly higher GFAP immunoreactivity (Fig. 8) in the TMT group as compared with the control group (p < 0.001) and pioglitazone treatment of the TMT group reduced GFAP immunoreaction in comparison with the TMT group (p < 0.01).

Fig. 7.

Histopathological quantitative analysis of hippocampal CA1 neurodegeneration and comparable photomicrographs. One-way ANOVA test indicated significant inter-group differences for degeneration of CA1 pyramidal neurons, as revealed upon Nissl staining (F_{3, 20} = 11.69, p < 0.001). No significant effect of donepezil was obtained regarding neurodegeneration. Data are shown as mean ± SEM (n = 6/testing group).

Fig. 8.

Quantitative analysis of hippocampal glial fibrillary acidic protein (GFAP) as a distinctive marker of astrocytes and comparable photomicrographs. Statistical analysis for GFAP immunoreactivity (IRA) through one-way ANOVA test disclosed significant inter-group variation (F_{3, 20} = 21.97, p < 0.001). Moreover, donepezil treatment of the TMT group did not produce significant reduction of the GFAP IRA. Data are shown as mean ± SEM (n = 6/testing group).

Discussion

The current study aimed to evaluate the neuroprotective effects of pioglitazone in a rat model of neurodegeneration induced by trimethyltin (TMT), a well-established neurotoxin for inducing selective hippocampal damage and mimicking some aspects of AD. Our results demonstrated that pioglitazone treatment significantly ameliorated behavioral, biochemical, and histological impairments induced by TMT administration. These findings underscore the potential of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, in mitigating neurodegeneration-associated pathology and cognitive dysfunction.

TMT exposure resulted in marked cognitive deficits, as evidenced by decreased performance in the novel object recognition (NOR) test and Barnes maze. Specifically, TMT-treated rats exhibited a significant reduction in recognition index and an increase in both the number of errors and latency during Barnes maze testing. These findings are consistent with prior reports describing the detrimental cognitive impact of TMT^16,30. Importantly, pioglitazone administration reversed these deficits, suggesting restoration of memory function. Given that cognitive impairments in AD are largely attributed to hippocampal damage, the cognitive improvement observed in pioglitazone-treated TMT rats may provide a compelling rationale for its further exploration as a disease-modifying agent in neurodegenerative conditions.

In line with behavioral findings, histological analysis revealed extensive neurodegeneration and neuronal loss in the CA1 region of the hippocampus in TMT animals. The observed neuronal loss was significantly mitigated in animals receiving pioglitazone, further supporting its neuroprotective role. Notably, pioglitazone is known to activate PPARγ pathway, which modulates gene transcription associated with anti-inflammatory and anti-apoptotic mechanisms⁴⁴.

Recent evidence indicates the important bidirectional interaction of oxidative stress and ER stress as key contributors to the pathogenesis of neurodegenerative diseases such as AD. In this respect, oxidative imbalance upsets ER protein folding process and calcium balancing and unresolved ER stress with its markers such as PERK, IRE1α, GRP78, and CHOP leads to overproduction of free radicals, prompting neuroinflammation, glial impairment, and subsequent neurodegeneration. These processes are considered as attractive targets for intervention strategies^45,46. When antioxidant defensive system could not effectively counteract excess free radicals, vicious cycle of oxidative damage begins. In addition, oxidative injury is associated with mitochondrial dysfunction and inflammation. Alongside, uncompensated ER stress can shift cells toward an apoptotic event^47,48. Our data showed that TMT markedly reduces the activities of key antioxidant enzymes, i.e. catalase and superoxide dismutase (SOD), while significantly increasing levels of malondialdehyde (MDA), a byproduct of lipid peroxidation, and nitrite, a reactive nitrogen species (RNS). These changes are indicative of heightened oxidative stress. Pioglitazone treatment effectively restored catalase and SOD activity and reduced MDA concentrations, suggesting a normalization of the oxidative status in the hippocampus. These findings align with earlier studies reporting that pioglitazone can attenuate oxidative damage in various models of central nervous system disorders^27,49. This study demystified protective effect of pioglitazone against TMT-instigated ER stress factors, which was shown by lower GRP78, IRE1α CHOP, and PERK. Consistent with such findings, pioglitazone can diminish reoxygenation-induced renal tubular cells through inhibition of oxidative stress load and ER stress³⁷ and PPAR-γ agonistic agent pioglitazone treatment lowers oxidative and ER stresses in N-nitro-L-arginine methyl ester-induced model of hypertension in the rats⁵⁰. However, there still exists contradicting evidence on the effect of pioglitazone on ER stress⁵¹, which entails further investigation. Our results also demonstrated that TMT significantly increases hippocampal TNF-α levels and MPO activity, both hallmark indicators of neuroinflammation. Concomitantly, IL-10, an anti-inflammatory cytokine, was significantly downregulated. Interestingly, pioglitazone treatment reduced TNF-α and MPO activity while restoring IL-10 levels, further indicating a potent anti-inflammatory action. These effects are likely mediated through PPARγ activation, which has been shown to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, a key regulator of pro-inflammatory gene expression⁵². The dual modulation of pro- and anti-inflammatory mediators by pioglitazone underscores its potential to rebalance the neuroinflammatory milieu characteristic of neurodegenerative states.

TMT exposure causes selective neurodegenerative alterations of the hippocampus due to its enhancement of inflammatory responses, linked to NLRP3 inflammasomes⁵³. Likewise, we observed higher hippocampal level of pyroptosis, as shown by higher NLRP3 and enhanced activity of caspase 1. Conversely, pioglitazone treatment of the TMT group was associated with lower intensity of pyroptosis. To support this finding, it has been proven that pioglitazone can mitigate sepsis-accompanying encephalopathy through partial suppression of cerebral activation of microglia, oxidative stress, NLRP3, and caspase 3⁵⁴.

Beyond classical inflammatory markers, astrocyte reactivity, assessed via GFAP expression, was significantly elevated in TMT-exposed animals, reflecting astrogliosis, a common feature in neurodegenerative disorders. Pioglitazone substantially reduced GFAP expression, suggesting attenuation of astrocytic activation. These findings are consistent with prior evidence indicating that pioglitazone can limit glial activation, potentially through downregulation of pro-inflammatory cytokines and mitigation of oxidative stress^55,56. As reactive astrocytes are involved in the progression of neurodegeneration through both neurotoxic and neuroprotective pathways, the ability of pioglitazone to modulate their activity may contribute significantly to its overall neuroprotective effects.

Tau pathology and aberrant amyloid precursor protein (APP) processing are pathological hallmarks of AD. Although the TMT model does not fully recapitulate AD pathology, it induces some biochemical alterations that overlap with the disease, including hyperphosphorylated tau (p-Tau) and increased expression of presenilin-1 (PSEN1), a component of the γ-secretase complex involved in amyloidogenic APP processing. Our study demonstrated that TMT administration significantly elevated both p-Tau and PSEN1 levels in the hippocampus. Pioglitazone treatment significantly reduced these elevations, pointing toward a modulatory effect on tau phosphorylation and amyloidogenic pathways. These findings align with earlier studies demonstrating that pioglitazone reduces tau hyperphosphorylation in diabetic rats⁵⁷ besides its mitigation of tauopathy and amyloid burden in AD-related models^23,58.

In this animal research, we did observe a significant increase of acetylcholinesterase (AChE) activity in the TMT group, which to some extent and significantly reversed back with pioglitazone treatment. AChE hyperactivity is responsible for cholinergic dysfunction in AD and its associated conditions and its modulation is a key therapeutic strategy⁵⁹. The reduction of AChE activity by pioglitazone may reflect preservation of cholinergic neurons or an indirect consequence of its anti-inflammatory and antioxidant effects. This adds another dimension to pioglitazone’s neuroprotective profile, suggesting potential synergy with existing cholinergic therapies in AD. In agreement with this finding, Rajabian et al.⁶⁰, have shown that treatment with pioglitazone is associated with improvement of learning and memory in a murine model of cholinergic deficit due to scopolamine, partly through its attenuation of AChE activity⁶⁰.

Mitochondrial dysfunction is an important pathogenic factor in the development of neurodegenerative disorders and neurological syndromes, as typified by noticeable cell death in involved brain regions⁶¹. Similarly, mitochondrial dysfunction is also postulated as a key factor in AD pathogenesis which is a promising goal for developing novel therapeutics(Ashleigh et al., 2023). In the present investigation, intraperitoneal TMT caused notable fall of hippocampal TFAM as a significant mitochondrial transcription factor with a pivotal role in mitochondrial maintenance and health as well as MMP as a valuable marker of mitochondrial function which its reduction obviously signifies threatened mitochondrial health. To demystify cellular targets of TMT in more detail, we also quantified hippocampal levels of PPARγ and PGC-1α, both of which have determinant roles in mitochondrial biogenesis and functionality and also with therapeutic worth for restoring mitochondrial tasks in neurological illnesses^62,63. As has been shown before^30,64, hippocampal levels of MMP, PGC-1α, and PPARγ levels notably drops in the TMT group. In contrast, pioglitazone treatment of the TMT group was accompanied by improvement of mitochondrial integrity-allied factors. Consistent with this finding, reversing and protective effect of pioglitazone on mitochondrial biogenesis and/or function has been proven in past studies^65–67.

Pioglitazone is a thiazolidinedione that can cross blood brain barrier (BBB) to attain suitable concentrations in the brain⁶⁸. Preliminary evidence has shown that pioglitazone at its sub-therapeutic and low dose which is ineffective for the treatment of type 2 diabetes mellitus can produce changes in the brain functional connectivity in rats and these connections are affected in early stages of AD⁶⁹. However, further researches are still required to investigate how the pioglitazone affects brain regions in relation to AD.

Several limitations of the present study should be considered. Firstly, the TMT model, while useful for studying hippocampal neurodegeneration and cognitive impairment, does not fully replicate the complex, progressive pathology of human AD. In this regard, although TMT administration is obviously associated with cognitive decline and neurodegeneration, however, it is not suggested as a direct causative neurotoxin for AD in the population. Since AD is known as a multifaceted and multifactorial pathological condition and genetic, age-related factors, and environmental state play pivotal roles in its pathogenesis, thus, TMT-provoked hippocampal injury could not replicate all aspects of AD pathology such as notable aggregation of Aβ plaques, chronic neuroinflammation, and even cortical spread as pathological hallmarks of AD in humans⁷⁰. Another limitation of our study was that only a single dose of pioglitazone (20 mg/kg) was used with a sub-chronic treatment period (3 weeks). Long-term efficacy of pioglitazone and its dose-response relations are strongly suggested for relevant studies. Additionally, forthcoming studies should explore the effects of pioglitazone in transgenic models that exhibit both amyloid and tau pathology. Lastly, further molecular assessments of downstream PPARγ target genes and signaling pathways are suggested. In this respect, although PPARγ activation was suggested as the underlying mechanism in this study, direct validation through application of antagonists, knockdown strategy, and/or gene expression assays would strengthen the findings^71,72.

To the best of our knowledge, this study is the first report showing that pioglitazone could mitigate pyroptotic marker including NLRP3 and caspase-1 and ER stress factors consisting of CHOP, IRE1α, and GRP78 in the TMT AD-related phenotype of neurodegeneration. In this study, we also compared the beneficial effect of anti-diabetic pioglitazone with FDA-approved donepezil as our positive control drug. Although donepezil administration was associated with some improvements in behavioral tasks for cognitive performance, however, it did not produce reversal and significant effects regarding some oxidative stress-, ER-stress-, and neuroinflammation-related factors, specific AD-associated markers including presenilin-1 and p-Tau, some pyroptotic and mitochondrial dysfunction factors, and hippocampal neurodegenerative changes. This clearly indicate the pioglitazone multi-targeted effects under conditions of neurodegeneration which warrants further research.

Together, findings of this study may build promising evidence for pioglitazone as a multifunctional neuroprotective agent in some models of neurodegeneration.

Author contributions

NA: Investigation, formal analysis, writing original draft, review and editing; MR: Conceptualization, resources, funding acquisition, methodology, supervision, formal analysis, validation, review and editing; MB: Conceptualization, formal analysis, writing original draft, review and editing; MK: Conceptualization, supervision, formal analysis, review and editing.

Funding

This research project was partially supported in 2024 by Shahed University (grant # 522458).

Data availability

Data presented and analyzed for the current study will be available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.