Animals and drug treatment

The heterozygote C57BL/6N-Trpc6em1/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.