基于“肝病实脾”理论探讨逍遥散对肝纤维化大鼠肝星状细胞铁死亡的影响  

Effect of Xiaoyao powder on ferroptosis of hepatic stellate cells in rats with liver fibrosis based on the theory of “treating liver by nourishing spleen”

作　　者：胡哲君 汤穆浛 张锐 金泓 严伦 何鸿志 曾凤兰 周志鹏 HU Zhejun;TANG Muhan;ZHANG Rui;JIN Hong;YAN Lun;HE Hongzhi;ZENG Fenglan;ZHOU Zhipeng(Guangzhou Red Cross Hospital,Guangzhou 510220,China)

机构地区：[1]广州市红十字会医院,广东广州510220

出　　处：《陕西中医》2024年第2期159-164,170,共7页Shaanxi Journal of Traditional Chinese Medicine

基　　金：广东省医学科学技术研究基金资助项目(A2020178);广州市卫生健康项目(2022A010034)。

摘　　要：目的:基于“肝病实脾”理论探讨逍遥散对肝纤维化大鼠肝星状细胞铁死亡的影响。方法:采用腹腔注射四氯化碳的方法建立肝纤维化大鼠模型,随机分为模型组、逍遥散组、水飞蓟宾葡甲胺组,每组12只,另选12只SD大鼠腹腔注射等剂量玉米油设为对照组,以逍遥散、水飞蓟宾葡甲胺分组处理后检测各组大鼠肝功能指标天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT);检测各组大鼠肝指数并以Masson染色检测各组大鼠肝纤维化,比较其肝胶原容积分数(CVF);以免疫组织化学染色检测各组大鼠肝星状细胞活化,比较其肝组织α-SMA阳性表达;通过16SrRNA基因测序检测各组大鼠肠道菌群改变。分离培养上述各组大鼠原代肝星状细胞(HSC),以试剂盒测定各组大鼠HSC铁死亡指标:铁含量、谷胱甘肽(GSH)及丙二醛(MDA)水平;以免疫印迹法检测各组大鼠HSC铁死亡标志蛋白[长链脂酰辅酶A合成酶4(ACSL4)、环加氧酶2(PTGS2)、谷胱甘肽过氧化酶4(GPX4)]表达。结果:与对照组相比,模型组大鼠拟杆菌目丰度、HSC铁含量、MDA水平、ACSL4与PTGS2蛋白表达均明显降低(P<0.05),血清AST与ALT水平、肝指数、肝CVF、肝组织α-SMA阳性比、ACE指数、梭菌目丰度、HSC中GSH水平与GPX4蛋白表达均明显升高(P<0.05);与模型组相比,逍遥散组、水飞蓟宾葡甲胺组大鼠拟杆菌目丰度、HSC铁含量、MDA水平、ACSL4与PTGS2蛋白表达均升高(P<0.05),血清AST与ALT水平、肝指数、肝CVF、肝组织α-SMA阳性比、ACE指数、梭菌目丰度、HSC中GSH水平与GPX4蛋白表达均降低(P<0.05);与逍遥散组相比,水飞蓟宾葡甲胺组大鼠各指标差异无统计学意义(P>0.05)。结论:逍遥散可通过诱导肝星状细胞铁死亡而抑制其活性,进而减轻肝纤维化大鼠肝组织肝纤维化,减轻其肝功能损伤,最终对大鼠发挥明显肝保护作用。Objective:To explore the effect of Xiaoyao powder on ferroptosis of hepatic stellate cells in rats with liver fibrosis based on the theory of “treating liver by nourishing spleen”.Methods:Rats model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride,and were randomly grouped into model group,Xiaoyao powder group,and Silibin meglumine group,with 12 rats in each group,another 12 SD rats were collected as the control group by intraperitoneal injection of equal doses of corn oil,after grouping and treating with Xiaoyao powder and Silibin meglumine,liver function indicators such as aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were detected in each group;the liver index of rats in each group was detected,liver fibrosis was detected using Masson staining,and their liver collagen volume fraction(CVF) was compared;the activation of hepatic stellate cells in rats of each group was detected using immunohistochemical staining,and the positive expression of α-SMA in their liver tissues was compared;the changes in gut microbiota of rats in each group was detected through 16SrRNA gene sequencing.The primary hepatic stellate cells(HSCs) of rats in the aforementioned groups were isolated and cultured,and the kits were applied to determine the iron death indicators of HSCs in each group:iron content,glutathione(GSH),and malondialdehyde(MDA) levels;immunoblotting was applied to detect the expression of long-chain acyl CoA synthetase 4(ACSL4),cyclooxygenase 2(PTGS2),and glutathione peroxidase 4(GPX4) in HSC iron death marker proteins of rats in each group.Results:Compared with the control group,the abundance of Bacteroidei,HSC iron content,MDA level,ACSL4 and PTGS2 protein expression in the model group were significantly reduced(P<0.05),the levels of serum AST and ALT,liver index,liver CVF,α-SMA positive ratio in liver tissue,ACE index,abundance of Clostridium,GSH level in HSC,and GPX4 protein expression were significantly increased(P<0.05);compared with the model group,

关 键 词：肝纤维化 肝病实脾 逍遥散 肝星状细胞 铁死亡 大鼠 

分 类 号：R256.4[医药卫生—中医内科学]