大黄黄连泻心汤加味对肥胖2型糖尿病大鼠内脏脂肪线粒体自噬及棕色化影响

Effect of Modified Dahuang Huanglian Xiexintang on Mitochondrial Autophagy and Browning of Visceral Fat in Obese Type 2 Diabetes Mellitus Rats

目的:观察大黄黄连泻心汤加味对肥胖2型糖尿病(T2DM)模型ZDF大鼠内脏脂肪组织线粒体自噬及棕色化的影响。方法:将40只ZDF大鼠高脂饲料诱导为肥胖T2DM模型,随机分为模型组、二甲双胍组(0.18 g·kg^(-1))、大黄黄连泻心汤加味高、中、低剂量组(2.16、1.08、0.54 g·kg^(-1)),每组8只,另取8只ZDF(fa/+)大鼠作为正常组,灌胃体积均为10 mL·kg^(-1),模型组和正常组灌服等体积纯净水,1次/d,持续12周。定期检测大鼠空腹血糖(FBG);干预12周后检测大鼠体质量、附睾脂肪质量、血清中葡萄糖(GLU)、糖化血清蛋白(GSP)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平;采用苏木素-伊红(HE)染色观察附睾脂肪组织病理形态学改变;透射电镜(TEM)观察脂肪细胞线粒体自噬情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测附睾脂肪低氧诱导因子-1α(HIF-1α)、腺病毒E1B19kD相互作用蛋白3(BNIP3)、微管相关蛋白1轻链3B(LC3B)、泛素结合蛋白1(p62)、解偶联蛋白1(UCP1)、碘代甲状腺素2(Dio2)、PR结构域结合因子16(Prdm16)mRNA表达,蛋白免疫印迹法(Western blot)检测附睾脂肪HIF-1α、BNIP3、LC3B、p62及UCP1蛋白表达。结果:与正常组比较,模型组大鼠附睾脂肪出现病理学改变;脂肪细胞线粒体固缩、自噬小体较多并发生线粒体自噬;大鼠体质量、附睾脂肪质量、FBG、GLU、GSP、TC、TG、LDL-C水平显著升高(P<0.01),HDL-C水平显著降低(P<0.01),附睾脂肪HIF-1α、BNIP3、LC3B mRNA及蛋白表达显著升高(P<0.01),p62、UCP1 mRNA及蛋白表达显著降低(P<0.01),Dio2、Prdm16 mRNA表达显著降低(P<0.01)。与模型组比较,各给药组大鼠附睾脂肪组织病变不同程度减轻;二甲双胍组和大黄黄连泻心汤加味高剂量组大鼠脂肪细胞胞浆中线粒体形态结构完整,嵴清晰,基质均匀,胞浆中可见少量自噬溶酶体和自噬小体;二甲双胍组、大黄黄连泻心汤加味高、中剂量组大鼠体质量及附睾脂肪质量均显著降低(P<0.01);各给药组大鼠附睾脂肪指数均明显降低(P<0.05);各给药组大鼠FBG均显著降低(P<0.01);二甲双胍组及大黄黄连泻心汤加味高、中剂量组大鼠血清中GSP、GLU、TG、LDL-C水平均明显降低(P<0.05,P<0.01);二甲双胍组和大黄黄连泻心汤加味高剂量组大鼠血清TC水平显著降低(P<0.01),各给药组大鼠血清HDL-C水平均明显升高(P<0.05,P<0.01);二甲双胍组和大黄黄连泻心汤加味高、中剂量组附睾脂肪HIF-1α、BNIP3、LC3B mRNA及蛋白表达明显降低,UCP1蛋白表达水平明显升高(P<0.05,P<0.01);二甲双胍组和大黄黄连泻心汤加味高剂量组p62、Dio2、Prdm16 mRNA及p62蛋白表达明显升高(P<0.05,P<0.01)。结论:大黄黄连泻心汤加味可能通过HIF-1α/BNIP3/LC3B途径抑制内脏脂肪组织线粒体自噬,促进脂肪棕色化,改善肥胖T2DM糖脂代谢。

Objective:To observe the effect of modified Dahuang Huanglian Xiexintang on mitochondrial autophagy and browning of visceral adipose tissue in obese type 2 diabetes mellitus(T2DM)model ZDF rats.Method:Forty ZDF rats were induced with a high-fat diet to establish an obese T2DM model.The rats were randomly divided into five groups:Model group,metformin group(0.18 g·kg^(-1)),and high,medium,and low dose groups of modified Dahuang Huanglian Xiexintang(2.16,1.08,0.54 g·kg^(-1)),with eight rats in each group.Additionally,eight ZDF(fa/+)rats were assigned to the normal group.All groups received an intragastric volume of 10 mL·kg^(-1),with the model and normal groups receiving the same volume of purified water once daily for 12 weeks.Fasting blood glucose(FBG)was regularly measured.After 12 weeks of intervention,the body weight,epididymal fat weight,and serum levels of glucose(GLU),glycated serum protein(GSP),triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)were measured.Hematoxylin-eosin(HE)staining was used to observe pathological changes in epididymal fat tissue.Transmission electron microscopy(TEM)was employed to observe mitochondrial autophagy in adipocytes.Real-time PCR was used to detect the mRNA expression of hypoxia-inducible factor-1α(HIF-1α),Bcl-2/adenovirus E1B 19 kDa interacting protein 3(BNIP3),microtubule-associated protein 1 light chain 3B(LC3B),p62/SQSTM1,uncoupling protein 1(UCP1),iodothyronine deiodinase 2(Dio2),and PR domain containing 16(Prdm16)in epididymal fat.Western blot was used to detect the protein expression of HIF-1α,BNIP3,LC3B,p62,and UCP1 in epididymal fat.Result:Compared with the normal group,the model group showed pathological changes in epididymal fat,with adipocyte mitochondrial condensation and numerous autophagosomes indicating mitochondrial autophagy.The model group also exhibited significantly increased body weight,epididymal fat weight,FBG,GLU,GSP,TC,TG,and LDL-C levels(P<0.01),significantly decreased HDL-C levels(P<0.01),significantly elevated mRNA and protein expression of HIF-1α,BNIP3,and LC3B(P<0.01),significantly reduced mRNA and protein expression of p62 and UCP1(P<0.01),and significantly reduced mRNA expression of Dio2 and Prdm16(P<0.01).Compared with the model group,all intervention groups showed varying degrees of improvement in epididymal fat pathology.The metformin group and high-dose modified Dahuang Huanglian Xiexintang group displayed intact mitochondrial morphology,clear cristae,uniform matrix,and few autophagosomes and autophagosomes in the adipocyte cytoplasm.The metformin group and high-and mediumdose groups of modified Dahuang Huanglian Xiexintang showed significantly reduced body weight and epididymal fat weight(P<0.01).The epididymal fat index was reduced in all intervention groups(P<0.05),and FBG was lowered in all intervention groups(P<0.01).Serum GSP,GLU,TG,and LDL-C levels were reduced in the metformin group and the high-and medium-dose groups of modified Dahuang Huanglian Xiexintang(P<0.05,P<0.01).The serum TC level was significantly reduced in the metformin group and high-dose group of modified Dahuang Huanglian Xiexintang(P<0.01),and HDL-C levels were significantly increased in all intervention groups(P<0.05,P<0.01).The mRNA and protein expression of HIF-1α,BNIP3,and LC3B were significantly reduced,and UCP1 protein expression was significantly increased in the metformin group and highand medium-dose groups of modified Dahuang Huanglian Xiexintang(P<0.05,P<0.01).The mRNA and protein expression of p62,Dio2,and Prdm16 were significantly increased in the metformin group and high-dose group of modified Dahuang Huanglian Xiexintang(P<0.05,P<0.01).Conclusion:Modified Dahuang Huanglian Xiexintang may inhibit mitochondrial autophagy and promote the browning of visceral adipose tissue through the HIF-1α/BNIP3/LC3B pathway,thereby improving glucose and lipid metabolism in obese T2DM rats.