DAV was purchased from Zhenyuan Deer Co., Ltd. (Jilin Province, P. R. China). VAP extracts were prepared by the Molecular Pharmacology Laboratory of the Research and Development Center at the Changchun University of Chinese Medicine.

The human neuroblastoma cell line SH-SY5Y was provided by the Pharmacology Laboratory of the College of Basic Medical Sciences at Jilin University, Jilin, P. R. China.

SH-SY5Y cells were cultured in RPMI 1640 medium containing 12% fetal bovine serum, 100 U/mL penicillin, and 100 $\mu$g/mL streptomycin under standard culture conditions of 37 ${}^{\circ }$C and 5% CO${}_2$. The cells were divided into the following groups: control group, model group, low-dose VAP group (VAP1), medium-dose VAP group (VAP2), and high-dose VAP group (VAP3). The VAP1, VAP2, and VAP3 groups were pretreated with 100, 250, and 500 $\mu$g/mL of VAP for 24 h, respectively, while the control and model groups were only exposed to the new culture medium for 24 h. Subsequently, 400 $\mu$mol/L H${}_2$O${}_2$ was added to all treatment groups for 4 h, while the control group was treated with an equivalent volume of culture medium for 4 h.

SH-SY5Y cells were seeded into 96-well plates at a density of 20,000 cells/well, and MTT was added after treating the cells as described in 2.2. Following a reaction period of 4 h, 150 $\mu$L of dimethyl sulfoxide was added to each well, the well contents were mixed, and the optical densities were measured at 495 nm using a microplate reader (Bio-Rad, USA).

Treated SH-SY5Y cells, as described in 2.2, were washed twice with cold phosphate-buffered saline (PBS). Apoptosis was detected using Annexin V-FITC/PI (BestBio, P. R. China) staining. A volume of 400 $\mu$L PBS binding buffer was added to re-suspend the cells at a concentration of approximately 1 $\times$ 10${}^6$cells/mL, and 5 $\mu$L of Annexin V-FITC staining solution was added to the cell suspension. The solution was gently mixed and incubated at room temperature in darkness for 15 min. Subsequently, 10 $\mu$L of PI staining solution was added; the solution was again gently mixed and incubated at room temperature in darkness for 5 min. The apoptosis rate was detected using a flow cytometer (BD, USA).

A total of 60 ICR mice (30 male and 30 female; 2-3 months of age; weight 25 $\pm$ 2 g) were purchased from Changchun Yisi Experiment Animal Technology Co., Ltd.

Mice were kept at a constant temperature (22-25 ${}^{\circ }$C) and relative humidity (40-50%) with standard feed and water. The 60 ICR mice were randomly allocated into the following groups: control group, model group, positive group (3 mg/kg memantine), low-dose VAP group (VAP1, 100 mg/kg VAP), medium-dose VAP group (VAP2, 200 mg/kg VAP), and high-dose VAP group (VAP3, 400 mg/kg VAP).

Mice of the positive group and the VAP groups received intragastrically memantine or VAP for 21 days, whereas saline was intragastrically administered to mice of the control and model groups. Apart from the control group, mice in all groups received from the seventh day intraperitoneal SCOP injections 1 h after memantine or VAP treatment.

After the drug treatment period of 21 days, each mouse was decapitated, and the brain was rapidly removed. Brain tissue samples of seven mice per group were stored at -80 ${}^{\circ }$C for enzyme-linked immunosorbent assay (ELISA), western blot, and PCR experiments. Brain tissue samples of three mice per group were placed in 4% paraformaldehyde for 24 h and then processed into paraffin sections for subsequent tissue staining.

Paraffin wax sections were dewaxed and washed. Subsequently, they were placed in hematoxylin staining solution for 5 min, washed with tap water, differentiated using 1% hydrochloric acid in alcohol for 1 min, washed with distilled water, blued in weak ammonia water for 30 s, washed with distilled water, and stained with eosin for 15 min. Finally, they were rinsed with distilled water and ethanol at different concentrations. The morphology and arrangement of hippocampal neurons in each experimental group were observed using an optical microscope (Olympus, Japan).

The sections were washed using PBS, and the TUNEL assay (Beyotime Biotechnology, P. R. China) was performed according to the manufacturer’s instructions. To each sample, 50 $\mu$L of the TUNEL staining solution was added, and the mixture was incubated at 37 ${}^{\circ }$C in darkness for 60 min, followed by three washing steps with PBS. Apoptosis of hippocampal neurons in the various groups was observed with a confocal laser scanning microscope (Olympus, Japan) using an excitation wavelength range of 450-500 nm and an emission wavelength range of 515-565 nm.

Murine brain tissue samples of each group were placed in saline at a 1: 9 ratio, homogenized and centrifuged. Adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined using the appropriate ELISA kits (Nanjing Jiancheng Bioengineering Institute, P. R. China) according to the manufacturer’s instructions.

After adding Radio-Immunoprecipitation Assay lysis buffer, brain tissues of mice were adequately lysed, and all samples were centrifuged at 14,000$\times$$g$ for 5 min to collect supernatants subsequently. Protein concentrations were quantified using the Coomassie brilliant blue assay (Sigma-Aldrich, USA). Processed samples were separated by 12% SDS-PAGE, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked in skimmed milk powder for 2 h and blotted with $\beta$-actin antibody (1 : 20000), glucocorticoid receptor (GR) antibody (1 : 1000), mineralocorticoid receptor (MR) antibody (1 : 400), corticotropin-releasing hormone (CRH) antibody (1 : 1000), B-cell lymphoma-2 Associated X-protein (Bax) antibody (1 : 1000), B-cell lymphoma-2 (Bcl-2) antibody (1 : 1000), or Cysteine-aspartic acid protease-3 (Caspase-3) antibody (1 : 1000) (all Proteintech, P. R. China). After washing with Tris-buffered saline/Tween buffer, PVDF membranes were incubated at room temperature for 2 h with a secondary peroxidase-conjugated antibody (Proteintech, P. R. China). The antibody signals were detected using the enhanced chemiluminescent substrate kit (Proteintech, P. R. China), and bands were visualized using a chemiluminescence imager (Aplegen, USA). The gray values of the bands were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) and calculated according to the following equation: relative expression = sample expression value/$\beta$-actin expression value.

Total RNA was isolated using Trizol reagent. cDNA was synthesized from total RNA (0.2-2 $\mu$g) using the Bioteke Super RT Kit (BioTeke Corporation, P. R. China). Real-time quantitative PCR (qPCR) was performed using DNA-binding dye SYBR Green (Roche, P. R. China). Amplification was performed using the following primers: $\beta$-actin forward primer 5’-CCAGCCGAGCCACATCGCTC-3’, reverse primer 5’-ATGAGCCCCAGCCTTCTCCAT-3’; Bcl-2 forward primer 5’-CAGCTGCACCTGACGCCCTT-3’, reverse primer 5’-CCCAGCCTCCGTTATCCTGGA-3’; Bax forward primer 5’-GATCAGCTCGGGCACTTTAG-3’, reverse primer 5’-TTGCTGATGGCAACTTCAAC-3’; Caspase-3 forward primer 5’-CTGACTGGAAAGCCGAAACTC-3’, reverse primer 5’-CGACCCGTCCTTTGAATTTCT-3’; GR forward primer 5’-GTGAAATGGGCAAAGGCCATAC-3’, reverse primer 5’-GAAGAGAAACGAGCAAGCATAG-3’; MR forward primer 5’-GGCTACCACAGTCTCCCTGA-3’, reverse primer 5’-AGAACGCTCCAAGGTCTGA-3’; and CRH forward primer 5’-CAGAACAACAGTGCGGGCTCA-3’, reverse primer 5’-GGAAAAAGTTAGCCGCAGCCT-3’. Using the 2${}^{-\mathrm{\Delta }\text{𝐶𝑡}}$ method (Livak and Schmittgen, 2001), relative expression levels of Bcl-2, Bax, Caspase-3, GR, MR, and CHR mRNA were calculated for each sample after normalization to the housekeeping gene $\beta$-actin.

All statistical analyses were performed using the GraphPad Prism software (Graph-Pad Software Inc). All data are expressed as mean $\pm$ SEM. Statistical comparisons between two groups were performed using Student’s $t$-tests; P 0.05 was considered statistically significant.

H${}_2$O${}_2$ was added to establish a neuron damage model and to assess the protective effect of pretreatment using different doses of VAP on damaged SH-SY5Y cells. The cell survival rate of SH-SY5Y cells was significantly lower in the model group when compared to the control group (83.58% $\pm$ 0.07%, P 0.05), indicating the successful establishment of the cell injury model. The cell survival rates of the VAP1, VAP2, and VAP3 groups were significantly higher than the model group (all P 0.05; Fig. 1A). Compared to the control group, the total apoptosis rate of the model group was significantly increased (P 0.05); the total apoptosis rate of the VAP3 group was significantly decreased in comparison to the model group (P 0.05, Fig. 1B).

Fig. 1.

Protective effects of VAP on H${}_{\mathrm{2}}$O${}_{\mathrm{2}}$-induced SH-SY5Y cell injury. (A) Effect of VAP on SH-SY5Y cell proliferation rate (n = 8). (B) Effect of VAP on SY5Y cell apoptosis (n = 3). (C) Effect of VAP on apoptosis proteins in damaged SH-SY5Y cells. (D) Effect of VAP on the relative expression of apoptotic proteins in damaged SH-SY5Y cells (n = 3). All data were analyzed by Student’s $t$-test and are presented as the mean $\pm$ SEM. 0.05 vs. control, 0.05 vs. model.

The expression levels of Caspase-3 and Bax had also increased (P 0.05), whereas the expression of Bcl-2 had reduced (P 0.05). By contrast, VAP administration inhibited the expression of Caspase-3 and Bax (P 0.05), and promoted the expression of Bcl-2 (P 0.05, Fig. 1C-D). This indicated that H${}_2$O${}_2$-induced injury caused increased apoptosis in SH-SY5Y cells and that VAP rescued this apoptotic process.

We observed the morphology of hippocampal neurons in each group of mice by HE staining, as well as the pre-protective effect of VAP on neurons. In the control group, the hippocampal regions of the mice comprised a large number of neurons with dense and neat arrangements, uniform distribution, intact structures, normal morphology, and distinct cell and nuclear membranes. In the model group, a decrease in the number of hippocampal neurons was observed, and cells exhibited a loose and disorderly arrangement of neurons, uneven staining, and distribution, and widened extracellular spaces, where the majority of cells exhibited abnormal morphology and showed obvious pyknosis. In comparison to the model group, the hippocampal neurons of the positive, VAP1, VAP2, and VAP3 groups exhibited denser and neater arrangements and normal cell morphology, with a small minority of cells exhibiting pyknosis (Fig. 2A).

Fig. 2.

Protective effects of VAP on hippocampal neurons in SCOP-injured mice. (A) Morphology of mouse hippocampal neurons. The red arrow points to the damaged neuron. (B) Effect of VAP on the apoptosis of hippocampal neurons in mice. (C) VAP regulates apoptosis proteins in the mouse brain. (D) Quantification of the relative expression of apoptosis proteins (n = 3). (E) Regulation of apoptosis genes in murine brain tissue by VAP administration (n = 8). All data were analyzed by Student’s $t$-test and are presented as the mean $\pm$ SEM. 0.05 vs. control, 0.05 vs. model.

TUNEL fluorescence staining revealed that compared to the control group, the numbers of apoptotic hippocampal neurons marked by green fluorescence were significantly increased in the model group. Compared to the model group, significant decreases in the numbers of apoptotic hippocampal neurons were observed in the positive, VAP1, VAP2, and VAP3 groups (Fig. 2B).

To further examine the effect of VAP on neuronal apoptosis, we detected the protein and gene products of Caspase-3, Bax, and Bcl-2 in mouse brain tissue. After SCOP injury, the expression of Bcl-2 decreased, while the expression of Caspase-3 and Bax increased at the gene and protein level (P 0.05). By contrast, after pretreatment with VAP, the expression of Bcl-2 increased, while the expression of Caspase-3 and Bax decreased at the gene and protein level (P 0.05); the ability of VAP to regulate apoptosis was better than that of memantine (Fig. 2C-E).

We detected the hormone regulation ability of VAP on the HPA axis release. ACTH and CORT concentrations increased in the brains of mice after SCOP injury (P 0.05), whereas they significantly reduced after pretreatment with VAP and memantine (P 0.05). The ability of 400 mg/kg VAP to regulate the two hormones was better than both 100 mg/kg and 200 mg/kg VAP (Fig. 3A and 3B).

CORT, secreted by the HPA axis, directly affects the expression of GR and MR on hippocampal neurons, thereby affecting the secretion of CRH on the HPA axis. The GR, MR, and CRH related proteins and genes in the brain tissue of mice were detected; results indicated that their expression increased in SCOP model mice (P 0.05), and decreased in the brains of mice pretreated with memantine and VAP (P 0.05) (Fig. 4). VAP may, therefore, regulate the expression of GR and MR in the hippocampus, whereas negative feedback regulates the conduction of the HPA axis and secretion of related hormones.

Fig. 3.

Influence of VAP on hormone levels related to the HPA axis. (A) Regulation of the ACTH content in the mouse brain by VAP treatment (n = 8). (B) Effect of VAP on the CORT content in the mouse brain (n = 8). All data were analyzed by Student’s $t$-test and are presented as the mean $\pm$ SEM. 0.05 vs. control, 0.05 vs. model.

Fig. 4.

Levels of proteins and mRNAs related to the HPA axis in the mouse brain. (A) VAP regulates the expression of HPA axis-related proteins in the mouse brain. (B) Quantification of the relative expression of HPA axis-related proteins (n = 3). (C) Effect of VAP on HPA axis-related genes in the mouse brain (n = 8). All data were analyzed by Student’s $t$-test and are presented as the mean $\pm$ SEM. 0.05 vs. control, 0.05 vs. model.