木犀草素通过下调CISD1抑制铁死亡参与蒙药德都古日古木-7治疗慢性肝损伤的机理研究

If chronic liver disease isn’t treated in time, it can induces liver fibrosis, cirrhosis and liver cancer. Traditional Mongolian Medicine Dedugurigumu-7 can protect the liver mitochondrial enenrgy metabolism trough its anti-lipid peroxidation function to play role in protecting CCl4 induced chronic liver injury. Our research found that the luteolin - a composition of Dedugurigumu-7 reduced the CISD1 and regulating the ferroptosis. In this research, the ferroptosis inhibition function and protecting effect through CISD1 regulation of Dedugurigumu-7 is verified. The cell model is used to verify the ferroptosis regulating function of luteolin; research the roles of CISD1 in ferroptosis and chronic liver disease inhibition. The expressions of DMT1, GPX4, FtMt protein and mRNA, and the expressions of CISD1 protein and mRNA in hepatic tissue were detected by WB and QPCR. The iron deposition and iron content in hepatocytes were detected by immunofluorescence. The expression of DMT1 and GPX4 was detected by immunofluorescence.The activities of MDA, CSH and GPX4 in the mitochondria of liver were measured by the method of JC-1. The mitochondrial ATP, AMP and ADP contents were detected by JC-1 assay. To provide a scientific basis for Mongolian medicine Dedugurigumu-7 treatment of chronic liver injury.

慢性肝病不及时治疗可诱发肝纤维化，并发肝硬化、肝癌危机生命。蒙药德都古日古木-7通过抗脂质过氧化，保护肝线粒体能量代谢，起到保护CCl4所致慢性肝损伤作用。研究发现该方和成分木犀草素可下调CISD1调节铁死亡。本课题通过动物模型验证该方抑制肝细胞铁死亡，调节CISD1保护肝损伤。细胞模型研究木犀草素调节肝细胞铁死亡作用；研究CISD1调控铁死亡功能，并探讨木犀草素通过CISD1调控铁死亡，抑制慢性肝损伤。WB和QPCR法检测肝组织铁死亡指标DMT1、GPX4、FtMt蛋白和mRNA表达、CISD1蛋白和mRNA表达；检测肝细胞内铁沉积和铁含量；免疫荧光检测铁死亡指标DMT1、GPX4蛋白表达定位；Elisa法测定肝线粒体MDA、CSH、GPX4活性；JC-1法检测肝线粒体膜电位变化和发光法测定线粒体ATP、AMP、ADP含量。为蒙药德都古日古木-7治疗慢性肝损伤提供科学研究依据。

慢性肝病不及时治疗可诱发肝纤维化，并发肝硬化、肝癌危机生命。德都古日古木-7味散（德都红花-7味散）具有明显的保肝降酶作用。本课题通过动物模型验证德都红花-7味散抑制肝细胞铁死亡，调节CISD1保护肝损伤。细胞模型研究木犀草素调节肝细胞铁死亡作用；研究CISD1调控铁死亡功能，并探讨木犀草素通过CISD1调控铁死亡，抑制慢性肝损伤。动物实验结果表明德都红花-7味散组中央静脉、汇管区周围及肝实质内肝细胞颗粒变性明显轻于模型组；小叶结构紊乱，多见纤维间隔形成与模型组比较较轻；与模型组比较德都红花-7味散组普鲁士蓝染色面积百分比下降，胶原面积下降，差异有统计学意义（P＜0.05）；与模型组比较德都红花-7味散组Bcl-x、Bcl-2蛋白表达下降，Bcl-2 、CISD1、DMT1mRNA表达下降，GPX4、FTMTmRNA表达升高，差异有统计学意义（P＜0.05）；木犀草素对细胞铁死亡的调控的研究结果表明：组织铁试剂盒检测铁含量，CCl4作用能引起组织铁含量、MDA含量的升高，GSH含量降低（P＜0.01），木犀草素降低细胞铁含量、MDA，升高T-GSH（P＜0.01）；WB检测模型组CISD1蛋白表达升高、FTMT、GXP4表达降低：与空白组比较模型组表达升高，木犀草素降低CISD1、升高FTMT、GXP4蛋白表达。 CISD1功能回复实验结果：表明过表达CISD1后降低了木犀草素对损伤细胞的恢复作用，CISD1基因的表达量和铁死亡相关基因DMT1、FTMT、GPX4、SLC7A11基因的表达水平相关，结果说明CISD1和铁死亡相关；CISD1基因的表达量和细胞损伤情况是正相关，同时表明过表达CISD1能降低木犀草素对损伤细胞的恢复作用。CISD1的表达水平能影响铁、T-GSH、MDA含量，木犀草素能减低铁的积累，促进T-GSH含量的积累抑制MDA含量的积累。通过本项目研究表明，上调 CISD1 可导致肝细胞铁死亡。蒙药德都古日古木-7 及成分木犀草素可通过下调 CISD1 抑制肝细胞铁死亡起到防治慢性肝损伤的作用。