芪灵扶正清解颗粒调控P53/AMPK信号通路诱导HepG2细胞铁死亡

Qiling Fuzheng Qingjie granule induces ferroptosis in HepG2 cells by regulating P53/AMPK signaling pathway

目的探究芪灵扶正清解颗粒是否调控P53/AMPK信号通路诱导Hepg2细胞铁死亡。方法①使用不同浓度芪灵扶正清解颗粒培养液培养HepG2细胞,CCK-8法筛选其最佳干预浓度;②将Hepg2细胞分为空白组、Fer-1组、芪灵组、Fer-1+芪灵组;③透射电镜观察HepG2细胞线粒体形态;④流式细胞术检测活性氧ROS含量;⑤生化法检测Fe2+含量;⑥免疫荧光检测P53、Slc7a11、Gpx4蛋白表达;⑦荧光定量PCR测定P53、Ampk、Gpx4mRNA水平;⑧蛋白印迹法检测Ampk、p-Ampk、P53、Beclin1、Acsl4、Slc7a11、Gpx4蛋白表达情况。结果①芪灵扶正清解颗粒能够抑制HepG2细胞增殖,以5mg/ml浓度的抑制作用最明显(P<0.05);②与空白组及Fer-1+芪灵组比较,芪灵组ROS和Fe^(2+)均升高(P<0.05),Ampk、p-Ampk、P53、Beclin1、Acsl4蛋白、ACSL4、P53mRNA表达水平及P53荧光信号强度升高(P<0.05),Slc7a11、Gpx4蛋白水平、Gpx4mRNA及荧光强度降低(P<0.05);Fer-1组ROS和Fe^(2+)均降低(P<0.05),Ampk、p-Ampk、P53、Beclin1、Acsl4蛋白、ACSL4、P53mRNA水平及P53荧光信号强度降低(P<0.05),Slc7a11、Gpx4蛋白水平、Gpx4mRNA及荧光强度升高(P<0.05)。结论芪灵扶正清解颗粒可通过调控P53/AMPK信号通路诱导HepG2细胞铁死亡。

Objective To investigate whether Qiling Fuzheng Qingjie granules induce ferroptosis in Hepg2 cells by regulating P53/AMPK signaling pathway.Methods①HepG2 cells were cultured with different concentrations of Qiling Fuzheng Qingjie granule culture medium,and the optimal intervention concentration was screened by CCK-8 method.Hepg.②cells were divided into blank group,Fer-1 group,Qiling group and Fer-1+Qiling group.③Mitochondrial morphology of HepG2 cells was ob⁃served by transmission electron microscopy;④The content of reactive oxygen species(ROS)was detected by flow cytometry.⑤Fe^(2+)content was detected by biochemical method;⑥The expressions of P53,Slc7a11 and Gpx4 were detected by immunofluores⁃cence.⑦The mRNA levels of P53,Ampk and GPx4 were detected by real-time PCR.⑧The protein expressions of Ampk,p-Ampk,P53,Beclin1,Acsl4,Slc7a11 and Gpx4 were detected by Western blot.Results Qiling Fuzheng Qingjie granules could inhibit the proliferation of HepG2 cells,and 5mg/ml Qiling Fuzheng Qingjie granules had the most obvious inhibitory effect(P<0.05);②Compared with the blank group and Fer-1+Qiling group,ROS and Fe2+in Qiling group were increased(P<0.05),the expression levels of Ampk,p-Ampk,P53,Beclin1,Acsl4 protein,ACSL4,P53 mRNA and p53 fluorescence sig⁃nal intensity were increased(P<0.05),Slc7a11 and Gpx4 protein levels,GPX4 mRNA and fluorescence intensity decreased(P<0.05);ROS and Fe^(2+)were decreased in Fer-1 group(P<0.05),the levels of Ampk,p-Ampk,P53,Beclin1,Acsl4 protein,ACSL4,P53 mRNA and p53 fluorescence signal intensity were decreased(P<0.05),Slc7a11 and Gpx4 protein levels,GPX4 mRNA and fluorescence intensity increased(P<0.05).Conclusion Qiling Fuzheng Qingjie granules can induce ferropto⁃sis in HepG2 cells by regulating P53/AMPK signaling pathway.