鸢尾苷元调控miR-103a-3p/SHC3通路对帕金森病模型细胞损伤的影响

Tectorigenin protects cell damage in Parkinson’s disease model by regulating miR-103a-3p/SHC3 pathway

目的探讨鸢尾苷元对帕金森病(Parkinson’s disease,PD)模型细胞损伤的影响,分析其是否与调控miR-103a-3p/含有Src同源结构域2的衔接蛋白3(SHC adaptor protein 3,SHC3)通路有关。方法用250 mmol/L的1-甲基-4-苯基吡啶离子(MPP^(+))诱导PC12建立PD细胞模型。PC12细胞分为对照组、PD组、PD+鸢尾苷元50μmol/L组、PD+鸢尾苷元100μmol/L组、PD+鸢尾苷元200μmol/L组、PD+anti-miR-NC组、PD+miR-103a-3p Inhibitor组、PD+鸢尾苷元+miR-103a-3p mimic组。CCK-8法检测各组细胞活力,流式细胞术分析各组细胞凋亡率,试剂盒检测各组细胞超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)水平以及乳酸脱氢酶(lactate dehydrogenase,LDH)释放量,RT-qPCR检测miR-103a-3p表达,Werstern blot检测SHC3蛋白水平。双荧光素酶报告实验分析miR-103a-3p和SHC3靶向关系。结果与对照组比较,PD组PC12细胞活力、SOD活性、SHC3蛋白表达显著降低(P<0.05),凋亡率、MDA水平、LDH释放量、miR-103a-3p表达显著升高(P<0.05)。与PD组比较,PD+鸢尾苷元50μmol/L组、PD+鸢尾苷元100μmol/L组、PD+鸢尾苷元200μmol/L组PC12细胞活力、SOD活性、SHC3蛋白表达显著升高(P<0.05),凋亡率、MDA水平、LDH释放量、miR-103a-3p表达显著降低(P<0.05)。与PD+anti-miR-NC组比较,PD+miR-103a-3p Inhibitor组PC12细胞活力、SOD活性、SHC3蛋白表达显著升高(P<0.05),凋亡率、MDA水平、LDH释放量显著降低(P<0.05)。与PD+鸢尾苷元组比较,PD+鸢尾苷元+miR-103a-3p mimic组PC12细胞活力、SOD活性、SHC3蛋白表达显著降低(P<0.05),凋亡率、MDA水平、LDH释放量显著升高(P<0.05)。miR-103a-3p与SHC3直接结合。结论鸢尾苷元可保护PD模型细胞凋亡和氧化损伤,其机制与抑制miR-103a-3p/SHC3通路有关。

Objective To investigate the effect of tectorigenin on cell model of Parkinson’s disease(PD),so as to further analyze whether its mechanism is related to the regulation of miR-103 a-3 p/SHC adaptor protein 3(SHC3) pathway.Methods PC12 cells were treated with 250 mmol/L 1-methyl-4-phenylpyridine ion(MPP^(+)) to establish a PD cell model.Then,the cells were divided into control group and experimental groups as follows:PD group,PD+50 μmol/L tectorigenin,PD+100 μmol/L tectorigenin,PD+200 μmol/L tectorigenin,PD+anti-miR-NC,PD+miR-103 a-3 p inhibitor,and PD+tectorigenin+miR-103 a-3 p mimic.Cell viability and apoptotic rate of each group were evaluated using CCK-8 assay and flow cytometry respectively.The activity of superoxide dismutase(SOD),content of malondialdehyde(MDA) and release of lactate dehydrogenase(LDH) of cells in each group were subsequently detected.Moreover,RT-qPCR and Western blotting were performed to determine the mRNA expression of miR-103 a-3 p and protein level of SHC3,respectively,and dual luciferase reporting assay was adopted to analyze the targeting relationship between miR-103 a-3 p and SHC3.Results As compared with the control group,the viability,SOD activity,and SHC3 expression of cells were notably reduced(P<0.05),while the apoptotic rate,MDA content,LDH release,and miR-103 a-3 p expression were significantly increased(P<0.05) in the PD group.When compared with the PD group,the viability,SOD activity,and SHC3 expression of cells in the PD+tectorigenin(50,100 and 200 μmol/L) groups were all notably improved(P<0.05),while the apoptotic rate,MDA content,LDH release and miR-103 a-3 p level were greatly declined(P<0.05).In addition,the cell viability,SOD activity and SHC3 level were higher,whereas the apoptotic rate,MDA content and LDH release were lower in the PD+miR-103 a-3 p inhibitor group than in the PD+anti-miR-NC group(P<0.05).Finally,the cell viability,SOD activity and SHC3 expression in the PD+tectorigenin+miR-103 a-3 p mimic group were notably decreased(P<0.05),while the apoptotic rate,MDA content,and LDH release were increased(P<0.05),as compared with the PD+tectorigenin groups.Dual luciferase reporting assay showed that SHC3 was a direct target of miR-103 a-3 p.Conclusion Tectorigenin protects PD model cells against apoptosis and oxidative damage,which may be related to the inhibition of miR-103 a-3 p/SHC3 pathway.