The experimental protocols were carried out according to the guidelines for animal experiments of the Shanghai Public Health Center Laboratory Animal Welfare and Ethics Committee (no. 2019-A026-01) to minimize animal suffering. C57BL/6 mice were from the Shanghai Jiesijie Experimental Animal Co., Ltd. (China), housed under a controlled environment (12 h light/dark cycle, 22 ± 2°C, and 55% ± 5% humidity), and provided with food and water ad libitum. MPTP·HCL (30 mg/kg, Selleck, United States) were administrated once a day for seven consecutive days to make the subacute mouse model of PD in this study. After two weeks of adaptive feeding, the mice were randomly divided into five groups, namely, normal group (N), MPTP group, MPTP + LECH (LE, 10 mg/kg/d) group, MPTP + MECH (ME, 20 mg/kg/d) group, and MPTP + HECH (HE, 30 mg/kg/d) group. Three doses of ECH treatment were administered the respective pre-treatment ECH (intraperitoneal injection, i.p.) every 24 h for 14 consecutive days, with the first day of administration designated as day 1. The mouse PD models for the three treatment groups and the MPTP group were generated by seven consecutive i.p. of MPTP (30 mg/kg, Selleck, United States) once a day from day 8 to day 14, as shown below.

The spontaneous locomotor activity of mice was measured with an open field test. Before each behavioral test, the experimental box was scrubbed with 75% ethanol solution to ensure odorless. Then, mice in the different groups were placed into the box, and their behavioral parameters were recorded with a video analyzer for 10 min. The animal behavioral analysis software EthoVisionXT12 (Noldus, the Netherlands) was opened to automatically record the animal movement path, total distance, and other information related to mouse movement.

Mouse motor coordination was evaluated with rotarod apparatus Rotamex-5 Rota Rod (Columbus Instruments, United States). Before the test, all mice were trained on the rotarod (12 rpm) to achieve stable performance. During the test, the mice were placed on the rotarod. Then, it was conducted at a uniformly accelerating speed from 4 to 30 rpm in 300 s. Recording was stopped when the mouse fell off the rotating rod. Repeat the rotarod test three times and take the average time of each mouse to measure its motor ability.

The pole test was conducted with a rough-surfaced wooden pole (1 cm in diameter, 55 cm in height) to evaluate the bradykinesia of mice. The pole was covered with black tape to prevent the mice from skidding, and a wooden ball (2 cm in diameter) was fixed on the top of the pole. In the beginning, the wooden pole was placed vertically, and a mouse was placed at the top of the pole. Then, it would climb down along the pole to the bottom, and the time used during this period was recorded. The climbing time of mice before ECH treatment (0 days) and after MPTP injection (14 days) was recorded, and the time difference between the two pole tests of the same mouse was calculated.

After fixing (4% paraformaldehyde) and dehydration (20% and 30% sucrose solution), brain tissues were sliced into sections with a freezing microtome (Leica Biosystems, Germany) from Bregma −2.00 mm to Bregma −3.52 mm (20 μm for each) and stored in −80°C refrigerator.

After the sections were restored to room temperature, they were treated with 0.3% Triton X-100 to penetrate cytomembrane and 0.3% hydrogen peroxide (H₂O₂) to block the endogenous peroxidase activity. Then, they were blocked with bovine serum albumin (BSA) for 30 min and incubated with TH antibody (1:100) overnight at 4°C. On the following day, they were incubated with secondary antibodies (1 h, 37°C) and stained with fresh DAB solution. After placing hematoxylin and hydrochloric acid alcohol, they were dehydrated and dried with gradient alcohol and xylene and sealed with neutral balsam. The images were obtained with the cellSens standard system (Olympus, Japan).

Immunofluorescence staining was performed with overnight incubation using diluted primary antibodies against IBA-1(1:100, Novus) at 4°C. On the following day, second antibodies were applied to sections and incubated for one hour at room temperature after washing with PBS (three times, 5 min). Then nuclei of cells were counterstained with Hoechst33343 for 2 min.

Total RNA was extracted from SN tissues and BV2 cells with TRIzol reagent according to the standard protocol, respectively, and its purity and concentration were measured with a nucleic acid analyzer (Thermo, United States). Then, it was reverse-transcribed with PrimeScript™ RT reagent Kit with gDNA eraser (Code no. RR047A, Takara, Japan), including genomic DNA remove reaction and reverse transcription reaction. The former system (10 μL) contained 5 × gDNA eraser buffer (2 μL), gDNA eraser (1 μL), total RNA (calculated according to concentration), and RNase-free dH₂O (up to 10 μL), and the latter (total 20 μL) included the reaction liquid from last step (10 μL), PrimeScript RT Enzyme Mix I (1 μL), RT Primer Mix *4 (1 μL), 5 × PrimeScript Buffer 2 (4 μL), and RNase-free dH₂O (4 μL). The RT-qPCR system was configured with TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (Code no. RR420A, Takara, Japan) on ice, and the RT-qPCR was run with CFX96 Real-Time PCR Detection System (Bio-Rad, United States). The mRNA levels of target genes were normalized to that of β-actin in the same sample. The primer sequences in RT-qPCR are shown in Table 1.

BV2 cells, provided by the State Key Laboratory of Medical Neurobiology, Fudan University, were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone) with 10% fetal bovine serum (FBS, HyClone, United States), 100 U/mL penicillin, and 100 g/ml streptomycin in a 5% CO₂ incubator (37°C).

Echinacoside was purchased from Selleck and dissolved into 5, 10, and 20 mg/L. LPS were from Sigma. STAT3 inhibitor, S3I-201, was purchased from Selleck. Opti-MEM was from Gibco. Antibodies used in this study such as those against tyrosine hydroxylase (TH), against α-synuclein, and against brain-derived neurotrophic factor (BDNF) were purchased from Cell Signaling Technology (CST, United States); that against ionized calcium-binding adapter molecule 1 (IBA-1, goat) was from Novus Bio; those against STAT3, p-STAT3(tyr705), p-STAT3(ser727), donkey anti-rabbit IgG (Alexa Fluor^® 594), and donkey anti-goat IgG (Alexa Fluor^® 488) were from Abcam (United States); and those against IL-6, p-JAK2 (tyr1007/1008), JAK2, and β-actin were from ABclonal (China). SDS-PAGE gel preparation and cocktail protease inhibitor were purchased from Servicebio (China). Universal antibody diluent and serum-free cell freezing medium were purchased from New Cell and Molecular Biotech (NCM, China). Protein Ladder and SuperSignal West Pico PLUS Chemiluminescent Substrate were purchased from Thermo (United States).

Cell survival was assayed with cell counting kit-8 (CCK8) according to the manufacturer’s instructions (Beyotime). BV2 microglial cells were plated into 96-well plates. After planning LPS and ECH intervention, CCK-8 solution was added into each well, followed by incubation at 37°C for 2 h. The absorbance of different wells was measured at 450 nm with a Microplate photometer (Thermo, United States).

The proteins of SN tissues and BV2 cells were extracted with lysates supplemented with protease inhibitor cocktail and phosphatase inhibitor, and the protein expression was measured with Western blot. Then, the concentrated gel and separated gel were prepared according to the instructions. The proteins were separated on 10%–12% SDS-PAGE (80V, 0.5 h; then 120 V, 1 h) and transferred to 0.22 μm PVDF membrane. Membranes were blocked with BSA (5%, 1 h, room temperature) and then incubated with primary antibodies (4°C, overnight). On the following day, secondary antibodies (1 h, room temperature) were applied to the membrane, followed by three-time washes with Tris-buffered saline and Tween 20 (TBST, 10 min each). Bands were visualized with ChemiScope 6000 Exp (Clinx Science Instruments, China) and normalized against β-actin with Image-Pro Plus software (Media Cybernetics, United States).

Statistical analyses of data were performed with the SPSS 19.0 software program (Chicago, IL, United States). The significance of differences between groups was evaluated with the one-way analysis of variance (ANOVA) and Dunnett’s t-test, and p < 0.05 was considered significant.

We assessed the establishment of neurobehavior in mice with the open field, rotarod, and pole tests. For the open field test, we recorded the autonomous activity tracks of mice with EthoVision XT12, as shown in Figure 1A. Compared with the control group, the motor complexity in the MPTP group was significantly reduced, resulting in decreases in the total distance (Figure 1B) and the number of line crossings (Figure 1C). However, treatment with ECH reversed all three actions.

We evaluated the exercise coordination ability of mice with the rotarod test. As shown in Figure 1D, the residence time in the model group was shorter than that in the control group. Of the three different treatment doses, only the high ECH doses prolonged the duration of MPTP mice on the rotarod.

Per the pole test findings, the descending time from the top to the bottom of the pole was markedly lengthier in the MPTP group than in the control group. However, this phenomenon was remedied by intervention with ECH (Figure 1E).

To assess the protective effect of ECH on the nigrostriatal DA system, we examined the expression of TH and α-synuclein in the SN. Mice in the MPTP-induced group showed reduced TH expression (Figures 2A,B) and increased α-synuclein deposition (Figure 2A,C) compared to those in the control group; however, these trends were reversed with ECH treatment. Consistent with Western blot findings, immune-histochemistry also demonstrated that mice in the MPTP group exhibited reduced TH-positive neurons in the SN by approximately 64% compared to mice in the control group, but treatment with ECH reversed this condition (Figure 2D).

Then, we performed the immunofluorescence staining of IBA-1 and observed significantly positive microglial staining in the SN of MPTP mice more than in the same areas of control mice (Figure 3A) indicating that microglia were activated in the MPTP group mice; however, ECH inhibited the activation of microglia.

Because pro-inflammatory cytokines play a crucial role in neuroinflammation-related PD, we conducted RT-qPCR on SN homogenates and found IL-1β (Figure 3B), TNF-α (Figure 3C), and IL-6 (Figure 3D) expressions markedly increased in the MPTP-induced mice compared to control mice. In contrast, treatment with medium and high ECH doses reduced the production of these cytokines to different degrees.

As the downstream of IL-6, we analyzed the activation of JAK2/STAT3 signaling and the phosphorylation of STAT3 (tyr705) with Western blot. As shown in Figure 4A, MPTP triggered considerable augmentations in IL-6, p-JAK2, and p-STAT3 (tyr705) expressions in the SN; however, ECH suppressed these elevations dose-dependently (Figures 4A–D). Notably, MPTP induced phosphorylation at the tyr705 site of STAT3, not the ser727 site, as the latter was not detected, as shown in Figure 4A, which is consistent with findings from a previous study (Sriram et al., 2004). In contrast, ECH activated STAT phosphorylation on ser727 (Figure 4E) and upregulated BNDF expression in the SN of mice (Figure 4F).

We investigated the impact of ECH on the activation of LPS-treated (1 μg/ml) BV2 microglial cells in vitro. To assess the effect of ECH on cell viability, we added ECH (5, 10, and 20 mg/L) into cell culture media alone or alongside LPS. As shown in Figures 5A,B, treatment with ECH and LPS did not alter cell viability compared to vehicle. Morphological changes were observed in BV2 cells under a light microscope (Figure 5C). BV2 cells in the LPS group were larger, harbored shorter processes, and displayed apparent swelling of the cell bodies compared to the control group. However, treatment with ECH significantly reversed these morphological changes. Figure 5D, which depicts the activation of BV2 cells via the immunofluorescence staining of IBA-1, exhibits a similar trend.

To determine ECH’s ability to affect LPS-induced neuroinflammation, we treated BV2 microglial cells with ECH (5, 10, and 20 mg/L) for 2 h, followed by LPS (1 μg/ml) for 6 h, and then determined their pro-inflammatory cytokine levels with RT-PCR. As shown in Figures 5E–G, ECH markedly decreased the mRNA level of LPS-induced pro-inflammatory cytokines IL-1β, TNF-α, and IL-6.

We examined the impact of ECH on IL-6/JAK2/STAT3 signaling in LPS-induced BV2 cells. As shown in Figure 6A, LPS administration led to the enhanced expression of IL-6, p-JAK2, and p-STAT3 (tyr705) proteins. Co-treatment with different doses of ECH saw a decline in the increased expressions of these three proteins dose-dependently; their Western blot analyses are shown in Figures 6B–D, respectively. Unlike in MPTP mice, p-STAT3 (ser727) was also activated in LPS-treated BV2 cells, followed by a persistent and dose-dependent upregulation after treatment with different ECH doses (Figure 6E). As a result, ECH treatments also amplified BDNF expression in the same trend as in MPTP mice (Figure 6F).

We conducted cellular immunofluorescence assays of p-STAT3 (tyr705) to observe the nuclear transcription of STAT3 directly. As shown in Figure 6G, compared with the control group, the STAT3 (tyr705) level in nuclei was significantly upregulated in LPS-induced BV2 cells; however, ECH significantly reduced these levels.

We used the inhibitor of STAT3, S3I-201, to examine the ECH’s regulation of IL-6/JAK2/STAT3 (tyr705) and BNDF/STAT3 (ser727). BV2 cells were pretreated with S3I-201 (100 μM) for 24 h and then treated with ECH (20 mg/L) for 2h, followed by LPS (1 μg/ml) for 6 h. IL-6, p-JAK2, and p-STAT3 expressions were determined with Western blot (Figure 6H). Treatment with “S3I-201 + ECH + LPS” further decreased LPS-stimulated IL-6, p-JAK2, and p-STAT3 (tyr705) protein expressions compared to treatment with “S3I-201 + LPS” (Figures 6I–K). Additionally, treatment with S3I-201 eliminated ECH-engendered p-STAT3 (ser727) (Figure 6L) and BDNF upregulation (Figure 6M).