Plant Material

Semen Strychni (batch number 201,022,891) was obtained from SanXiang Co. Ltd. (Changsha, China). All herbs were authenticated by Professor Yu-hua Wang (Department of Pharmacy, the Second Xiangya Hospital, Central South University). Voucher specimens of the two herbs were deposited at the Department of Pharmacy, the Second Xiangya Hospital.

Extraction of Herbs

After the raw Semen Strychni seeds were crushed, a proper amount of powder was taken and soaked in 75% acidic ethanol (pH = 5, 1:12, W/V) for 12 h and then heated to reflux thrice, each for 1 h. The extract was filtered using a 0.45 μm microporous filter membrane under hot conditions. Then, the filtrate was combined and concentrated by a rotary evaporator until all ethanol was volatilized. The filtrate pH was adjusted to 6.5 with 0.5 mol/L NaOH. The extract was mixed with 1% carboxymethylcellulose sodium (CMC-Na) solution and water to prepare 50 mg raw Semen Strychni/0.5%CMC-Na mL. The composition of the Semen Strychni extract was detected by high-performance liquid chromatography coupled with tandem mass spectrometry and ultraviolet detector (HPLC-UV-MS).

Animals Experiments

The experimental animals were SPF-grade male SD rats purchased from Hunan SJA Laboratory Animal Co., Ltd., with the license number SCXY (Xiang) 2019-0004. The rats were raised in the Department of Laboratory Animal Science of Central South University, and the experimental unit’s animal use license number is SYXK (Xiang) 2015-0017. This study was reviewed and approved by the Laboratory Animal Management Committee of the Department of Laboratory Animal Science of Central South University (acceptance number: 2020sydw0821). Forty-eight rats were used to assess the hippocampal injury induced by Semen Strychni and randomly divided into four groups (n = 12) as follows:

Blank group (Control): Rats were intraperitoneally injected with 0.5% CMC-Na solution with a volume of 3.5 mL/kg. After the injection, 0.5% CMC-Na solution was given by intragastric administration at a volume of 5 mL/kg.

Strychni poisoning group (SS): Rats were intraperitoneally injected with the extract of Semen Strychni (0.175 g/kg). Immediately after the injection, 0.5% CMC-Na solution was given by intragastric administration with a volume of 5.0 mL/kg.

Low-dose GA detoxification group (SS + LGA): Rats were intraperitoneally injected with the extract of Semen Strychni (0.175 g/kg), and GA (75 g/kg) was then given by gavage.

High-dose GA detoxification group (SS + HGA): Rats were intraperitoneally injected with the extract of Semen Strychni (0.175 g/kg), and GA (150 g/kg) was then given by gavage.

Semen Strychni and GA were given for four days.

Sample Collection

At the time of dissection, the blood was collected from the heart and then centrifuged (4 °C, 3500 rpm) for 10 min. The supernatant was extracted to obtain serum. The wholly separated brain tissues of rats were randomly selected, soaked, and cleaned with normal saline several times and then put into a centrifuge tube with paraformaldehyde.

Histopathological ExaminationNissl Staining

The rat brains were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin was then sectioned, dewaxed, and stained with toluidine blue after hydration. The sections were placed in xylene solution for 5 min to make them transparent and then mounted with neutral gum. Images of the rat hippocampus were collected under a microscope and analyzed.

TUNEL Staining

After paraffin embedding, sectioning, dewaxing, and other steps in the same way as above, proteinase K working solution was added dropwise for antigen retrieval, followed by membrane rupture, labeling with TdT: dUTP at 1:9, DAPI counterstaining, and anti-fluorescence quenching. After mounting with an anti-mounting medium, the tissue sections were observed under a fluorescence microscope, and images were collected.

FJB Staining

After dewaxing and hydrating the paraffin sections, FJB dilution was prepared according to the ratio of 50% glacial acetic acid to 1:400 for staining. The nuclei were stained with DAPI and then placed in xylene solution to seal the sections with transparent neutral resin. The tissue sections were then observed under a fluorescence microscope, and images were collected.

ELISA

The collected blood was centrifuged at 2500 rpm for 15 min at 4 °C, and serum was collected for ELISA detection. AChE, MAO, HMGB1, TNF-α, and IL-1β were quantitatively detected according to the instructions.

Western Blotting

Brain hippocampus tissues were centrifuged at 12,000 g for 10 min following homogenization in RIPA lysis buffer (1:10, w/v). The super-natants were collected. The whole protein samples were obtained by this method. The nucleus and cytoplasm protein were obtained by using the nucleus and cytoplasmic extraction kit. (Boster Biological Technology Co.,Ltd) All protein samples were determined by BCA protein assay kit. Make appropriate dilution of serum and tissue samples based on the protein concentration. Then the samples were mixed with 5× loading buffer, then were boiled at 100◦C for 5 min and stored at − 70◦C until further analysis.

Proteins were run on 10% or 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA for 60–90 min at room temperature and incubated with antigen-specific primary antibodies overnight at 4 °C. Blots were then incubated with species-specific secondary antibodies for 60 min at room temperature. Proteins were visualized by incubation with a chemiluminescent substrate kit. The primary antibodies used in this study were anti-HMGB1 (10829-1-AP, Proteintech, USA), anti-p-serine (ab9332, Abcam, UK), anti-NF-κB (10745-1-AP, Proteintech, USA), anti-p-38 (14064-1-AP, Proteintech, USA), anti-PP2A (13482-1-AP, Proteintech, USA), and anti-PKC (21991-1-AP, Proteintech, USA).

Immunofluorescent Staining

The paraffin sections were dewaxed and hydrated, and autofluorescence quenchers were added dropwise to the tissue sections after antigen retrieval. BSA solution was added dropwise for blocking. The primary antibody was added overnight at 4 °C. The secondary antibody was added and reacted at room temperature for 50 min. DAPI staining solution was added dropwise on the tissue to counterstain the nuclei. Slides were mounted with an anti-fluorescence quenching mounting medium. The tissue sections were then observed under a fluorescence microscope, and images were collected.

Co-immunoprecipitation

Total protein was extracted from each group and stored at − 80 °C. HMGB1 antibody was added to the protein supernatant, and the antigen–antibody mixture was shaken slowly at 4 °C and incubated overnight. Protein A/G agarose beads were taken, and IP lysis buffer was added to them and centrifuged at 3000 rpm to retain the pellet. The cell lysate incubated with the antibody overnight was added to the pretreated protein A/G agarose beads to couple the antibody to the protein A/G agarose. After co-immunoprecipitation, centrifugation was performed at 3000 rpm for 3 min at 4 °C, the supernatant was aspirated, and the pellet was collected. Expression of p-HMGB1 with PP2A and PKC was assessed using western blotting analysis.

Statistical Analysis

SPSS 18.0 software was used for statistical analysis of data. Data are presented as mean ± standard error (mean ± SEM). One-way analysis of variance (ANOVA) was used for comparison between multiple groups, and the LSD test was used for comparison between two groups. When the p-value was less than 0.05, the difference was regarded as statistically significant. The graphing software used was GraphPad Prism 8.0.