山栀苷对6-羟基多巴胺损伤的SH-SY5Y细胞的保护作用及其机制

Protective effect of shanzhiside on SH-SY5Y cells injured by 6-hydroxy⁃dopamine and its mechanism

目的:观察山栀苷对6-羟基多巴胺(6-OHDA)诱导的SH-SY5Y细胞氧化损伤的作用,并探讨其作用的分子机制。方法:以6-OHDA诱导的SH-SY5Y细胞为帕金森病体外细胞模型,分别以5、50、100和200μmol/L山栀苷作用于受损细胞,另设空白组及50μmol/L栀子苷阳性对照组。CCK-8法检测细胞活力;倒置显微镜下观察细胞形态学改变;流式细胞术检测细胞内活性氧(ROS)的荧光强度;分子对接技术评价山栀苷、栀子苷与Kelch样ECH相关蛋白1(Keap1)的结合情况;Western blot检测Keap1、核因子E2相关因子2(Nrf2)和血红素加氧酶1(HO-1)蛋白表达水平;RT-qPCR检测HO-1的mRNA水平。结果:(1)与空白组比较,模型组细胞活力降低,细胞数量减少,形态改变明显,ROS水平显著升高,Nrf2和HO-1蛋白表达均显著下降,HO-1的mRNA水平显著降低(P<0.05);(2)与模型组比较,山栀苷预处理2 h可提高6-OHDA损伤SH-SY5Y细胞的活力,其中100μmol/L山栀苷组的细胞活力最高(P<0.05),还可显著降低ROS水平,增加Nrf2和HO-1蛋白表达,提高HO-1的mRNA水平(P<0.05);各组Keap1蛋白表达的差异无统计学意义(P>0.05);(3)与受体Keap1蛋白的结合能力:阳性配体>山栀苷>栀子苷。结论:山栀苷对6-OHDA损伤的SH-SY5Y细胞具有保护作用,其机制可能是通过调控Keap1/Nrf/HO-1通路抑制氧化应激反应。

AIM:To investigate the effect of shanzhiside on oxidative damage of SH-SY5Y cells induced by 6-hydroxydopamine(6-OHDA)and its possible mechanism.METHODS:Parkinson disease cell model was established by treating neuroblastoma SH-SY5Y cells with 6-OHDA(100μmol/L).The SH-SY5Y cells were divided into treatment groups(treated with 5,50,100 and 200μmol/L shanzhiside),positive control group(treated with 50μmol/L geniposide),model group and blank group.CCK-8 assay was used to detect the cell viability.Inverted microscopy was used to observe the morphological changes of the cells.Flow cytometry was used to detect fluorescence intensity after intracellular reactive oxygen species(ROS)staining.Molecular docking technique was used to evaluate the binding of shanzhiside or gardenitin to Kelch-like ECH-associated protein 1(Keap1).Western blot was used to detect the protein expression levels of Keap1,nuclear factor E2 related factor 2(Nrf2)and heme oxygenase-1(HO-1)in SH-SY5Y cells.The mRNA level of HO-1 was detected by RT-qPCR.RESULTS:Compared with control group,the cells in model group showed significantly decreased viability,morphological changes,increased ROS levels,and decreased Nrf2 and HO-1 protein levels and HO-1 mRNA levels(P<0.05).Compared with model group,the cells in shanzhiside groups displayed increased viability,decreased ROS levels,and increased Nrf2 and HO-1 protein levels and HO-1 mRNA levels(P<0.05).Treatment with 100μmol/L shanzhiside had the most significant cell protective effect(P<0.05).The protein expression of Keap1 was not statistically changed(P>0.05).The molecular docking results showed that the binding ability of shanzhiside with Keap1 protein was better than that of gardenitin.CONCLUSION:Shanzhiside confers a significant protective effect against the injury of SH-SY5Y cells induced by 6-OHDA,which may be related to inhibition of oxidative stress by regulating the Keap1/Nrf/HO-1 signaling pathway.