丹参酮ⅡA通过Nrf2信号通路抑制肝组织铁死亡对非酒精性脂肪肝大鼠肝脏的保护作用

TanshinoneⅡA inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non⁃alcoholic fatty liver disease

探讨丹参酮ⅡA(tanshinoneⅡA,TSⅡA)对非酒精性脂肪肝(non⁃alcoholic fatty liver disease,NAFLD)大鼠肝脏的保护作用,及其通过核因子E2相关因子2(nuclear factor E2⁃related factor 2,Nrf2)信号通路对肝细胞铁死亡的调控机制。通过高脂饲料连续喂养12周构建NAFLD大鼠模型,造模成功后分为模型组,TSⅡA低、高剂量组,抑制剂组,另设对照组。通过酶联免疫吸附试剂盒检测血清超氧化物歧化酶(superoxide dismutase,SOD)和脂质过氧化产物丙二醛(malondialdehyde,MDA)含量;通过生化分析检测血清中天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、丙氨酸氨基转移酶(alanine aminotransfe⁃rase,ALT)、总胆固醇(total cholesterol,TC)、甘油三酯(triglycerides,TG)含量;苏木素⁃伊红(HE)染色检测肝组织病理损伤;通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal⁃deoxynucleoitidyl transferase⁃mediated nick end labeling,TUNEL)试剂盒检测肝组织的细胞凋亡;通过油红O染色检测各组大鼠肝组织脂质沉积;通过MitoSOX染色检测各组大鼠肝组织中氧自由基(reactive oxygen species,ROS)含量;通过普鲁士蓝染色检测肝组织中铁沉积;通过Western blot法检测肝组织中Nrf2、血红素加氧酶⁃1(heme oxygenase⁃1,HO⁃1)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、铁死亡抑制蛋白1(ferroptosis suppressor protein 1,FSP1)、B淋巴细胞瘤⁃2(B cell lymphoma⁃2,Bcl⁃2)和Bcl⁃2相关X蛋白(Bcl⁃2 associated X protein,Bax)的表达。结果显示,TSⅡA能明显降低大鼠血清中MDA、AST、ALT、TC和TG含量,升高SOD活性;下调肝组织细胞凋亡率,下调组织脂质沉积、ROS含量以及铁沉积;上调肝组织Nrf2、HO⁃1、FSP1、GPX和Bcl⁃2的表达,抑制肝组织Bax的表达;而抑制剂(ML385)可部分逆转TSⅡA对肝组织的保护作用。综上,TSⅡA能抑制大鼠NAFLD后肝细胞的铁死亡,下调肝组织ROS含量,降低肝组织中脂质蓄积,这可能与激活Nrf2信号通路有关。

This study investigated the protective effect of tanshinoneⅡA(TSⅡA)on the liver in the rat model of non⁃alcoholic fatty liver disease(NAFLD)and the mechanism of TSⅡA in regulating ferroptosis via the nuclear factor E2⁃related factor 2(Nrf2)signaling pathway.The rat model of NAFLD was established with a high⁃fat diet for 12 weeks.The successfully modeled rats were assigned into model group,low⁃and high⁃dose TSⅡA groups,and inhibitor group,and normal control group was set.Enzyme⁃linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD)and malondialdehyde(MDA)in the serum of rats in each group.A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST),alaninl aminotransferase(ALT),total cholesterol(TC),and triglycerides(TG).Hematoxylin⁃eosin(HE)staining was used to detect pathological damage in liver tissue.Terminal⁃deoxynucleoitidyl transferase⁃mediated nick end labeling(TUNEL)was employed to examine the apoptosis of the liver tissue.Oil red O staining,MitoSOX staining,and Prussian blue staining were conducted to reveal lipid deposition,the content of reactive oxygen species(ROS),and iron deposition in liver tissue.Western blot was employed to determine the expression of Nrf2,heme oxygenase⁃1(HO⁃1),glutathione peroxidase 4(GPX4),ferroptosis suppressor protein 1(FSP1),B cell lymphoma⁃2(Bcl⁃2),and Bcl⁃2 associated X protein(Bax)in the liver tissue.The result showed that TSⅡA significantly reduced the content of MDA,AST,ALT,TC,and TG in the serum,increased the activity of SOD,decreased the apoptosis rate,lipid deposition,ROS,and iron deposition in the liver tissue,up⁃regulated the expression of Nrf2,HO⁃1,FSP1,GPX,and Bcl⁃2,and inhibited the expression of Bax in the liver tissue of NAFLD rats.However,ML385 partially reversed the protective effect of TSⅡA on the liver tissue.In conclusion,TSⅡA could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.