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Objective: To establish an oxygen and glucose deprivation model (OGD) of SH-SY5Y cells, and investigate the effects of Xuesaitong Injection on the cell survival, apoptosis rate of SH-SY5Y cells and the mRNA and protein expressions of Lingo-1. Methods: The model was established using 1640 sugar-free medium and three-gas incubator. The cell survival rate was determined by CCK8 method to determine the optimal time for hypoxia and the optimal concentration of Xuesaitong Injection. The apoptosis rate of SH-SY5Y cells was detected by Annexin V-FITC/PI double staining. The mRNA and protein expressions of Lingo-1 were determined by qRT-PCR and Western blotting, respectively, and the effect of Xuesaitong on Lingo-1 expression was finally determined. Results: In this study, the optimal hypoxia time for the establishment of the OGD model of SH-SY5Y cells was 16 h and the optimum concentration of Xuesaitong Injection was 640 mg/L. The apoptosis rate of SH-SY5Y cells was significantly reduced, and the expression levels of lingo-1 mRNA and protein were decreased in the Xuesaitong group compared with the model group under the condition of this concentration. Conclusion: The apoptosis rate was significantly increased and Lingo-1 was highly expressed when SH-SY5Y cells were damaged by oxygen and glucose deprivation. Xuesaitong can significantly reduce the apoptosis of SH-SY5Y cells induced by oxygen and glucose deprivation, and inhibit the high expression of lingo-1, which has the anti-apoptosis and significant neuroprotective effect.
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[ABSTRACT]AIM:ToexaminetheeffectsofhighconcentrationofextracellularATPonhumanneuroblastoma SH-SY5Y cell injury.METHODS: Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time.The cell viability was detected by CCK-8 assay.The variation of autophagic vacuoles was observed with monodansylcadaverine staining .The cell apoptosis was analyzed by Hoechst 33258 staining.Meanwhile, apoptotic rate was detected by flow cytometry .The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ ( LC3-Ⅱ) were determined by Western blotting .RESULTS:Compared with control group , the survival rate of SH-SY5Y cells was signifi-cantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h).The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment , trended to decrease over time and returned to control level at 6 h.The protein expression of LC3-Ⅱwas significantly increased at 1 h with ATP treatment , which was consistent with the time points of increasing auto-phagic vacuoles .LC3-Ⅱexpression level gradually decreased at 2~3 h with ATP treatment , and returned to control level at 6 h.Compared with control group , the apoptotic rate and the expression level of caspase-3 were enhanced synchronously . The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION:High concentration of extracellular ATP induces the autophagy and apoptosis of SH -SY5Y cells.The in-creased autophagy shows up , followed by the climax of apoptosis until 6 h.With the prolonged duration of ATP , apoptosis is the main process in the cells .
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Objective To investigate the roles of hypoxia regulated genes/protein in chemical hypoxia preconditioning in differentiated SH-SY5Y cells.Methods Differentiated SH-SY5Y cells were randomly divided into control group,chemical hypoxic preconditioning group(50 ?mol/L CoCl2 preconditioning for 3 h,normal culture for 1 h,then in 2%O2 for 28 h) and hypoxia group(in 2%O2 for 28 h).RT-PCR was used to examine the mRNA expressions of VEGF,GLUT-1,EPO,LDH-A.Further evaluation of the potential neuroprotective effect of VEGF was done by investigating the effect of recombinant human VEGF on subsequent hypoxia injury.Results The mRNA levels of GLUT-1,EPO increased in the preconditioning group as compared with those in the hypoxia group(P