逍遥散及其拆方对Erastin诱导的LO-2肝细胞系铁死亡的影响

Effects of Xiaoyao Powder and its disassembled formulas on Erastininduced ferroptosis in hepatocyte LO-2 cell line

目的 探讨逍遥散及其拆方对Erastin诱导肝细胞铁死亡的影响及机制。方法 使用铁死亡诱导剂Erastin诱导肝细胞LO-2铁死亡,用逍遥散及各功效拆方(逍遥散去疏肝药组、逍遥散去健脾药组、逍遥散去养血药组)的含药血清进行干预,阳性对照药使用铁死亡抑制剂Ferrostatin-1。通过CCK-8检测细胞活力,ELISA法检测细胞上清液中谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)水平,比色法检测细胞中丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(reduced glutathione,GSH)、Fe^(2+)等含量,荧光酶标仪检测脂质过氧化活性氧(reactive oxygen species,ROS),RT-PCR检测细胞中谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)、铁调素(hepcidin)、膜铁转运蛋白(ferroportin,FPN1)mRNA表达,Western blot检测细胞中骨形态发生蛋白6(bone morphogenetic protein6,BMP6)、铁调素调节蛋白(hemojuvelin,HJV)、Smad同源物4(mothers against decapentaplegic homolog 4,SMAD4)蛋白表达。结果 erastin诱导肝细胞铁死亡后,细胞活力较对照组显著下降(P<0.05),细胞外上清液中ALT、AST含量显著上升(P<0.05),细胞中MDA、ROS、Fe^(2+)、PTGS2与hepcidin mRNA表达及BMP6/HJV/Smad4信号通路蛋白显著上升(P<0.05),GSH含量及GPX4、FPN1 mRNA表达显著下降(P<0.05)。除逍遥散去养血药组在Smad4蛋白表达方面与模型组无显著差异(P>0.05),逍遥散及各功效拆方及阳性对照药Ferrostatin-1均能显著逆转上述指标的变化(P<0.05)。与整方组疗效比较,逍遥散去疏肝药组AST、MDA、GSH、ROS、Fe^(2+)、BMP6、hepcidin、FPN1指标具有显著差异(P<0.05);除逍遥散去健脾药组在Smad4蛋白表达方面与整方组无显著差异(P>0.05),逍遥散去健脾药组及逍遥散去养血药组对所检测指标均有显著改善(P<0.05)。结论 逍遥散可抑制Erastin诱导的肝细胞铁死亡,其机制可能与调控BMP6/HJV/SMAD4信号通路及其下游hepcidin-ferroportin轴有关,而逍遥散各功效拆方均在一定程度上参与了逍遥散抑制肝细胞铁死亡的过程。

Objective To explore the effects of Xiaoyao Powder and its disassembled formulas on Erastin-induced ferroptosis in hepatocytes and the related mechanism.Methods Ferroptosis of hepatocyte LO-2 was induced by Erastin and intervened by the medicated serum of Xiaoyao Powder and its disassembled formulas(Xiaoyao Powder without liver-soothing medicines,Xiaoyao Powder without spleen-strengthening medicines,Xiaoyao Powder without blood-nourishing medicines).Ferroptosis inhibitor Ferrostatin-1 was used as the positive control drug.The cell viability was evaluated by CCK-8.The levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the cell supernatant were examined by ELIS A,and the content of malondialdehyde(MDA),reduced glutathione(GSH),and Fe~(2+) in the cells were determined by colorimetry.Fluorescence enzyme-linked immunosorbent assay was used to check lipid peroxidation-related reactive oxygen species(ROS),RT-PCR was used to examine the mRNA expressions of glutathione peroxidase 4(GPX4),prostaglandin-endoperoxide synthase 2(PTGS2),hepcidin,and ferroportin(FPN1) in the cells,and Western blot was used to check the expressions of bone morphogenetic protein 6(BMP6),hemojuvelin(HJV),and mothers against decapentaplegic homolog 4(Smad4) protein in the cells.Results After ferroptosis induced by Erastin,the viability of hepatocytes decreased significantly compared with the control group(P<0.05);the content of ALT and AST in the extracellular supernatant increased significantly(P<0.05);the contents of MDA,ROS,and Fe~(2+),the mRNA expressions of PTGS2 and hepcidin,and the protein level of BMP6/HJV/Smad4 signaling pathway in the cells increased significantly(P<0.05),while the content of GSH and the m RNA expressions of GPX4 and FPN1 decreased significantly(P<0.05).Except for the group of Xiaoyao Powder without blood-nourishing medicines showing no significant difference in Smad4 protein expression compared with the model group(P>0.05),groups of Xiaoyao Powder and its disassembled formulas,as well as the positive control drug Ferrostatin-1 showed significant reversed changes in the aforementioned indicators(P<0.05).There were significant differences in AST,MDA,GSH,ROS,Fe~(2+),BMP6,hepcidin and FPN1between Xiaoyao Powder without liver-soothing medicines group and Xiaoyao Powder group(P <0.05).Compared with Xiaoyao Powder,the group of Xiaoyao Powder without spleen-strengthening medicines showed no significant difference in Smad4 protein expression(P >0.05),while both groups of Xiaoyao Powder without spleen-strengthening medicines and Xiaoyao Powder without blood-nourishing medicines showed significant differences in the detected indicators(P <0.05).Conclusion Xiaoyao Powder can inhibit Erastin-induced ferroptosis in hepatocytes,and its mechanism may be related to the regulation of BMP6/HJV/Smad4 signal pathway and its downstream hepcidin-ferroportin axis.To some extent,each disassembled formula of it involves in the process of inhibiting ferroptosis in hepatocytes.