葛根芩连汤对2型糖尿病合并非酒精性脂肪肝大鼠模型铁死亡干预作用研究

Effect of Gegen Qinlian Decoction on the intervention of ferroptosis in rat model with T2DM combined with NAFLD

目的探讨葛根芩连汤对2型糖尿病合并非酒精性脂肪肝大鼠模型铁死亡途径的影响。方法30只SD大鼠随机抽取10只作为正常组,不干预,剩余20只大鼠给予高脂饲料喂养联合腹腔注射2次链脲佐菌素(streptozotocin,STZ)建立2型糖尿病合并非酒精性脂肪肝大鼠模型。最后将16只成模大鼠随机分为模型组和中药组,每组各8只。中药组给予葛根芩连汤4.095 g/kg灌胃,正常组和模型组以等量生理盐水灌胃,连续给药3周。给药结束后,检测各组大鼠空腹血糖;检测血清中肝功能相关指标天门冬氨酸氨基转氨酶(aspartate transaminase,AST)和丙氨酸氨基转氨酶(alanine aminotransferase,ALT);全自动生化分析仪检测血清超氧化物歧化酶(superoxide dismutase,SOD)活性;苏木精—伊红(hematoxylin-eosin,HE)染色观察肝脏组织病理变化;Western Blot和实时荧光定量PCR(RT-qPCR)检测谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、酰基辅酶A合成酶长链家族成员4(Acyl-CoA synthetase long chain family member 4,ACSL4)的蛋白和mRNA表达。结果(1)血糖和体质量检测结果:与正常组比较,模型组大鼠体质量下降,而血糖明显升高;与模型组比较,中药组大鼠体质量回升,而血糖明显下降(P<0.05)。(2)HE染色结果:与正常组比较,模型组肝索排列较为紊乱,小叶结构破坏明显,肝血窦扩大,肝细胞出现重度空泡变性;与模型组比较,中药组肝组织外形无明显增大,肝索排列有所改善,肝细胞脂肪变性明显减轻,脂滴空泡现象减少,大部分肝细胞接近正常形态。(3)生化检测结果:与正常组比较,模型组大鼠血清中ALT和AST水平升高(P<0.05),SOD水平降低(P<0.05);与模型组比较,中药组大鼠血清中ALT和AST水平降低(P<0.05),SOD水平升高(P<0.05)。(4)Western Blot和PCR(qRT-PCR)检测结果:与正常组比较,模型组大鼠肝组织的ACSL4的蛋白和mRNA表达增加(P<0.05),GPX4的蛋白和mRNA表达减少(P<0.05);与模型组比较,中药组大鼠肝组织的ACSL4蛋白和mRNA表达减少(P<0.05),GPX4的蛋白和mRNA表达增加(P<0.05)。结论葛根芩连汤明显改善2型糖尿病合并非酒精性脂肪肝大鼠肝损伤,其保护作用机制与调控氧化应激因子SOD、铁死亡相关因子ACSL4和GPX4表达水平从而抑制铁死亡有关。

Objective To investigate the effect of Gegen Qinlian Decoction on the pathway of ferroptosis in rat models of type 2 diabetic mellitus combined with nonalcoholic fatty liver disease.Methods 30 SD rats were randomly divided into three groups:the normal group,the model group,and the traditional Chinese medicine group.Rats in the normal group were not intervened,and the remaining two groups were given a high-fat diet combined with 2 intraperitoneal injections of streptozotocin to establish a T2DM combined NAFLD model.Finally,16 model rats were randomly divided into the model group and the traditional Chinese medicine group,with 8 rats in each group.Rats in the Chinese medicine group was administered Gegen Qinlian Decoction at a dose of 4.095 g/kg by gavage,while rats in the normal group and the model group were administered an equal volume of saline by gavage,with continuous administration for 3 weeks.After the end of administration,the fasting blood glucose of rats was detected.Detection of liver function-related indicators in serum,aspartate transaminase(AST)and alanine aminotransferase(ALT).Automatic biochemical analyzer to detect serum superoxide dismutase(SOD)activity.The method of HE staining was used to observe the histopathological changes of the liver.The methods of Western Western Blot and real-time quantitative polymerase chain reverse PCR(qRT-PCR)were used to detect the protein and mRNA expressions of glutathione Peroxidase 4(GPX 4),acyl-CoA synthetase long-chain family member 4(ACSL4).Results(1)Compared with the normal group,the rats in the model group lost weight and blood glucose was increased significantly.Compared with the model group,the rats in the traditional Chinese medicine group regained body weight and decreased significantly(P<0.05).(2)The results of HE staining showed that compared with the normal group,the model group had a disordered hepatic cord arrangement,obvious destruction of the lobular structure,enlargement of hepatic sinusoids,and severe vacuolar degeneration of hepatocytes.(3)Compared with the model group,the shape of liver tissues in the traditional Chinese medicine group were not increased significantly,the arrangement of liver cords was improved,the steatosis of hepatocytes was significantly decreased,the phenomenon of lipid droplet vacuoles was reduced,and most of the liver cells were close to normal shape.Compared with the normal group,the serum ALT and AST levels in the model group were increased(P<0.05)and SOD levels were decreased(P<0.05).Compared with the model group,the serum levels of ALT and AST in the traditional Chinese medicine group were decreased(P<0.05)and the SOD level were increased(P<0.05).(4)Western blot and qPCR experiments showed that compared with the normal group,the expression of ACSL4 protein and mRNA in rat liver tissues in the model group were increased(P<0.05),and the expression of GPX4 protein and mRNA were decreased(P<0.05).Compared with the model group,the expression of ACSL4 protein and mRNA in rat liver tissues in the traditional Chinese medicine group were decreased(P<0.05),and the expression of GPX4 protein and mRNA were increased(P<0.05).Conclusion Gegen Qinlian Decoction ameliorated liver injury in rats with T2DM combined with NAFLD significantly,and its protective mechanism is related to the regulation of the expression levels of oxidative stress factor SOD,ferroptosis-related factors ACSL4 and GPX4,thereby inhibiting ferroptosis.