摘要
目的:基于核因子红系2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)轴探讨大黄黄连泻心汤加味对2型糖尿病(T2DM)大鼠肝脏氧化应激损伤的影响及其作用机制。方法:6只ZDF(fa/+)大鼠为正常组,30只ZDF(fa/fa)大鼠为造模组,采用高脂饲料喂食诱导建立T2DM大鼠模型,造模成功后随机分为模型组、二甲双胍组(0.18 g·kg^(-1))及大黄黄连泻心汤加味低、中、高剂量组(0.54、1.08、2.16 g·kg^(-1)),每组6只。药物干预12周后测定大鼠体质量、肝质量、空腹血糖(FBG)、口服葡萄糖耐量试验(OGTT)水平、全自动生化仪检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,苏木素-伊红(HE)染色观察肝脏组织病理形态变化,酶联免疫吸附测定法(ELISA)检测肝脏组织超氧化物歧化酶(SOD)、活性氧(ROS)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)水平,免疫组化检测肝脏Nrf2的表达,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测肝脏组织Nrf2、HO-1 mRNA及蛋白表达水平。结果:与正常组比较,模型组大鼠体质量、肝质量、肝指数均显著升高(P<0.01);与模型组比较,二甲双胍组及大黄黄连泻心汤加味中、高剂量组大鼠体质量、肝质量、肝指数均显著降低(P<0.01)。与正常组比较,模型组大鼠TC、TG、LDL含量均显著升高(P<0.01),HDL含量显著降低(P<0.01);与模型组比较,二甲双胍及大黄黄连泻心汤加味各剂量组TC含量均显著降低(P<0.01),二甲双胍及大黄黄连泻心汤加味各剂量组TG含量均明显降低(P<0.05),大黄黄连泻心汤加味中、高剂量组LDL含量明显降低(P<0.05),二甲双胍及大黄黄连泻心汤加味各剂量组HDL含量明显升高(P<0.05)。与正常组比较,模型组大鼠ALT、AST活性显著升高(P<0.01);与模型组比较,二甲双胍及大黄黄连泻心汤加味各剂量组ALT活性均明显降低(P<0.05),AST活性均显著降低(P<0.01)。与正常组比较,所有时间点模型组大鼠FBG显著升高(P<0.01);与模型组比较,8、10、12周二甲双胍及大黄黄连泻心汤加味各剂量组FBG明显降低。OGTT结果显示,与正常组比较,所有时间点的模型组大鼠血糖均显著升高(P<0.01);与模型组比较,所有时间点的二甲双胍组血糖显著降低(P<0.01),90、120 min大黄黄连泻心汤加味中、高剂量组血糖均显著降低(P<0.01)。HE病理显示,正常组大鼠肝细胞结构清晰,排列规律;模型组大鼠肝细胞组织紊乱,可见脂肪空泡,大量细胞出现变形坏死状态;二甲双胍及大黄黄连泻心汤加味各剂量组大鼠肝组织结构转好,少量坏死细胞。与正常组比较,模型组大鼠肝脏SOD、GSH-Px显著降低(P<0.01),肝脏ROS、MDA显著升高(P<0.01);与模型组比较,二甲双胍及大黄黄连泻心汤加味各剂量组大鼠肝脏SOD、GSH-Px显著升高(P<0.01),MDA显著降低(P<0.01),二甲双胍及大黄黄连泻心汤加味中、高剂量组大鼠肝脏ROS明显降低(P<0.05)。与正常组比较,模型组大鼠肝脏Nrf2、HO-1 mRNA表达显著降低(P<0.01);与模型组比较,二甲双胍及大黄黄连泻心汤加味中、高剂量组大鼠肝脏Nrf2、HO-1mRNA表达明显升高(P<0.05)。免疫组化结果显示,与正常组比较,模型组大鼠肝脏Nrf2、HO-1阳性表达明显降低(P<0.05);与模型组比较,二甲双胍及大黄黄连泻心汤加味各剂量组大鼠肝脏Nrf2、HO-1阳性表达升高,细胞核周围棕黄色颗粒明显增多(P<0.05)。Weatern blot结果显示,与正常组比较,模型组大鼠肝脏Nrf2、HO-1蛋白表达显著降低(P<0.01);与模型组比较,二甲双胍及大黄黄连泻心汤加味各剂量组大鼠肝脏Nrf2、HO-1蛋白表达显著升高(P<0.01)。结论:大黄黄连泻心汤加味能明显改善T2DM大鼠一般情况及肝脏组织的病理学变化,可能通过调控Nrf2/HO-1轴改善肝脏氧化应激损伤。
Objective:To explore the effects and mechanisms of modified Dahuang Huanglian Xiexintang on hepatic oxidative stress injury in type 2 diabetes mellitus(T2DM)rats based on the nuclear factor erythroid 2 related factor 2(Nrf2)/heme oxygenase-1(HO-1)axis.Method:Six ZDF(fa/+)rats were as assigned to the blank group,and 30 ZDF(fa/fa)rats were used to induce the T2DM model by feeding a high-fat diet.After successful modeling,the rats were randomly divided into the model group,metformin group(0.18 g·kg^(-1)),and low,medium,and high dose groups of modified Dahuang Huanglian Xiexintang(0.54,1.08,2.16 g·kg^(-1)),with six rats in each group.After 12 weeks of drug intervention,the body mass,liver mass,fasting blood glucose(FBG),and oral glucose tolerance test(OGTT)levels were measured.Serum total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL),low-density lipoprotein cholesterol(LDL),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)levels were detected using an automatic biochemical analyzer.The pathological changes of liver tissue were observed by hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect the activity of superoxide dismutase(SOD),reactive oxygen species(ROS),glutathione peroxidase(GSH-Px),and the level of malondialdehyde(MDA)in liver tissues.Immunohistochemistry was used to detect the expression of Nrf2 in the liver.Real time quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect the mRNA and protein expression levels of Nrf2 and HO-1 in liver tissues.Result:Compared with the normal group,the model group showed a significant increase in body mass,liver mass,and liver index(P<0.01).Compared with the model group,the metformin group and the medium and high dose groups of modified Dahuang Huanglian Xiexintang showed a significant decrease in body weight,liver mass,and liver index(P<0.01).Compared with the normal group,the model group showed significantly increased TC,TG,and LDL levels(P<0.01),and significantly decreased HDL levels(P<0.01).Compared with the model group,the metformin group and all doses of modified Dahuang Huanglian Xiexintang showed significantly reduced TC levels(P<0.01),and significantly reduced TG levels(P<0.05).The medium and high dose groups of modified Dahuang Huanglian Xiexintang showed significantly reduced LDL levels(P<0.05).The metformin group and all doses of modified Dahuang Huanglian Xiexintang showed significantly increased HDL levels(P<0.05).Compared with the normal group,the model group showed significantly increased ALT and AST activities(P<0.01).Compared with the model group,all doses of modified Dahuang Huanglian Xiexintang and the metformin group showed significantly reduced ALT activities(P<0.05)and significantly reduced AST activities(P<0.01).Compared with normal group,the model group showed significantly increased FBG at all time points(P<0.01).Compared with the model group,the metformin group and all doses of modified Dahuang Huanglian Xiexintang showed significantly reduced FBG at 8,10,12 weeks.The OGTT results showed that compared with the normal group,the model group had significantly increased blood glucose at all time points(P<0.01).Compared with the model group,the metformin group showed significantly reduced blood glucose at all time points(P<0.01),and the medium and high dose groups of modified Dahuang Huanglian Xiexintang showed significantly reduced blood glucose at 90,120 min(P<0.01).HE pathology showed clear and regular liver cell structure in the normal group,while the model group showed disordered liver cell structure with visible fat vacuoles and a large number of deformed necrotic cells.The liver tissue structure improved in the metformin group and all doses of modified Dahuang Huanglian Xiexintang,with fewer necrotic cells.Compared with the normal group,the model group showed significantly reduced SOD and GSH-Px levels(P<0.01),and significantly increased ROS and MDA levels(P<0.01).Compared with the model group,the metformin group and all doses of modified Dahuang Huanglian Xiexintang showed significantly increased SOD and GSH-Px levels(P<0.01),and significantly reduced MDA levels(P<0.01).The medium and high dose groups of modified Dahuang Huanglian Xiexintang showed significantly reduced ROS levels(P<0.05).Compared with the normal group,the model group showed significantly reduced Nrf2 and HO-1 mRNA expression levels(P<0.01).Compared with the model group,the metformin group and the medium and high dose groups of modified Dahuang Huanglian Xiexintang showed significantly increased Nrf2 and HO-1 mRNA expression levels(P<0.05).Immunohistochemistry showed that compared with the normal group,the model group had significantly reduced positive expression of Nrf2 and HO-1(P<0.05).Compared with the model group,the metformin group and all doses of modified Dahuang Huanglian Xiexintang showed increased positive expression of Nrf2 and HO-1,with a significant increase in brown-yellow granules around the cell nucleus(P<0.05).Western blot results showed that compared with the normal group,the model group had significantly reduced protein expression of Nrf2 and HO-1(P<0.01).Compared with the model group,the metformin group and all doses of modified Dahuang Huanglian Xiexintang showed significantly increased protein expression of Nrf2 and HO-1(P<0.01).Conclusion:Modified Dahuang Huanglian Xiexintang can significantly improve the general condition and pathological changes of liver tissues in T2DM model rats.This improvement is likely achieved through ameliorating hepatic oxidative stress injury via regulating the Nrf2/HO-1 axis.
作者
马成军
闫丰喆
杨丽霞
梁永林
朱向东
安冬
高艳奎
MA Chengjun;YAN Fengzhe;YANG Lixia;LIANG Yonglin;ZHU Xiangdong;AN Dong;GAO Yankui(Gansu University of Chinese Medicine,Lanzhou 730000,China;Shaoxing People's Hospital,Shaoxing 312035,China;Ningxia Medical University,Yinchuan 750004,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第24期121-130,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
甘肃省教育厅产业支撑项目(2021CYZC-03)
甘肃省优秀博士生项目(23JRRA1218)
国家自然科学基金项目(82060914)。
作者简介
第一作者:马成军,在读硕士,从事中医药预防糖尿病的研究,E-mail:542715053@qq.com;通信作者:杨丽霞,博士,主任医师,硕士生导师,从事中医药防治糖尿病的研究,E-mail:yanglixia-415@163.com。
