Chronic lipopolysaccharide exposure promotes cognitive impairments by activating TRPC6-AIM2 inflammasome signaling and the regulation of ginsenoside Rg1 in Trpc6^−/− mice

Abstract

Background

Chronic neuroinflammation is a pivotal pathogenesis in neurodegenerative diseases (NDDs). Transient receptor potential canonical protein 6 (TRPC6) has an essential role in the maintenance of calcium homeostasis in cells. Our previous study indicated that TRPC6 signaling is involved in Aβ deposition and NLRP1 inflammasome activation in type 2 diabetes mellitus-associated cognitive dysfunction. However, whether TRPC6 signaling contributes to chronic lipopolysaccharide (LPS)-induced neuroinflammatory injury and the mechanism remain unclear.

Methods

In this study, male mice received intraperitoneal injections of LPS (200 µg/kg) for 21 days to induce a chronic neuroinflammation model. The open field test, hole-board test, and Morris water maze were conducted to evaluate cognitive function. The H&E and Nissl staining was employed to examine neuronal injury. The immunofluorescence, western blotting, or q-PCR were used to analyze TRPC6, AIM2 inflammasome expression, and Nrf2 activation. The fluorescent probes and calcium imaging were performed to assess ROS accumulation and calcium dysregulation in LPS-induced HT22 neuron cells.

Results

Chronic LPS exposure induced behavioral deficits in locomotion, exploratory behavior, and learning and memory, and neuronal damages with less expressions of PSD95 and Synaptophysin in mice. Mechanistically, LPS exposure significantly increased ROS production, TRPC6 expression and calcium overload, and induced AIM2 inflammasome activation in vivo or in vitro. While Trpc6 knockout could significantly improve LPS-induced cognitive dysfunction and neuronal injuries, inhibit TRPC6-mediated calcium overload, and downregulate the expressions of AIM2, caspase-1, IL-1β, IL-6, caspase-3 and Bax in vivo or in vitro. Additionally, Rg1 treatment significantly inhibited calcium overload and AIM2 inflammasome activation in LPS-induced HT22 cells. More importantly, Rg1 significantly activated Nrf2 signaling and reduced ROS production in LPS-induced mice or HT22 cells.

Conclusions

Trpc6 knockout can improve chronic LPS-induced neuroinflammation and injury by inhibiting TRPC6-AIM2 inflammasomes. While Rg1 treatment can alleviate LPS-induced neuroinflammation and injury not only by inhibiting TRPC6-AIM2 inflammasomes activation but also activating Nrf2 signaling.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12993-025-00290-1.

Introduction

Chronic neuroinflammation has a pivotal impact in neurodegenerative diseases including Parkinson’s disease (PD), multiple sclerosis and Alzheimer’s disease (AD). Pro-inflammatory cytokines, chemokines, and reactive oxygen species (ROS) are released and trigger inflammation because of the continued activation of microglia and astrocytes in the central nervous system (CNS) [1, 2]. These inflammatory mediators induce profound disruptions in neuronal function, including synaptic impairment, axonal degeneration, and neuronal apoptosis [3]. Conversely, damaged neurons amplify inflammation by activating inflammasome signaling, creating a vicious cycle. Lipopolysaccharide (LPS) has been reported significantly elevated in blood and brain tissue in patients with neurodegenerative diseases [4]. Therefore, LPS often serves as a well-established model to induce inflammation-mediated neurodegeneration [5]. Chronic LPS exposure could promote neuronal inflammation and injury through activating AIM2 inflammasome [6]. Despite advances in understanding neuroinflammation, the mechanisms underlying AIM2-mediated inflammation remain unclear, and effective therapeutic strategies are lacking. Exploring precise mechanisms and developing effective therapeutic strategies remain a challenge.

Transient receptor potential canonical protein 6 (TRPC6), a calcium-permeable cation channel, plays a fundamental role in maintaining intracellular calcium homeostasis [7]. Calcium overload is closely associated with pathological processes in AD, such as β-amyloid (Aβ) aggregation, tau protein phosphorylation. Conversely, Aβ can lead to an imbalance in calcium homeostasis by activating calcium channels or affecting the endoplasmic reticulum calcium pump [8]. TRPC6 maintains neuronal function by regulating calcium homeostasis in the CNS, and maintains dynamic intracellular calcium homeostasis in neurons and glial cells by modulating calcium influx, and exhibits neuroprotective effects in neurodegenerative diseases [9, 10]. Additionally, high levels of calcium ions in the mitochondria can induce reactive oxygen species (ROS) accumulation and triggering cell death. LPS exposure has also been shown to perturb key signaling pathways vital for cellular homeostasis, such as imbalance of redox and calcium homeostasis induced by activating TRPC6 signaling [11, 12]. Therefore, we supposed that TRPC6 might play a pivotal role in LPS-induced neuroinflammation.

ROS can act both as signaling molecules by affecting target proteins and as oxidative stressors by damaging cellular components. Given the broad action and overlapping signaling pathways of calcium ions and ROS, there is increasing evidence that the interaction between them is important for the development and progression of neurodegenerative disease [13]. Additionally, LPS has been reported to suppress the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, which plays a master role in regulation of the antioxidant response [14]. Activation of Nrf2 promotes the transcription of antioxidant enzymes such as heme oxygenase-1 (HO-1) and glutathione peroxidase, facilitating ROS elimination and inflammation resolution [12]. And suppression of Nrf2 induced by LPS diminishes the CNS’s capacity to counteract oxidative stress and inflammation, further intensifying neuronal injury [15]. However, the interrelationship between TRPC6 and Nrf2 pathways under LPS-induced neuroinflammation is still not fully elucidated.

Ginsenoside Rg1, a principal bioactive constituent of Panax ginseng, is well recognized for its robust anti-inflammatory, antioxidant, and neuroprotective effects [16]. Experimental studies indicate that Rg1 can attenuate neuroinflammatory processes by inhibiting microglial activation and downregulating the production of pro-inflammatory cytokines [17, 18]. Rg1 can enhance Nrf2 signaling, promoting the transcription of antioxidant defense enzymes and mitigating oxidative damage [19]. Additionally, the regulatory effects of Rg1 on calcium signaling further underscore its potential to restore cellular homeostasis under pathological condition [20, 21]. Despite its promising pharmacological profile, the role of Rg1 in ameliorating LPS-induced neuroinflammation, particularly in Trpc6-deficient conditions, has not been elucidated. This study is designed to explore the mechanism of chronic LPS exposure in promoting neuroinflammation by activating TRPC6-AIM2 inflammasome, and evaluate the therapeutic effects of Rg1 on LPS-induced chronic neuroinflammation in Trpc6^−/− mice. This study may provide a novel mechanism and strategy for mitigating inflammation-related neurodegenerative diseases.

Methods

Animals and drug treatment

The heterozygote C57BL/6N-Trpc6^em1/Cya mice (Trpc6^+/−, S-KO-05573) were acquired from Cyagen Biosciences Inc. All mice were placed in routinely laboratory conditions. Male Trpc6^−/− and wild-type (WT) offspring, aged 8 weeks, were selected for the experiment. The groups are as follows: WT group, WT + LPS (200 µg/kg) group, Trpc6^−/− group, Trpc6^−/− + LPS group, and Trpc6^−/− + LPS + Rg1 (10 mg/kg) group. LPS from E. coli 055: B5 was purchased from Sigma (L2880, Sigma Aldrich, St. Louis, US). LPS was dissolved in saline at a concentration of 20 µg/mL. The mice were injected intraperitoneally with LPS (0.1 ml/10 g body weight) daily for three weeks. Ginsenoside Rg1 (purity ≥ 98%, verified by HPLC, CAS:22427-39-0) was obtained from Desite (Chengdu, China). Rg1 was dissolved in distilled water at a concentration of 1 mg/mL, and administered by intragastric gavage once daily at a dose of 10 mg/kg/day for three weeks. For the WT and Trpc6^−/− groups, the same amount of vehicle was utilized. All animal experiments approved by the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20232095). For the rigor and repeatability of the experiment, all data were analyzed blind.

Cell culture and treatments

HT-22 cells, a mouse hippocampal cell line, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HT-22 cells were routinely cultured. Grouped as follows: control group; LPS (10 µg/ml) group, LPS + TRPC6 inhibitor (BI749327, 0.1 μm, MCE, USA) group and LPS + Rg1 (10 µM) group. The relevant indicators were tested after 24 h of drug-acting cells.

Open-field test (OFT)

The OFT is a method to evaluate autonomous motor behavior, inquiry behavior and tension. Prior to testing, each mouse (n = 8) was placed in the box for 120s to acclimate. Following this, mice was recorded for 180s by ANY-maze system (Stoelting, USA). Key metrics, including total distance traveled (m), line crossings, the number of rearings (stand-ups), and entries into the central zone (indicative of exploratory behavior) were calculated by the system.

Hole-board test (HBT)

The HBT is commonly used to assess exploratory behavior in unfamiliar environments in mice. The device consisted of a roofless square box with 16 evenly distributed holes (4 × 4) on the floor and a Visu-Track system camera (Xinran, Shanghai, China). Each mouse (n = 8) was introduced into the box at one corner, and its activity was recorded for 5 min. The number of head dips and the total head-dipping time were analyzed as indicators of exploratory behavior.

Morris water maze (MWM) test

The MWM test is a classic method used to evaluate spatial learning, memory ability and sense of direction in laboratory animals. Each mouse (n = 8) was sequentially placed in the four quadrants of the pool, and the time to locate the hidden underwater platform (escape latency) was recorded. If a mouse failed to find the platform within 60 s, it was guided to the platform, where it remained for 15 s. Training was conducted over four consecutive days. On the fifth day, the platform was removed, and each mouse was placed in the first quadrant. Behavior was recorded for 90 s to evaluate the memory function.

Histological examination

Mice (n = 4) were injected with PBS and 4% paraformaldehyde from heart, and then the brain tissue was dissected and dehydrated, and paraffin embedded. Brain tissues were sectioned into 4-µm slices using a microtome (Leica, Nussloch, Germany). Morphological changes were assessed using H&E and Nissl staining. Data were acquired using a digital imaging scanning system (Pannoramic MIDI, 3D HISTECH, Hungary).

Measurement of ROS production

In vivo, to evaluate ROS generation, the superoxide anion fluorescent probe DHE (Beyotime Biotechnology, China) was used. Mice (n = 4) were injected with DHE solution (100 µM, 0.1 ml/10 g) via the tail vein and allowed to circulate for 30 min. The Sect. (10 μm) were stained with Hoechst 33,258 (C1017, Biotechnology Co., Ltd., China) and were photographed with the digital imaging scanning system. Quantification of average fluorescence intensity was performed with image J 1.44p software from three randomized regions of hippocampal CA1, CA3 and cortical region (400×).

In vitro, ROS generation was detected using the ROS Detection Kit (Beyotime Biotechnology, China) according to the instructions. DCFH-DA staining solution (10 µM) was added to 24-well cell plates in culture medium and incubated at 37 °C for 30 min. The fluorescence densities of 5 random fields (200 ×) were detected by using a Micro Content Imaging System (Molecular Devices, USA) to show the ROS production. The experiment was conducted four times independently.

Calcium imaging

After 24 h of drug stimulation, the HT-22 cells were treated with Fura-2 AM solution for 30 min. The fluorescence was subsequently detected using an Olympus digital calcium imaging system (IX73, DG-4PLUS/OF30, Japan). The basal levels of [Ca²⁺]_i were detected in the first 5 min. Subsequently, BAPTA (1mM, MedChemExpress, USA) was added for 5 min and CaCl₂ (2mM) was added for another 5 min to change the extracellular Ca²⁺ concentration. The change of [Ca²⁺]_i was calculated using the following formula: [Ca²⁺]_i change = (F340/F380) before treatment - (F340/F380) after treatment. The experiment was repeated 3 times.

Western blot

The lysate (Beyotime Biotechnology, China) was added to the brain tissue or HT-22 cells, and the supernatant was extracted after lysis by grinding in an ice bath. The protein concentration was determined by BCA kit (Beyotime Biotechnology, China). After SDS-PAGE gel electrophoresis, the membrane was transferred to PVDF membrane (Millipore, USA), closed with 5% skimmed milk powder for 1 h, and proteins on PVDF membranes were incubated with the corresponding primary antibody solution (refer to Supplementary Table 1) for 14 h on a shaker at 4℃. The next day, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (1:10000, Biosharp). The protein blot was displayed using ECL developer (BL520B, Biosharp). An imaging equipment (ChemiDoc, Bio-Rad, USA) was used to capture the bands.

Quantitative real-time PCR (qPCR)

AIM2 mRNA expression was quantified by qPCR. Total RNA was isolated from the cortex and hippocampus (n = 3). cDNA synthesis was performed using the Reverse Transcription Kit (Takara Bio, RR037A, Japan). The RTPCR kit (Takara Bio, RR820A, Japan) was employed for mRNA quantification. The PCR amplification conditions included 40 cycles: 95°C for 30 seconds, followed by 95°C for 5 seconds and 60°C for 30 seconds. Ct values were obtained for each sample, and the relative expression of target genes was determined using the 2^−△△Ct method. Primer sequences were as follows: AIM2 (Forward: 5’-AGGC TGC TAC AGA AGT CTG TCC-3’, Reverse: 5’-T CAG CAC CGT GAC AAC AAG TGG-3’) and β-actin (Forward: 5’-C TAC CTC ATG AAG ATC CTG ACC-3’, Reverse: 5’-C ACA GCT TCT CTT TGA TGT CAC-3’).

Enzyme-linked immunosorbent assay (ELISA)

Samples and standards were added to the plate and incubated at 37℃ for 0.5 h. After that, enzyme hydrolysis reagent (50 µl) was added and incubated at 37 °C for 0.5 h. Then add the color development solution (100 µl) and incubate at 37℃ for 0.5 h. Finally, the termination solution (50 µl) was added and the absorbance was detected at 450 nm by Thermo Fisher Scientific (USA).

Immunofluorescence

The sections (n = 4) were subjected to antigen repair followed by endogenous peroxidase blockade for one hour, and primary antibodies of AIM2 (1:100, Affinity, Cincinnati, USA) or p-Nrf2 (1:100, Affinity, Cincinnati, USA) were added and then refrigerated at 4℃ overnight. On the second day, sections were incubated with FITC-coupled secondary antibody (1:200, Proteintech, SA00003-2) for one hour. Finally, sections were stained with Hoechst 33,258 for 9 min, and were scanned using the Pannoramic MIDI. The Image J software was used to quantify the average fluorescence density of three random fields (400 ×) per region in the cortex, hippocampal CA1 and CA3 regions.

Statistical analysis

Experimental data were analyzed using Graph Pad Prism 9.1. Data were shown as mean ± standard deviation (SD). The data were first tested for normality distribution (P > 0.05). Then, one-way ANOVA was performed on all data, and then Tukey’s test was applied to compare the differences between groups. Differences were considered statistically significant at P < 0.05.

Results

Chronic LPS exposure induces behavioral dysfunction and Trpcknockout and Rg1 treatment ameliorate the dysfunction in mice

Neurodegenerative diseases are often characterized by impaired spontaneous activity, exploration, and cognition. Thus, we employed the OFT, HBT and MWM to evaluate these behaviors. In the OFT, LPS-treated mice exhibited a significant reduction in the moving distance, the mean speed, the line crossings and number of stand up on both Day 11 and Day 21 compared with WT group (Fig. 1A–B). Similarly, the HBT showed a marked decline in head dipping and time spent in head dipping (Fig. 1C–D). Importantly, Trpc6 knockout significantly improved all these behavioral parameters relative to the WT + LPS group, restoring both locomotion and exploration (Fig. 1A–D). Moreover, Rg1 treatment in Trpc6^−/− mice resulted in further enhancement of spontaneous activity and exploratory behavior compared to Trpc6^−/−+LPS mice (Fig. 1A-D). These results suggest that chronic LPS exposure induces motor and exploratory deficits and Trpc6 knockout alone or combination with Rg1 can improve these motor and exploratory deficits.

Fig. 1.

Chronic LPS exposure induces behavioral dysfunction and Trpc6 knockout and Rg1 treatment ameliorate the dysfunction in mice. (A) The movement track of mice in OFT; (B) The moving distance; The mean speed; The line crossings; Number of standing up; (C) Representative traces in HBT; (D) Head dipping; )Time spent in head dipping. The data are expressed as mean ± SD, n = 8

Next, the MWM was employed to assess learning and memory. The results indicated that there were significant impairments in learning and memory functions in LPS-treated mice, which showed prolonged escape latency and shorter swim distances in positioning navigation, prolonged times to enter the platform zone, fewer platform crossings, and reduced time spent in the target quadrant in spatial exploration compared to WT control group (Fig. 2A-F). However, Trpc6 knockout significantly mitigated impairments in cognitive functions in mice. Notably, Rg1 treatment further significantly improved LPS-induced deficits in learning, and memory in Trpc6^−/− mice (Fig. 2A-F). The findings make known that chronic LPS exposure induces behavioral dysfunction and Trpc6 knockout and Rg1 treatment can ameliorate LPS-induced behavioral dysfunction in mice.

Fig. 2.

Chronic LPS exposure induces behavioral dysfunction and Trpc6 knockout and Rg1 treatment ameliorate the dysfunction in mice. (A) The movement track in the space exploration experiment; (B) The mean escape latency (s); (C) The swimming distance (m); (D) The swimming time in the quadrant of platform (s); (E) The latency of first entry to the platform (s); (F) The number of crossing the platform. The data are expressed as mean ± SD, n = 8

Chronic LPS exposure induces neuronal damage and Trpcknockout and Rg1 treatment ameliorate LPS-induced neuronal damage in mice

Neuronal dysfunction is intricately linked to pathological damage. To evaluate neuronal injury, H&E staining and Nissl staining were performed. H&E staining revealed no apparent pathological abnormalities in control mice. In contrast, LPS-treated mice displayed significant neuronal damage in the hippocampal CA3 region and cortical areas, characterized by nuclear pyknosis and deep nuclear staining (Fig. 3A). Remarkably, these neuronal injuries were substantially alleviated in both Trpc6^−/− mice and Trpc6^−/−+Rg1 mice (Fig. 3A), showing that Trpc6 knockout and Rg1 treatment mitigate the LPS-treated neuronal injury. Nissl staining is a classic histological staining method in neuropathology, mainly used to display Nissl bodies inside neuronal cells. When neurons are damaged, Nissl bodies first disintegrate and dissipate, and cytoplasmic staining becomes lighter or even pale [22]. In this study, the results of Nissl staining showed that there were abundant Nissl bodies in the cytoplasm of neurons in the cortex and hippocampus of the control group mice, with darker staining, while the neurons in the LPS exposed group showed lighter staining, indicating a decrease in Nissl bodies. However, compared with the WT model group, the Trpc6 knockout and combined Rg1 groups had more Nissl bodies in brain tissue neurons with darker staining (Fig. 3B). Additionally, we examined the expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYN), which are closely associated with synaptic functionality. The results indicated that LPS-treated mice exhibited dramatically decrease in LPS-treated mice comparing with WT control group (Fig. 3C-E). However, Trpc6 knockout could markedly increase their levels and Rg1 administration could further increase their expression in Trpc6^−/− mice (Fig. 3C-E). These findings suggest that LPS exposure induces neuronal injury, and Trpc6 knockout can ameliorate LPS-induced neuronal injury in mice, while Rg1 can further mitigate neuronal and synaptic injuries in LPS-treated Trpc6^−/− mice.

Fig. 3.

Chronic LPS exposure induces neuronal damage and Trpc6 knockout and Rg1 treatment ameliorate LPS-induced neuronal damage in mice. (A) The neuronal pathological change in the cortex, hippocampus, CA1 and CA3 (H&E); (B) The Nissl bodies change in the cortex, hippocampus, CA1 and CA3 (400×); (C-E) The bands and statistical analysis of PSD95 and synaptophysin in hippocampus tissues. The data are expressed as mean ± SD, n = 4

LPS exposure induces neuroinflammation and Trpcknockout and Rg1 treatment ameliorate LPS-induced neuroinflammation in mice

The activation of inflammasomes has been shown to promote neuroinflammation. AIM2 is a pivotal contributor to inflammation. To investigate LPS-induced neuroinflammation, we first examined the expression levels of AIM2 protein and mRNA. The results demonstrated that LPS induction markedly upregulated AIM2 levels of protein and mRNA (Fig. 4E-G). In contrast, Trpc6 knockout could significantly decrease the protein and mRNA expression of AIM2, which was further significantly reduced by Rg1 administration in Trpc6^−/− mice (Fig. 4E-G). We further measured the AIM2 expression by immunofluorescence. The results showed that in WT control group, AIM2 was lowly expressed. However, LPS induction significantly upregulated AIM2 expression in the hippocampus and cortex (Fig. 4A-D). While Trpc6 knockout could significantly decrease the expression of AIM2, which was further reduced by Rg1 in LPS-treated Trpc6^−/− mice (Fig. 4A-D).

Fig. 4.

LPS exposure induces neuroinflammation and Trpc6 knockout and Rg1 treatment ameliorate LPS-induced neuroinflammation in mice. (A) The expression of AIM2 in the cortex, hippocampus CA1, and CA3 (400×); (B-D) The mean density of AIM2 in the cortex, hippocampal CA1, and CA3. (E-F) The bands and statistical analysis of AIM2, in hippocampus tissues. (G) The relative mRNA expression of AIM2. The data are expressed as mean ± SD, n = 3 or n = 4

To confirm the LPS-induced neuronal inflammation and injury, we further detected the expression levels of IL-6, IL-1β, Caspase-1, Caspase-3, Bcl-2 and Bax with WB, and the levels of IL-6 with IL-1β with ELISA in brain tissues. The WB results indicated that LPS induction significantly increased the expression levels of IL-6, IL-1β, Caspase-1, Caspase-3 and Bax, while decreased the expression of Bcl-2 compared to WT controls (Fig. 5A-G). However, Trpc6 knockout could significantly reverse the changes of these inflammation and apoptosis related proteins, which were further improved by Rg1 in LPS-induced Trpc6^−/− mice (Fig. 5A-G). The results of ELISA showed a similar result for IL-6 and IL-1β to their WB results (Fig. 5H-I). In addition, the in vitro results of WB also confirmed that the AIM2, IL-1β, ASC, Caspase-1 and TRPC6 protein levels were significantly upregulated in LPS-induced HT22 cells (Supplementary Fig. 1A-G). In contrast, BI749327 and Rg1 significantly decreased the expressions of AIM2, IL-1β, ASC, Caspase-1 and TRPC6 proteins. Collectively, these findings suggest that LPS exposure induces neuronal inflammation and injury by activating inflammasome of AIM2, and promoting apoptosis related proteins in brain tissue. And Trpc6 knockout could ameliorate LPS-induced neuronal inflammation and injury by restraining AIM2 inflammasome and apoptosis, which were further reduced by Rg1 in Trpc6^−/− mice.

Fig. 5.

LPS exposure upregulates inflammation and apoptosis-related proteins and Trpc6 knockout and Rg1 treatment ameliorate neuronal inflammation and injury in mice. (A-G) The western blot bands and statistical analysis of IL-6, p17, p20, Cleaved-Caspase3, Bcl-2, Bax in hippocampus tissues, n = 4. (H-I) The Elisa of IL-6 and IL-1β, n = 6 or 8. The data are expressed as mean ± SD

Rgtreatment activates Nrf2 signaling and ameliorates LPS-induced oxidative stress injury, while Trpc6 knockout has no effect

ROS are intricately associated with inflammatory responses, forming a vicious cycle that exacerbates oxidative stress and inflammation. To assess ROS accumulation, we used DHE fluorescent probes. The results indicated that exposure to LPS significantly increased ROS levels (Supplementary Fig. 2A-D). Trpc6 knockout had no significant effect on ROS generation. However, Rg1 therapy markedly reduced ROS production in LPS-induced Trpc6 knockout mice (Supplementary Fig. 2A-D).

The Nrf2 signaling plays multifaceted roles in antioxidant defense through up-regulating the antioxidants, for instance, HO-1. To investigate this pathway, we firstly performed immunofluorescence staining to observe the expression of p-Nrf2. The results showed a non-significant increase in p-Nrf2 expression in hippocampus and cortex following LPS induction (Fig. 6A). And Trpc6 knockout did not produce a notable change in p-Nrf2 expression as compared with WT + LPS group. However, Rg1 treatment significantly enhanced p-Nrf2 expression and promoted its nuclear translocation in LPS exposure Trpc6 knockout mice (Fig. 6A-D). Subsequently, WB analysis was employed to quantify Nrf2 expression and its downstream target HO-1. The results demonstrated that LPS induction could increase the expression of p-Nrf2/Nrf2 and HO-1 (both P < 0.01). And Trpc6 knockout showed minimal impact on this phenomenon as compared with WT + LPS group. Notably, Rg1 treatment could further enhance the expression of p-Nrf2/Nrf2 and HO-1 in LPS-induced Trpc6 knockout mice (Fig. 6E-H). These findings suggest that chronic LPS exposure can slightly activate the Nrf2 signaling pathway but can markedly increase ROS production, while Rg1 can significantly activate the Nrf2 signaling pathway and reduce ROS production. And Trpc6 knockout gave no obvious impact on ROS generation and Nrf2 signaling, which may be the upstream signaling of TRPC6.

Fig. 6.

Rg1 treatment activates Nrf2 signaling in LPS-induced mice. (A) The expression of p-Nrf2 in the cortex, hippocampus CA1, and CA3 (400× & 630×); (B-D) The mean density of p-Nrf2 in the cortex, hippocampal CA1, and CA3; (E-H) The western blot bands and statistical analysis of Nrf2, p-Nrf2, HO-1, and TRPC6 in hippocampus tissues. The data are expressed as mean ± SD, n = 4

LPS stimulation induces ROS production and calcium overload, and BIand Rg1 treatment inhibit LPS-induced calcium overload in HT-22 cells

In vitro, we further performed DCFH-DA fluorescence staining to investigate the ROS production, which also closely involves in calcium overload. The results revealed that, in comparison to control group, LPS stimulation markedly raise intracellular ROS levels (Fig. 7A-B). Notably, Rg1 treatment drastically reduced ROS production in LPS-stimulated HT-22 cells (P < 0.01, Fig. 7A–B), whereas BI749327 only showed a no-significant decrease (P = 0.0968). Calcium imaging demonstrated that the basal [Ca²⁺]_i levels were greatly elevated in the LPS-treated group compared to controls (Fig. 7C-D). In contrast, BI749327 and Rg1 treatment could significantly reduce the basal [Ca²⁺]_i levels compared to the LPS group (Fig. 7C-D). Following BAPTA (1 mM) and CaCl₂ treatment, the Δ[Ca²⁺]_i levels were markedly higher in the LPS group compared to controls, whereas BI749327 and Rg1 treatment could significantly reduce the Δ[Ca²⁺]_i levels in comparison to the LPS group (Fig. 7C, E and F). In a nutshell, the results make clear that LPS stimulation induces ROS accumulation and calcium overload in HT-22 cells. Rg1 effectively reduces ROS production and calcium overload, while BI749327 can significantly inhibit LPS-induced calcium overload but has no effect on ROS production.

Fig. 7.

LPS stimulation induces ROS production and calcium overload in HT-22 cells. (A) The results of ROS generation (DCFH-DA, 200×). (B) The mean density of ROS in HT-22 cells. (C) Representative ratios detected by calcium imaging in HT-22 cells (F340/F380); The labels “1 mM “, “0 mM " and “2 mM " represent the extracellular calcium concentrations under basal conditions, following BAPTA-mediated calcium chelation, and after CaCl₂ supplementation, respectively. (D-F) The relative basal [Ca²⁺]_i and relative Δ[Ca²⁺]_i after BAPTA (1 mM) or CaCl₂ (2 mM) treatment in HT-22 cells. The data are expressed as mean ± SD, n = 4

Discussion

Neurodegenerative diseases (NDDs), including Alzheimer’s disease, Parkinson’s disease, and other cognitive disorders, has been a significant public health challenge due to their progressive and debilitating nature [23, 24]. These diseases are characterized by neuronal degeneration, synaptic dysfunction, and eventual cognitive and motor decline, severely impacting patients’ quality of life [25]. Despite advancements in understanding the molecular and cellular mechanisms underlying NDDs, the detailed mechanism remains unclear and no therapeutic strategies have been developed that can effectively halt or reverse their progression. In the present study, we found that chronic LPS exposure significantly increased the expression of TRPC6 and AIM2, and induced neuronal ROS accumulation, calcium overload and neuroinflammation, eventually promoting neuronal injury and cognitive dysfunction. While Trpc6 knockout could significantly reverse LPS-induced TRPC6-AIM2 activation. Moreover, Rg1 treatment can further improve these changes in LPS-induced Trpc6^−/− mice. Additionally, Rg1 treatment can significantly upregulate the p-Nrf2 and HO-1 in LPS-induced Trpc6^−/− mice, and significantly reduces ROS production in vivo and in vitro. Whereas, in LPS-induced mice, Trpc6 knockout has no obvious impact on neuronal ROS level and Nrf2 signals transduction. Our study suggests that TRPC6-AIM2 inflammasome signaling plays a critical role in NDDs, and Rg1 may attenuate LPS-induced chronic neuroinflammation not only by activating the Nrf2 signaling and scavenging ROS production, but also by inhibiting TRPC6 and calcium overload, and suppressing AIM2 inflammasome and neuroinflammation.

One of the most critical pathological contributors to NDDs is chronic neuroinflammation, which serves as both a cause and consequence of disease progression [26]. Chronic LPS exposure is considered to be an important factor by inducing neuroinflammation and exacerbating NDDs [27]. Although intraperitoneal administration of LPS induces systemic inflammation, numerous studies have demonstrated that repeated LPS injections facilitate the translocation of peripheral endotoxins across the blood-brain barrier, resulting in sustained microglial activation and inflammasome signaling within brain tissue [28–30]. Chronic LPS exposure can effectively mimic certain pathological features of neuroinflammation observed in Alzheimer’s and Parkinson’s diseases, including microglial activation, enhanced Aβ accumulation, tau hyperphosphorylation, and dopaminergic neuronal loss [31–33]. In the present study, we established a chronic neuroinflammation model through daily intraperitoneal injection of low-dose LPS over a 21-day period. Calcium overload is an important mediator signaling of neuroinflammation, which further aggravates neuronal damage by activating pro-inflammatory factors. TRPC6 is a calcium-permeable non-selective cation channel, which is involved in many physiological processes and also plays an important role in regulator of calcium homeostasis and inflammation. Increasing studies have suggested that calcium overload is a hallmark of neurodegenerative diseases, leading to cognitive dysfunction and progressive neuronal loss [34]. Our previous study indicated that TRPC6 and calcium overload are closely involved in cognitive impairment in type 2 diabetes [20]. Therefore, we supposed that TRPC6-mediated calcium overload might involve in LPS-induced neuronal damage by promoting neuroinflammation. In this study, our results showed that chronic LPS exposure significantly increased TRPC6 expression, calcium overload and inflammatory factors of IL-1β and IL-6 in brain tissues, impaired the learning and memory abilities of mice, and led to obvious structural damage and functional impairment of neurons. Meanwhile, we found that Trpc6 knockout and Rg1 treatment was able to significantly ameliorate LPS-induced learning memory deficits and neuronal injury in mice by attenuating calcium overload and inflammatory responses. These data indicate that Trpc6 knockout and Rg1 treatment could ameliorate LPS-induced neuronal injury by attenuating neuronal calcium overload and inflammatory responses. However, as compared with Trpc6 knockout alone, combination of Trpc6 knockout and Rg1 could further improve LPS-induced cognitive deficits and neuronal injury, suggesting that Rg1 may have other mechanisms to protect against LPS-induced neuronal damage.

Currently, the role of inflammasomes signaling pathways has received increasing attention in NDDs. By activation, inflammasomes can recruit apoptosis-associated speck-like proteins (ASC) and form a molecular platform, which further activates caspase-1 and promotes maturation and release of IL-6 and IL-1β [35]. Among them, NLRP1 and NLRP3 inflammasomes are reported to be widely expressed in brain tissues and involve in the occurrence and development of NDDs [36, 37]. In addition, increasing studies suggest that AIM2, a non-NLR family inflammasome known to recognize ectopic double-stranded DNA, is also expressed in neurons and is closely involved in the development of neuronal inflammation and injury [38]. The recent study has reported that AIM2 inflammasome also involves in LPS-induced neuroinflammation, and inhibition of AIM2 can improve age-related neuronal inflammation and cognitive dysfunction [39]. Our latest research also shows that Rg1 therapy can activate Nrf2, inhibit oxidative stress and AIM2 inflammasome, and alleviate neuronal ferroptosis caused by chronic LPS exposure [6]. These studies suggest that activation of AIM2 inflammasome also plays a key role in NDDs, but the detailed mechanism still needs to be clarified. It has been reported that calcium overload can further exacerbate inflammation by enhancing inflammasome activation, forming a vicious cycle [40]. However, it remains unclear whether TRPC6 participates in AIM2 activation and whether Rg1 treatment can mitigate neuroinflammation by suppressing the TRPC6-AIM2 signaling pathway in chronic LPS-induced neuroinflammatory conditions. In this study, we found that chronic LPS induction significantly upregulated the expression of AIM2 and downstream proteins of Caspase-1, IL-1β and IL-6 in brain tissues. Meanwhile, Trpc6 knockout significantly inhibited the activation of AIM2 inflammasome, and Trpc6 knockout combination with Rg1 could further inhibit AIM2 inflammasome and exhibited stronger neuroprotective effects than Trpc6 knockout alone. The results in vitro also indicated that TRPC6 inhibitor BI749327 and Rg1 treatment could significantly suppress TRPC6 and AIM2 inflammasome in LPS-induced HT-22 cells. These findings indicate that chronic LPS exposure exacerbates neuroinflammation through the activation of AIM2 inflammasomes, thereby promoting neuronal inflammation and injury. TRPC6 signaling appears to play a pivotal role in modulating AIM2 inflammasome activity in chronic neuroinflammation, suggesting that this mechanism may underlie the neuroprotective effects of Rg1 in ameliorating neuroinflammation.

Oxidative stress and inflammation are intricately linked processes that contribute to neuronal damage and accelerate the progression of NDDs [41, 42]. ROS accumulation has an important role in the progression of chronic inflammation, which in turn can induce uncontrolled production ROS, resulting a vicious cycle to promote the development of diseases [43]. The Nrf2 signaling pathway plays a pivotal role in breaking this cycle by regulating cellular redox homeostasis. Nrf2 activation leads to the upregulation of key antioxidant enzymes, including heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) [44]. However, LPS exposure not only led to significant accumulation of ROS, but also inhibited Nrf2 activation, further exacerbating oxidative stress damage to neurons [45]. In this study, we found that chronic LPS exposure significantly increased ROS production but had no obvious influence on Nrf2/HO-1 signaling. Meanwhile, in LPS-treated mice, we found that Trpc6 knockout had no obvious influence on ROS production and Nrf2/HO-1 signaling, suggesting that the ROS-Nrf2/HO-1 may be an upstream signaling of TRPC6. However, Rg1 treatment effectively alleviated the levels of LPS-induced ROS production in vivo and in vitro, and also promoted p-Nrf2 expression and nuclear translocation, further upregulating the expression of antioxidant enzyme of HO-1 in LPS-induced Trpc6^−/− mice. These data suggest that Rg1 treatment not only inhibits TRPC6 signaling, but also activates Nrf2 signaling, reducing ROS generation and oxidative stress damage in LPS-induced chronic neuroinflammation.

Collectively, although our findings reveal a correlation between TRPC6 signaling and AIM2 activation in chronic LPS-induced neuronal injury and behavioral deficits, and demonstrate that Rg1 mitigates LPS-induced neuronal damage via inhibition of the TRPC6-AIM2 pathway, certain experimental limitations remain in this study. One limitation of the current study is the lack of experimental groups treated with Rg1 or the TRPC6 inhibitor alone, both in vivo and in vitro, which precludes a comprehensive evaluation of their individual effects on normal animals and cells. Additionally, chronic LPS model involves systemic administration of a bacterial endotoxin that activates TLR4 signaling in immune and non-immune cells throughout the body [46], which may confound behavioral outcomes. Moreover, the complexity of human NDDs, involving multiple pathogenic pathways, cannot be fully recapitulated by systemic LPS exposure. Furthermore, given the constitutive deletion of TRPC6, it is possible that TRPC3 or other calcium channels may partially compensate [47], which should be investigated in future studies. Although the neuroprotective properties of ginsenoside Rg1 have been previously documented in various models of neuroinflammation and neurodegeneration, our study provides additional mechanistic and translational insights. Specifically, we demonstrate that Rg1 not only inhibits TRPC6-mediated calcium overload and AIM2 inflammasome activation, but also attenuates neuronal damage even in Trpc6 knockout mice, suggesting the involvement of TRPC6-independent protective mechanisms. Anyway, these findings provide a foundation for future studies using more refined models.

Conclusion

In summary, the present study revealed that oxidative stress, calcium overload and inflammation are jointly involved in the pathological process of NDDs. Chronic LPS exposure increases ROS production and inhibits Nrf2 activation, which further induce TRPC6 upregulation and calcium overload, resulting in activation of AIM2 inflammasome, ultimately leading to neuronal inflammation and damage. While Rg1 treatment can significantly inhibit ROS oxidative stress by regulating Nrf2 signaling, and significantly attenuate calcium homeostasis and inflammatory response by downregulating TRPC6-AIM2 inflammasome signaling, therefore ameliorating LPS-induced behavioral deficits and neuronal damage in mice.

Electronic supplementary material

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Author contributions

WZ Li, Y Su, and WP Li have designed this research. YL Fu, H Zhang, and X Zhu did the research. YL Fu drafted the manuscript. HY Liang and L Fan had analyzed the data outcomes. WZ Li, Y Su, and WP Li certified the raw data. All authors reviewed and confirmed the final version of the manuscript.

Funding

This study was supported by the Nature Science Foundation of Anhui Province (2208085MH219); and the National Natural Science Foundation of China (81970630).

Data availability

Data can be obtained from the corresponding author.

Declarations

Ethics approval and consent to participate

Animal experiments involved in the approval of the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20232095).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.