铁死亡参与四氯化碳致肝纤维化发病过程的研究

目的 探讨铁死亡是否参与CCl4致肝纤维化发病过程。方法 小鼠分为对照组和CCl4组。采用腹腔注射法造模，每隔2 d给药1次，共8次，造模完成后隔2 d处死。通过HE、MASSON和天狼星红染色评价CCl4致小鼠肝损伤及纤维化情况；铁离子检测试剂盒检测小鼠肝组织铁离子含量；MDA检测试剂盒检测小鼠肝组织MDA含量;免疫荧光法观察小鼠肝组织中MDA含量；谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)评价小鼠肝组织脂质过氧化情况；通过Western Blot和RT - qPCR评价谷胱甘肽过氧化物酶4(GPX4)、铁蛋白(Ferritin)的蛋白和mRNA表达水平。结果 通过HE染色、MASSON染色和天狼星红染色发现，CCl4引起小鼠肝细胞排列紊乱，炎症细胞浸润，纤维化隔片形成。而对照组没有显著的病理变化；CCl4组小鼠肝组织铁离子含量(2.83±0.81) μmol/g较对照组小鼠肝组织铁离子含量(1.54±0.19) μmol/g增加(t = 5.415，P＜0.001)；CCl4组MDA荧光强度高于对照组；CCl4组MDA含量(0.74±0.13) nmol/mg较对照组MDA含量(0.21±0.06) nmol/mg增加(t = 15.650，P＜0.001)；GSH/GSSG在CCl4组(1.29±0.19)较对照组(1.90±0.16)降低(t = 10.580，P＜0.001)；在CCl4组GPX4蛋白相对表达水平(0.27±0.07)较对照组(0.42±0.10)降低(t = 3.000，P = 0.013)。CCl4组Ferritin蛋白相对表达水平(0.18±0.06)较对照组(0.30±0.06)降低(t = 3.571，P = 0.005)；CCl4组Ferritin mRNA相对表达水平(1.83±0.60)较对照组(0.84±0.30)降低(t = 4.420，P＜0.001)。CCl4组GPX4 mRNA相对表达水平(1.10±0.45)较对照组(3.54±0.87)降低(t = 7.385，P＜0.001)。结论 铁死亡参与了四氯化碳致肝纤维化的发病过程。

Study on the role of ferroptosis in the pathogenesis of liver fibrosis induced by carbon tetrachloride

Objective To explore whether ferroptosis is involved in the pathogenesis of liver fibrosis induced by CCl4. Methods Mice were divided into control group and CCl4 group. The model was formed by intraperitoneal injection, and the drug was given every 2 days for a total of 8 times. The mice were killed every 2 days after the completion of the model. Evaluation of liver injury and fibrosis induced by CCl4 in mice was done by HE, MASSON and Sirius red staining, and detection of iron content in mouse liver tissue by iron ion detection kit. The content of MDA in liver tissue of mice was detected by MDA detection kit and immunofluorescence. The protein and mRNA expression of glutathione peroxidase 4 (GPX4) and ferritin were evaluated by Western Blot and RT-qPCR. Results Through HE staining, MASSON staining and Sirius red staining, it was found that CCl4 caused disordered arrangement of hepatocytes, infiltration of inflammatory cells and formation of fibrotic septum. There were no significant pathological changes in the control group. The iron content in liver tissue of mice in CCl4 group [(2.83±0.81) μmol/g] was significantly higher than that in control group [(1.54±0.19) μmol/g] (t=5.415, P<0.001). The fluorescence intensity of MDA in CCl4 group was significantly higher than that in control group. The content of MDA in CCl4 group [(0.74±0.13) nmol/mg] was significantly higher than that in control group [(0.21±0.06) nmol/mg] (t=15.650, P＜0.001). GSH/GSSG in CCl4 group (1.29±0.19) was significantly lower than that in control group (1.90±0.16) (t=10.580, P＜0.001). The relative expression level of GPX4 protein in CCl4 group (0.27±0.07) was significantly lower than that in control group (0.42±0.10) (t=3.000, P=0.013). The relative expression level of Ferritin protein in CCl4 group (0.18±0.06) was significantly lower than that in control group (0.30±0.06) (t=3.571, P=0.005). The relative expression level of Ferritin mRNA in CCl4 group (1.83±0.60) was significantly lower than that in control group (0.84±0.30) (t=4.420, P＜0.001). The relative expression level of GPX4 mRNA in CCl4 group (1.10±0.45) was significantly lower than that in control group (3.54±0.87) (t=7.385, P＜0.001). Conclusion Ferroptosis is involved in the pathogenesis of liver fibrosis induced by carbon tetrachloride.