氧化苦参碱联合复方茵陈颗粒对大鼠肝BRL-3A细胞凋亡的抑制作用及其机制

Inhibitory effect and mechanism of oxymatrine plus Compound Yinchen Granules on apoptosis of BRL-3A cells in rats

目的:探讨氧化苦参碱(OMT)联合复方茵陈颗粒(FYK)对脂多糖(LPS)诱导的大鼠肝BRL-3A细胞凋亡的抑制作用及其机制,为该联合用药抑制肝细胞凋亡提供更多的实验依据。方法:采用LPS诱导大鼠肝BRL-3A细胞建立细胞凋亡模型。应用CCK-8法测定BRL-3A细胞活力;流式细胞术检测细胞凋亡率;Western blot法检测BRL-3A细胞中TLR4、P38、P-P38、JNK及P-JNK蛋白的表达;RT-q PCR法检测BRL-3A细胞中Bax、Bcl-2及Caspase-3 m RNA的表达。结果:CCK-8法结果显示,OMT可以减弱由LPS引起的细胞活力降低(P<0.05),OMT+FYK-M+LPS组及OMT+FYK-H+LPS组细胞活力高于OMT组(P<0.05);OMT预处理后细胞凋亡率低于LPS组(P<0.05),OMT+FYK-M+LPS组及OMT+FYK-H+LPS组细胞凋亡率低于OMT组(P<0.05)。Western blot结果显示,LPS组TLR4、P-P38/P38、P-JNK/JNK的表达较正常组增加(P<0.05);OMT可以下调TLR4、P-P38/P38、P-JNK/JNK的表达(P<0.01,P<0.05),OMT+FYK-M+LPS组及OMT+FYK-H+LPS组TLR4、P-P38/P38低于OMT组(P<0.05)。OMT+FYK-H+LPS组P-JNK/JNK低于OMT组(P<0.05)。RT-q PCR结果显示:LPS组Bax/Bcl-2及Caspase-3 m RNA表达较正常组明显增高(P<0.01)。OMT能够降低Bax/Bcl-2及Caspase-3 m RNA的水平(P<0.05)。OMT+FYK-M+LPS组、OMT+FYK-H+LPS组Bax/Bcl-2及Caspase-3 m RNA表达低于OMT组(P<0.05,P<0.01)。结论:OMT联合FYK预处理可通过TLR4/P38/JNK信号通路抑制BRL-3A细胞凋亡。

Objective： To investigate the inhibitory effect and mechanism of oxymatrine （OMT） plus Compound Yinchen Granules （CYG） on apoptosis of BRL-3A cells in rats induced by LPS, and provide more experimental basis for the inhibitory effect of hepatocyte apoptosis. Methods： The apoptosis model of BRL-3A cells was established by LPS. Cell activity was determined by CCK-8 method; cell apoptosis was detected by flow cytometry; Western blot was used to detect protein levels of TLR4, P38, P-P38, JNK and P-JNK; RT-qPCR was used to detect the expression of Bax, Bcl-2 and Caspase-3 mRNA. Results： The results of CCK-8 showed that OMT pretreatment up-regulated the cell viability induced by LPS （P〈0.05）. OMT＋CYG-M and OMT＋CYG-H preconditioning could further improve the cell viability （P〈0.05）. Cell apoptotic rate of BRL- 3A cells in OMT group was lower than that of LPS group （P〈0.05）. OMT＋CYG-M and OMT＋CYG-H preconditioning could further decrease cell apoptotic rate （P〈0.05）. Western blot test showed that the expression of TLR4, P-P38/P38, and P-JNK/ JNK increased in LPS group （P〈0.05）, which were down-regulated by OMT preconditioning （P〈0.01, P〈0.05）. OMT＋CYG-M and OMT＋CYG-H preconditioning could further decrease the expression of TLR4, P-P38/P38 （P〈0.05）, OMT＋CYG-H preconditioning could further decrease the expression of P-JNK/JNK （P〈0.05）. RT-qPCR showed that the expression of Bax/Bcl-2 and mRNA Caspase-3 significantly increased in LPS group （P〈0.01）, which was reduced by OMT pretreatment （P〈0.05）. OMT＋CYG-M and OMT＋CYG-H preconditioning could further down-regulate the expression of BaxlBcl-2 and Caspase-3 mRNA （P〈0.05, P〈0.01）： The expression in OMT＋CYG-H＋LPS group was lower than that in OMT＋CYG-M＋LPS group （P〈0.05）. Conclusion： OMT＋CYG pretreatment could inhibit the apoptosis of BRL-3A cells through TLR4/P38/JNK signaling pathway.