黄芩苷通过上调lncRNA H19表达来减轻MPP^(+)诱导的PC12细胞神经毒性

Baicalin reduced the neurotoxicity of PC12cells induced by MPP^(+) by up-regulating lncRNA H19

目的探讨黄芩苷是否通过上调长链非编码RNA(Long noncoding RNA,lncRNA)H19表达来减轻1-甲基4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP^(+))诱导的PC12细胞神经毒性。方法分别采用0.5、0.75、1、2 mmol/L MPP^(+)和10、20、50和70μmol/L黄芩苷作用PC12细胞,后续试验使用水平分别选择0.75 mmol/L MPP^(+)和50μmol/L黄芩苷;以0.75 mmol/L MPP^(+)损伤PC12细胞,体外模拟帕金森病,并加入黄芩苷;采用四甲基偶氮唑盐(Methyl thiazolyl tetrazolium,MTT)进行细胞活力测定,Annexin V-异硫氰酸荧光素(Fluorescein isothiocyanate isomer,FITC)/碘化丙啶(Propidium iodide,PI)双染法进行凋亡定量分析,蛋白质印迹分析(Western blot)检测Pro-天冬氨酸特异性半胱氨酸蛋白酶(Cysteinyl aspartate specific proteinase,Caspase)-3,Cleaved-caspase-3,B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)蛋白和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)的相对表达水平,实时定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qPCR)进行lncRNA H19的相对表达水平检测;在PC12细胞中转染lncRNA H19小干扰RNA(lncRNA H19 siRNA,si-H19),并加入MPP^(+)和黄芩苷,考察PC12细胞增殖、凋亡等变化。结果与对照组比较,MPP^(+)降低PC12细胞的存活率、Bcl-2蛋白、lncRNA H19相对表达水平,提高PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。与MPP^(+)组比较,黄芩苷显著提高MPP^(+)诱导的PC12细胞的存活率、Bcl-2蛋白、lncRNA H19相对表达水平,降低PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。转染si-H19后黄芩苷和MPP^(+)诱导的PC12细胞的存活率、Bcl-2蛋白相对表达水平降低,PC12细胞的凋亡率、Cleaved-caspase-3,Bax蛋白相对表达水平升高(P<0.05),而Pro-caspase-3蛋白相对表达水平无明显变化(P>0.05)。结论黄芩苷上调lncRNA H19表达,提高MPP^(+)诱导的PC12细胞活力和降低其凋亡,减轻MPP^(+)诱导的PC12细胞神经毒性。

Objective To investigate whether baicalin reduces the neurotoxicity of PC12 cells induced by 1-methyl-4-phenylpyridine ion(MPP^(+))by up-regulating long non-coding RNA(lncRNA)H19.Methods PC12 cells were treated with0.5,0.75,1,2 mmol/L MPP^(+)and10,20,50,and70μmol/L baicalin,respectively,and the concentrations used in subsequent experiments were 0.75 mmol/L MPP^(+)and 50μmol/L baicalin,respectively.PC12 cells were injured with 0.75 mmol/L MPP^(+)to simulate Parkinson's disease in vitro,and baicalin was added.MTT method was used for cell viability determination,phosphatidyl binding protein V(Annexin V)-FITC/propidium iodide(PI)double staining method was applied for apoptosis quantitative analysis,and western blot analysis was performed to detect pro-asparagus Caspase(caspase)-3,cleaved-caspase-3,Bcl-2 related X protein(Bax)and B-cell lymphoma/leukemia-2(Bcl-2)protein expression,real-time Quantitative PCR was employed to detect the expression of lncRNA H19.Transfect si-H19 into PC12 cells,and add MPP^(+)and baicalin to investigate the changes of PC12 cell proliferation and apoptosis.Results Compared with the control group,MPP^(+)reduced PC12 cell survival rate,Bcl-2 protein expression level,lncRNA H19 expression,increased apoptosis rate,cleaved-caspase-3 protein,and Bax protein expression levels.The difference was statistically significant(P<0.05),the expression level of pro-caspase-3 protein was not significantly different(P>0.05).Compared with the MPP^(+)group,baicalin significantly increased the survival rate,Bcl-2 protein expression level,and lncRNA H19 expression of PC12 cells induced by MPP^(+),and decreased its apoptosis rate,cleaved-caspase-3 protein and Bax protein expression levels.The difference was It was statistically significant(P<0.05),and the expression level of pro-caspase-3 protein was not statistically significant(P>0.05).After transfection of si-H19,the survival rate and Bcl-2 protein expression level of PC12 cells induced by baicalin and MPP^(+)decreased,and the apoptosis rate,cleaved-caspase-3 protein and Bax protein expression levels increased,and the difference was statistically significant(P<0.05),the expression level of pro-caspase-3 protein was not significantly different(P>0.05).Conclusion Baicalin could up-regulate the expression of lncRNA H19,increase the viability of PC12 cells induced by MPP^(+)and reduce their apoptosis,and reduce the neurotoxicity of PC12 cells induced by MPP^(+).