Echinacoside protects against MPTP/MPP⁺-induced neurotoxicity via regulating autophagy pathway mediated by Sirt1

Echinacoside (ECH, CAS Number 82854–37-3), 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP, M0896), 1-Methyl-4-phenylpyridinium iodide (MPP⁺, D048), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, M2128) and Chloroquine diphosphate salt (CQ, C6628) were obtained from Sigma-Aldrich. EX527 (HY-15452) was purchased from MedChemExpress. DMEM and foetal bovine serum were purchased from Gibco.

Ten-week-old Male C57BL/6 J mice weighing 25-28 g were used in this Experiments. The animals were kept under a conditions of 12/12 h light/dark cycle, 21 ± 2 °C and were given free access to food and water. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Nanjing University of Chinese medicine. The experimenters were blinded to the assignments of the mice.

After being housed in animal facilities for 1 week to acclimate, mice were randomly divided into the four groups (10 mice per group): group A (vehicle control group, an equal volume of normal saline(NS)); group B (MPTP model group, treated with an equal volume of saline); group C (treated with 30 mg/kg Echinacoside (Chengdu Research Institute of Biology of the Chinese Academy of Sciences, HPLC≥98%,82,854–37-3); groups D (positive control group, treated with 30 mg/kg Selegiline (Finland Orion Corporation). Four groups of animals were pre-trained for behavioral test every other day in the next week. All groups were administered the respective pre-treatment compounds orally (p.o.) every 24 h for 7 consecutive days, with the last day of administration designated as day 0. Then the mouse PD models for groups B, C, D were made by injecting MPTP (sigma, M0896) at a dose of 30 mg/kg twice a week. Probenecid (250 mg/kg) was injected intraperitoneally 1 h following subcutaneous injection of MPTP to delayed its metabolism, and 1 h later respective treatment compounds were oral administration. Groups A received the same administration schedule except an equal volume of saline was injected instead of MPTP. All the animals were sacrificed at the 36th day post the behavioral test (Fig. 1).

To assess bradykinesia and motor coordination, the pole test was implemented according to previously published methods (Drucker-Colín and García-Hernández 1991). Selecting a steel pipe (55 cm long, 1 cm in diameter) tightly wrapped in white antiskid tape. Placing a spherical protrusions (2 cm in diameter) on the top of the pipe as attachment points of mice. The pipe should be fixed on the plastic foam base and placed in a cage with thick dressing. Putting mice on the upside of the spherical protrusions and recording the total time (T-total) of climbing to the bottom.

PC12 cells were cultured in 10% foetal bovine serum DMEM, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37 °C under a containing 5% CO₂ humidified atmosphere. When the cells enter logarithmic growth phase, they were seeded into 96-well plates at a density of 1 × 10⁴/well for 24 h. In order to determine the non-toxic dosages of ECH, PC12 cells were incubated with ECH (12.5-300 μM) for another 24 h. To study the effect of ECH on cell viability in MPP⁺-treated PC12 cells, the cells were pre-cultivated in serum-free DMEM with different concentrations of ECH (12.5-300 μM) for 1 h. Subsequently, MPP⁺ was added to the wells at a final concentration of 1 mM and incubated for another 24 h. CQ or EX527 was added 2 h before ECH.

Cell viability was measured using an MTT assay (Sladowski et al. 1993; Lv et al. 2017). After the initial incubation, a concentration of 0.5 mg/ml MTT was added to each well, then incubating for further 4 h at 37 °C. After removing the supernatant, the formazan product at bottom was solubilized sufficiently with 100 μl dimethyl sulfoxide (DMSO). The solutions was measured at 570 nm optical density using Epoch microplate assay reader (Bio-Tek).

PC12 cells were seeded into 24-well plates for 24 h before changing the culture medium and random grouping. ECH(100 mM)was pre-treated 1 h following the CQ which was pre-dosage of 0.05 μM, and 30 min later incubating with MPP+ (1 mM) for 24 h, change for serum-free DMEM medium and add Hoechst 33342 solution with a final concentration of 10 μg/mL for 5 min at room temperature. Subsequently, abandon the culture medium and add formaldehyde solution with a concentration of 4% for another 10 min. Then wash the cells 2 times with 1 mL PBS solution, each time for 5 min. Finally, observe the size and shape of cell nucleus under ultraviolet filter of Olympus fluorescence microscope (excitation wavelength is 360 nm and scattering wavelength is 420 nm) to determine and reflect apoptosis.

After the left ventricle perfusion, the mice were quickly taken to the brains and placed in a 4% paraformaldehyde. After the gradient dehydration of sucrose solution, brains was buried with OCT adhesive, and the frozen slicer was used as a continuous slice of brain tissue (30 μm thick). Slices of interest were selected from the SN-VTA according to the atlas of Paxinos and Franklin (2004). Frozen sections from the substantia nigra were incubated with anti-TH antibody (Sigma, SAB4200697, 1: 1000) in blocking solution (0.3% v/v Triton X-100 and 10% v/v BSA in PBS) for 12 h. Next, the slices were washed three times in 0.01 M PBS for 10 min each time. A second incubation with goat anti-mouse IgG H&L (HRP) (Abcam, ab6789, 1:2000) was performed for 2 h at 25 °C. After several washings with PBS, the antibody complex was detected using a modification of the ABC system (Kit Vectastain ABC, Vector Laboratory Inc.). The midbrain dopaminergic cell groups were plotted from TH-immunostained slices. TH-immunoreactive (TH-ir) neurons were stereologically quantified using Image-Pro Express 6 (Media Cybernetics). The mean number of TH positive neurons in each hemisphere, obtained through the quantification of four alternating slices, was considered to be representative of the SNpc neuronal cells in each animal. The selected areas were digitized with a microscope (DX45; Olympus). The images were taken using an Olympus camera (DP72; Olympus) at an original magnification of 100 × .

Substantia nigra and PC12 cells were prepared in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was measured by BCA assay (Pierce). 20 μg of total protein from each sample was separated by SDS-PAGE (8%, 10% or 15%) and transferred to a PVDF membrane. After incubating in 0.01 M TBS, 0.1% Tween-20 and 5% non-fat dry milk powder for 1 h to block any remaining protein binding sites, membranes were incubated overnight at 4 °C using the following primary antibodies: the mouse antibody against TH (Sigma, SAB4200697, 1:2000), alpha-synuclein (Abcam, ab1903, 1:1000), β-tubulin (Proteintech,10,094–1-AP, 1:2000); the rabbit antibody against LC3, P62, Beclin-1, p-PI3K, PI3K, Sirt1 (Cell Signaling Technology, #4108, #5114, #3738, #4228, #4249, #9475, 1:1000) and FoxO1 (Abcam, ab52857, 1:1000). After three 5-min washes in TBST, membranes were incubated with the goat anti-mouse IgG H&L (HRP)/goat anti-rabbit IgG H&L (HRP) (Abcam, ab6789, ab6721, 1:2000) for 1 h at room temperature. All antibody incubations and washing steps were carried out in TBST. The immunoreactive blots were visualized using the supersignal chemiluminescence detection system (Thermo Fisher Scientific Inc.) and quantified with densitometry using ImageJ software.

Data were analyzed using SPSS Version 20.0 (IBM). All experiments were performed at least three times. Comparisons between two groups were performed using a two-sample Student’s t test, and comparisons between multiple groups were performed using a one-way ANOVA followed by Tukey’s post hoc test. All data are presented as mean ± SEM, and statistical significance was accepted at the 5% level unless otherwise indicated.

We used the pole test to evaluate bradykinesia and coordination in mice. At the seventh day after the last induction of MPTP, we detected that the return time and the total time were both significantly prolonged, and the effect was significantly reversed with ECH and selegiline treatment (Fig. 2a, b). Our results suggested that ECH reversed the behavioral impairments induced by MPTP. Then we used immunohistochemistry staining for TH-positive neurons to observe the loss of dopaminergic neurons in four groups of mice. As shown in Fig. 2c and d, MPTP inducement reduced the number of nigral TH positive neurons remarkably. Compared with the MPTP group, the administration of ECH and selegiline significantly increased the number of nigral TH positive neurons.

Western blotting analysis indicated that MPTP-induced a marked α-synuclein protein increase in striatum tissue compared to saline control (7 days after last injection). However, the MPTP-lesioned animals treated with ECH and selegiline inhibited the accumulation. Meanwhile, after MPTP treatment, the expression of autophagy-related proteins such as LC3-II, Beclin-1 and p-PI3K reduced while the expression of P62 which is negatively related to autophagy significantly increased (p < 0.05), showing that the level of autophagy in neurons reduced after modeling. ECH could reverse such change by increasing the expression of LC3-II, Beclin-1 and p-PI3K and reducing the expression of P62 (Fig. 3). Taken together, our results suggested that the mechanism of ECH in alleviating loss of DA neurons and increase of α-synuclein in SNpc induced by MPTP is related to up-regulation of autophagy.

In this study, we showed ECH has an effect of protection on neurons. We further tested the cell viability of PC12 cells treated with different concentrations of ECH to determine non-toxic dosages, as shown in Fig. 4a. There was no significant difference been observed in the viability of PC12 cells with concentrations of 0-100 μM of ECH treatment. However, cell viability was significantly lower at a concentration of 300 μM than the other groups of concentrations. To determine whether ECH had neuro-protective effects against the administration of MPP⁺, PC12 cells were pre-incubated with the concentrations of 0-300 μM ECH for 1 h followed by exposing to 1 mM MPP⁺ for 24 h. The MTT assay showed that treating PC12 cells with MPP⁺ alone resulted in a 40% reduction in the number of surviving cells. Co-treatment with 50 or 100 μM ECH showed a reduction in MPP⁺-induced cytotoxicity. PC12 cell viability increased in an ECH-dose-dependent manner compared with the cells treated with MPP⁺ only (Fig. 4b). This indicates that ECH reduced MPP⁺-induced cytotoxicity. Consequently, we chose to use concentrations of 100 μM of ECH in the following experiment. To confirm the enhancement of autophagy by ECH, we treated MPP⁺-induced PC12 cells with ECH and ECH plus CQ (chloroquine, a lysosome inhibitor). Then the protective effect of ECH on MPP⁺ induced PC12 cell damage was reduced (Fig. 4c, d). Western blotting detection indicated that the expressions of autophagy proteins such as LC3-II and Beclin-1 and p-PI3K were significantly reduced (p < 0.01, p < 0.01, p < 0.05) while the expression of P62 which is negatively related to autophagy was significantly increased (p < 0.05) in the PC12 cell after being incubated with 1 mM MPP⁺ for 24 h. 100 μM ECH treatment increased the expression of LC3-II, Beclin-1 and p-PI3K (p < 0.05, p < 0.01, p < 0.05) while reduced the expression of p62 (p < 0.05). (Figure 4e‑j) The result above indicated that ECH reversion of MPP⁺ induced PC12 cell damage was related to the up-regulation of autophagy.

Structure-based drug design was adopted to evaluate whether Sirt1 can bind to ECH and forecast their docking pattern and docking energy. Preparations including adding hydrogens and charges were performed to Sirt1 and ECH respectively before docking the ligand ECH to the receptor protein Sirt1. Result of the molecular docking revealed that the docking energy of ECH to Sirt1 was −12.5 kcal/mol, and the micromolecule and protein have 9 hydrogen bonds, forming a stable docking pattern (Fig. 5a). ECH molecules falls inside the active pocket of protein Sirt1 (Fig. 5b). The calculation forecasted that ECH molecules could directly bind to protein Sirt1. The result above indicated that ECH and Sirt1 have affinity, and ECH was likely to regulate the pharmacological effect of Sirt1. Furthermore, we use EX527 (specific Sirt1 inhibitor) to validate this conjecture. We found that the protective effect of ECH on MPP⁺ induced PC12 cell damage was reduced by EX527 (Fig. 5c). Followed by the western blotting, Sirt1 and FoxO1 (the downstream protein of Sirt1) protein expression were significantly down-regulated after MPP⁺ exposure on PC12, and ECH reversed this phenomenon. However, when EX527 was applied, the effect of ECH was reduced. Meanwhile, the effect that ECH raised LC3 was also reduced. (Fig. 5d‑g). The experiment showed that the enhancing autophagy effect of ECH may be achieved by regulating Sirt1.