Cell culture and drug treatment
Human neuroblastoma cell line SH-SY5Y was obtained from Sun Ye San University (Guangzhou, China), and cultured in DMEM/H medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum(Gibco, Grand Island, NY, USA ) and 1% glutamine at 37℃ in a humidified incubator with 5% CO2. We changed the culture medium every other two days, and subcultured the cells when the density reached 80%. (1) To study the effect of MPP+ or ART on cell viability, we treated the cells with different concentrations of MPP+ or ART for 24h and performed CCK-8 assay; (2) To study the protective effect of ART on MPP+-induced cytotoxicity, we treated the cells with PBS, ART, MPP+, ART+MPP+ as indicated concentrations for 24h. ART(20μM) pre-treated the cells for 4h, and then added MPP+ for 24h. (3) To study the changes of autophagy, we treated the cells with PBS, ART, MPP+, ART+MPP+ and 3-MA+MPP+ for 24h. ART(20μM), 3-MA (5mM) pre-treated the cells for 4h, and then added MPP+ for 24h. MPP+ was purchased from Sigma (CAS No. 36913-39-0, USA). ART was purchased from DASF (CAS No. 71963-77-4, Nanjing, China). Autophagy inhibitor 3-methyladenine (3-MA ) was purchased from sigma (M9281, USA).
Cell viability was measured by CCK-8 assay (Solarbio, CA1210, China) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded in 96-wells culture plates for 24h, and treated with MPP+ or ART for another 24h, then 100μl culture medium containing 10mM CCK-8 was added to each well and incubated at 37℃ for 2h. The absorbance at 450nm was measured by a multi-mode microplate reader (EnSpire, PerkinElmer, Singapore). The cell viability of the control group was set to 100%, and the cell viability of the other groups were compared with that of the control group.
Intracellular Reactive Oxygen Species (ROS) assay
Levels of intracellular ROS were measured by ROS assay kit (Beyotime Biotechnology, S0033, China) according to the manufacturer’s instructions. After drug treatment, SH-SY5Y cells were incubated with serum-free fresh medium which was contained 10μM DCFH-DA at 37℃ for 20min, then washed the cells with serum-free culture medium for three times. Add 1ml fresh medium into each well, the fluorescence was observed using the GFP channel by an inverted fluorescence microscopy (Nikon, Japan) and the fluorescence intensity was analyzed by ImageJ.
Superoxide dismutase (SOD) activity assay
Intracellular SOD activity was measured by total SOD activity detection kit (Beyotime Biotechnology, S0101, China) according to the manufacturer’s instructions. After drug treatment, washed the cells once with PBS, and centrifuged to collect the cells. Lysed the cells fully with SOD sample preparation solution and collected the supernatants at 12, 000 × g for 5min at 4°C. Detected their absorbance at 450nm and 600nm (reference wavelength) by a multi-mode microplate reader and calculated the SOD activity.
Glutathione (GSH) assay
The content of GSH was measured by the glutathione assay kit (Beyotime Biotechnology, S0052, China) according to the manufacturer’s instructions. After drug treatment, washed the cells once with PBS, and centrifuged to collect the cells. Prepared the samples and standards and detected their absorbance at 412nm. Made the standard curve and calculated the contents of GSH in the samples according to the standard curve.
Malondialdehyde (MDA) assay
Intracellular MDA levels were measured by MDA detection kit (Beyotime Biotechnology, S0131, China) according to the manufacturer’s instructions. After drug treatment, collected the cells. Lysed the cells and collected the supernatants at 10, 000 × g for 10min. Prepared the standards, then detected the absorbance of samples and standards at 532nm and 450nm (reference wavelength). The contents of MDA in the samples were quantified based on the standard curve.
Mitochondrial membrane potential (MMP) assay
To monitor mitochondrial integrity, mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, C2006, China) was used according to manufacturer’s instructions. Briefly, after drug treatment, SH-SY5Y cells were incubated with JC-1 working solution for at 37 °C 20min and washed twice with JC-1 buffer. Then observed the red fluorescence and green fluorescence through the GFP channel and TRITC channel by an inverted fluorescence microscopy (Nikon, Japan), and the fluorescence intensity was analyzed by ImageJ.
Western blot assay
Cell lysates were prepared and the protein concentrations were quantified with BCA protein assay kit (CWBIO, CW2011S, China). Equal protein samples were separated wite 12% SDS-PAGE and transferred to PVDF membranes. After blocked with 5% skim milk at room temperature for 1h, the membranes were incubated at 4℃overnight with primary antibody (1:1000) against cleaved caspase-3 (Cell Signaling Technology, Aps175, USA), caspase-3 (Abcam, ab90437, UK), LC3 (Abcam, ab128025, UK), Beclin1(BD, 612113, USA), P62(Abcam, ab56416, UK) or β-actin 1:5000 (CWBIO, CW0096M, China). The next day, membranes were washed with TBST for three times, and incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody (CWBIO,1:5000) at room temperature for 1h. The blots were detected by cECL western blot kit (CWBIO, CW0049M, China) and images were analyzed by ImageJ.
SH-SY5Y cells were seeded on slides in 12-well culture plates. After drug treatment, cells were fixed with ice methanol for 5min, and blocked with 1% bovine serum albumin (BSA) for 30min, then incubated with primary antibody against LC3(1:200), P62(1:100) respectively at 4℃for overnight. The next day, slides were incubated with Alexa Fluor 488 (Bioss Antibodies, bs-0295G-AF488, China) or Alexa Fluor 555 (Bioss Antibodies, bs-0296G-AF555, China) secondary antibody (1:500) at 37℃ for 1h and incubated with DAPI (BOSTER, AR1177) for 5min. The fluorescence was observed by a fluorescence microscope (OLYMPUS, BX53).
Results were expressed as the mean±standard error of mean (mean ± SEM). Comparison among multiple groups were performed with One-way ANOVA followed by Tukey post- hoc test and P <0.05 was considered statistically significant.