Echinocystic acid (EA, >98.0% purity; Shanghai Yuan-ye, St. Louis, Shanghai, China), lipopolysaccharide (LPS, Sigma, St. Louis, MO, United States), dimethyl sulfoxide (DMSO), LY294002 (a PI3K inhibitor), and MK2206 (an Akt inhibitor) were obtained from Sigma Aldrich (St Louis, MO, United States). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, United States). Penicillin–streptomycin (PS) solutions were obtained from Invitrogen (Carlsbad, CA, United States). Trypsin was obtained from Beyotime Institute (Biotech, Beijing, China).

BV2 (murine microglia cell line), SN4741 (murine dopaminergic neuron cell line), and SHSY5Y (human neuroblastoma cell line) were obtained from Shanghai Binsui Biological Technology (Shanghai, China). The cultured conditions of cells were as follows: 89% DMEM+10% FBS+1% penicillin–streptomycin (PS) solutions, 95% air, 5% carbon dioxide (CO2), and a temperature of 37°C. The cells were digested with 0.25 and 0.05% trypsin, and passaged every 2 days. The cells were seeded into a 6-cm diameter cell culture dish during the research period. When the density was 70–80%, the medium was replaced with serum-free DMEM, and then EA was added to the petri dish and incubated for 1 h. Later, the cells were exposed to LPS (100 ng/ml) for 1, 12, and 24 h. During the experiment, SN4741and SHSY5Y cells were cultured in a conditioned medium.

Cells were seeded into 96-well plates at a density of 1–2 × 10⁴ cells per well and cultured overnight in a cell incubator. When the density was moderate, different concentrations of EA and solvent DMSO were added to the culture wells and then cultured. After 24 h, the culture medium was replaced with CCK8 dilution (Beyotime Inst Biotech, Beijing, China) and incubated for 2 h. After that, cell viability was measured with a microplate reader at 450 nm.

Cells were seeded into 96-well plates at a density of 1–2 × 10⁴ cells per well and cultured overnight in a cell incubator. Different concentrations of EA and solvent DMSO were added to the culture wells, and then the cells were cultured. After 24 h, the release of LDH in the medium was determined using an LDH assay kit (Beyotime Inst Biotech, Beijing, China) according to the manufacturer’s instructions.

According to the manufacturer’s protocols, cells and tissue RNAs were extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, United States). After determining the concentration, 2 μg RNA was reverse-transcribed into cDNA using a reverse transcription kit (Sigma-Aldrich, St. Louis, MO, United States). Then quantitative PCR (qPCR) was performed using the SYBR Green QuantiTect RT-PCR Kit (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s protocols. The mRNA levels of inflammatory mediators were evaluated relative to β-actin according to the 2^−ΔΔCT method. The primers involved in the experiment are presented in Table 1.

When the cells seeded into a 24-well plate were 70–80% in density, the medium was changed to serum-free DMEM. Then the cells were incubated with EA (1 h, 16 μM) and stimulated with LPS (100 ng/ml, 24 h). After that, the expression of IL-6 and TNF-α in the supernatant was measured using the ELISA kits, according to the manufacturer’s protocols (BioLegend, San Diego, CA, United States).

Total protein of BV2 cells and mice midbrain tissue was extracted using P0013 Lysis Solution (Beyotime Ist. Beijing, China). Protein concentration was measured using a BCA reagent (Beibo Biological Technology, Shanghai, China). The 30 µg protein was loaded and separated by 13% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes (Biyuntian Biological Reagent, Beijing, China). After blocking with 5% skim milk for 2 h at room temperature, the PVDF membranes were incubated with primary antibodies [COX-2 (1:5,000), iNOS (1:5,000), p-Akt (1:5,000) (Abcam, Cambridge, United Kingdom), p-JNK1/2 (1:5,000), p-ERK1/2 (1:5,000) (Cell Signal Technology, MA, United States), p-p38 (1:5,000), p-NF-κB p65 (1:5,000), JNK1/2 (1:5,000), ERK1/2 (1:5,000), p38 (1:5,000), Akt (1:5,000), NF-κB p65 (1:5,000), and β-actin (1:5,000) (Santa, CA, United States)] for 12 h at 4°C and then washed three times (20 min per time) with TBST solution. Thereafter, the membranes were incubated with the following secondary antibodies: goat anti-rabbit (1:5,000) and goat anti-mouse (1:5,000) (Santa, CA, United States) for 2 h, and then washed three times (20 min per time) with TBST solution. All the primary antibodies and secondary antibodies were dissolved in a 5% BSA solution. Next, the membranes were incubated with ECL Luminous Liquid (Amersham Pharmacia Biotech, Philadelphia, United States), and the target proteins were presented with the ScanLater Western Blot Detection System (Meigu Molecular Instruments, Shanghai, China).

Forty male C57BL6 mice (25–30 g) were purchased from the Experimental Animals Center of Norman Bethune Medical College of Jilin University (Changchun, PR China). Animals were kept at 22–24°C in an artificial 12/12 h day/night cycle condition. All the animals were provided with adequate food and drink. Animal models were induced with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP, Sigma-Aldrich, MO, United States). The mice were randomly divided into three groups during the experiment: saline, MPTP, and MPTP + EA. MPTP (30 mg/kg, dissolved in saline) was injected intraperitoneally for 7 d. EA (dissolved in saline) was administered intragastrically once a day for 17 days. After administration, the mice were tested through behavioral tests. After that, the mice were euthanized.

The number of dopaminergic neurons and microglia activation in the substantia nigra of the mouse midbrain were detected through immunohistochemical staining. In brief, after being euthanized, the midbrains of mice were soaked in a 4% paraformaldehyde solution for 24 h, and then processed using the following procedure: alcohol (70, 80, 90% each for 1 h, 100% for 2 h), xylene (20 min), and then dipping wax (40 min). Then the tissues were sectioned at 5 µm per slice. Thereafter, the immunohistochemistry staining was performed with an UltrasensitiveTM S-P kit (MBX Biotechnologies, Fuzhou, China), according to the manufacturer’s protocols. The dopaminergic neurons were marked with the anti-TH and anti–IBA-1 antibodies (1:200, Abcam, Cambridge, United Kingdom). IHC images were taken under an optical microscope. Total TH-positive cells and IBA-1–positive cells were counted by five researchers blind to the experimental design, and the average of these scores was reported.

Differences in data were analyzed using SPSS 19.0 software (IBM). All data were represented as means ± SD. Differences of groups were calculated adopting a one-way ANOVA plus multiple testing method and controlling the within-group error. Differences were considered statistically significant at p < 0.05 and p < 0.01.

To determine whether EA affects the growth of BV2 cells, we tested the effect of different concentrations of EA (0–32 μM) on the survival rate of BV2 cells and LDH release. The results of CCK8 showed that EA no more than 16 μM did not affect the survival rate of BV2 cells (Figure 1C). The results of LDH showed that EA no more than 16 μM did not affect the LDH release of BV2 cells (Figure 1D). These results demonstrated that EA no more than 16 μM did not show potential toxic effects on BV2 cells.

When inflammation occurs, immune cells release a large amount of pro-inflammatory factors (IL-6 and TNF-α) and pro-inflammatory enzymes (iNOS and COX-2), which are considered one of the causes of damage to the surrounding tissues and neurons. To clarify the anti-inflammatory effect of EA, we treated cells with EA (16 μM) for 1 h and stimulated them with LPS (100 ng/ml) for another 12 (mRNA) or 24 h (protein). The mRNA expression of pro-inflammatory mediators [iNOS (Figure 2A), COX-2 (Figure 2B), IL-6 (Figure 2C), and TNF-α (Figure 2D)] was measured by the quantitative PCR method, and the protein expression was measured by ELISA (IL-6 (Figure 2H), TNF-α (Figure 2I), and Western blot [iNOS (Figures 2E,F) and COX-2 (Figures 2E,G)] technology. Results showed that EA inhibited the mRNA and protein expression of inflammatory mediators.

The PI3K/Akt is an intracellular signal transduction pathway responsible for cell proliferation and survival. In the experiment, we investigated the effect of EA on the PI3K/Akt signal pathway. We treated BV2 cells at different times (0, 15, 30, and 60 min) with EA (16 μM) and measured the phosphorylation changes of PI3K/Akt signal pathways. Results showed that EA promoted the activation of PI3K (Figures 3A,B) and Akt (Figures 3A,C) pathway in the BV2 cells. Thereafter, we treated BV2 cells with PI3K pathway inhibitors (LY294002, 5 μM) for 6 h and measured the activation of the PI3K/Akt pathway. Results showed that LY294002 could block the activation of PI3K (Figures 3D,E) and Akt (Figures 3D,F) pathway by EA to a certain extent. Similarly, Akt pathway inhibitor (MK2206, 10 μM for 6 h) treatment inhibited the activation of the Akt pathway by EA (Figures 3G,H).

In further studies, we treated BV2 cells with pathway inhibitors (LY294002 or MK2206) for 6 h and measured the expression of pro-inflammatory mediators. The results showed that treatment with pathway inhibitors inhibited the regulatory effect of EA on inflammatory mediators [iNOS (Figure 3K), COX-2 (Figure 3L), IL-6 (Figure 3I), and TNF-α (Figure 3J)] in LPS-exposed BV2 cells. These results demonstrated that EA inhibits inflammation through the PI3K/Akt signal pathway.

Studies have revealed that NF-κB and MAPK signal pathways play a crucial role in the process of inflammation, and their activation can regulate the transcription of inflammatory mediators. In order to clarify the mechanism of EA regulating inflammatory mediators, we studied the effect of EA on NF-κB and MAPK pathway activation. We treated the cells with EA (16 μM) for 1 h and then stimulated with LPS (100 ng/ml) for another 1 h. Then we detected the phosphorylation levels of NF-κB p65, IκBα, ERK, JNK, and p38 and their expression by Western blot. Results proved that EA inhibited the phosphorylation of NF-κB p65 (Figures 4A,B), IκBα (Figures 2 A,C), ERK (Figures 4 E,F), JNK (Figures 4E,G), and p38 (Figures 4 E,H), and the degradation of IκBα (Figures 4 A,D). These results demonstrated that EA inhibited the activation of NF-κB and MAPK signal pathways in LPS-exposed BV2 cells.

To study the neuroprotective effect of EA on neural cells, we cultured two types of neuron cell lines (SN4741 and SHSY5Y). We incubated BV2 cells with EA (16 μM) and LPS (100 ng/ml) for 2 h and then changed the medium. After 24 h, the medium was collected and mixed with fresh medium in a 1:1 ratio as the conditioned medium (Figure 5A). When the density of SN4741 and SHSY5Y cells was 50–60%, the medium was changed to a conditional medium, and then the survival rate of cells was detected through CCK8 experiment. Results showed that EA eases microglia-mediated neuron death in SN4741 (Figure 5B) and SHSY5Y cells (Figure 5C).

The main clinical manifestation of PD is decreased or loss of locomotor activity. We detected the effects of EA on weight loss and locomotor activity in MPTP-induced PD mice. Results showed that EA (5 mg/kg) improved MPTP-induced body weight loss (Figure 6B). Open-field experiments showed that EA increased the total distance mice traveled in the open field (Figures 6 C,D). The pole climbing experiment showed that EA reduced the climbing experiment duration in mice (Figure 6E). The rod rotation test showed that EA increased the duration of mice on the rod rotation fatigue meter (Figure 6F). These results demonstrated that EA improves MPTP-induced body weight loss and locomotor activity decreasing.

The main pathological feature of PD is the damage of dopaminergic neurons in the substantia nigra of the midbrain. To clarify the role of EA, we examined the effect of EA (5 mg/kg) on the damage of dopaminergic neurons in the substantia nigra of the midbrain in mice. The results showed that EA improved MPTP-induced loss of dopaminergic neurons (Figures 7A,B). TH is a rate-limiting enzyme synthesized by dopaminergic neurons. We also detected the protein expression of TH in the midbrain by Western blot (Figures 7 C,D). The results demonstrated that EA inhibited the reduction of TH protein induced by MPTP.

The pathogenesis of PD is accompanied by excessive activation of microglia. In order to study the effect of EA, we tested the effect of EA on the activation of microglia. Immunohistochemical results showed that EA inhibited the number of IBA-1–positive cells in the substantia nigra (Figures 7E,F). Western blot results showed that EA inhibited the protein expression of IBA-1 in the mouse midbrain (Figures 7G,H). The results demonstrated that EA inhibited the activation of microglia.

To further investigate the mechanism by which EA exerts its neuroprotective effect, we investigated the effects of EA (5 mg/kg) on mediators and inflammatory pathways in a PD mouse model. The results of fluorescence quantitative PCR showed that EA inhibited the mRNA expression of pro-inflammatory mediators (iNOS (Figure 8A), COX-2 (Figure 8B), IL-6 (Figure 8C), and TNF-α (Figure 8D). The results of Western blot showed that EA activated PI3K/Akt (Figures 8E–G) and inhibited NF-κB (Figures 8E,H–J) and MAPK (Figures 8K–N) signal pathways. These results demonstrated that EA inhibits MPTP-induced inflammation in the MPTP-induced mouse PD model.