摘要
目的:观察益气活血养阴清热方对糖尿病肾病(DN)大鼠肾组织损伤及单磷酸腺苷活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/UNC-51样激酶1(ULK1)(AMPK/mTOR/ULK1)信号通路的影响,探讨其治疗DN的可能作用机制,为临床应用益气活血养阴清热方治疗DN提供客观的理论依据.方法:将90只SPF级雄性SD大鼠采用随机数字表法分为正常对照组和造模组,分别给予普通饲料、高糖高脂饲料喂养.6 w后造模组大鼠一次性腹腔注射链脲佐菌素(STZ)建立DN模型,模型制备成功后随机分为模型对照组、益气活血养阴清热方7.7、15.3、30.6 g/kg组、厄贝沙坦0.017 g/kg组.给药后检测大鼠空腹血糖(FBG)、24h尿蛋白(24h-UTP)、肌酐(Scr)、尿素氮(BUN)含量;HE染色、Masson染色、碘酸六胺银(PASM)染色观察肾组织病理变化;透射电镜观察肾组织超微结构变化;免疫组化法(IHC)检测大鼠肾组织自噬相关蛋白微管相关蛋白1轻链3 Ⅱ(LC3 Ⅱ)、肌球蛋白样BCL2结合蛋白(Beclin 1)表达水平;Western blot法检测大鼠肾组织AMPK、p-AMPK、mTOR、p-mTOR、ULK1、p-ULK1蛋白表达;Real-time PCR法检测大鼠肾组织Ampk、Mtor、Ulk1 mRNA表达.结果:与正常对照组比较,模型对照组大鼠FBG、Scr、BUN、24 h-UTP含量显著升高(P＜0.01),肾小球增大,基底膜增厚,系膜基质增生,毛细血管扩张明显,足突融合,LC3Ⅱ、Beclin 1蛋白表达水平显著降低(P＜0.01);AMPK、p-AMPK、ULK1、p-ULK1蛋白表达明显下调(P＜0.05或P＜0.01),p-mTOR、mTOR蛋白表达显著上调(P＜0.01);Ampk、Ulk1 mRNA表达显著下调(P＜0.01),MtormRNA表达显著上调(P＜0.01);与模型对照组比较,益气活血养阴清热方30.6 g/kg组大鼠FBG、Scr、BUN、24 h-UTP含量显著降低(P＜0.01);肾小球增大减轻,肾组织病理变化改善,同时可见明显的自噬溶酶体,LC3Ⅱ、Beclin 1 蛋白表达显著升高(P＜0.01);AMPK、p-AMPK、ULK1、p-ULK1 表达显著上调(P＜0.01),mTOR、p-mTOR 表达显著下调(P＜0.01);Ampk、Ulk1 mRNA表达显著上调(P＜0.01),Mtor mRNA表达显著下调(P＜0.01).结论:益气活血养阴清热方能够缓解DN大鼠肾组织损伤,其机制可能与调控AMPK/mTOR/ULK1信号通路介导的肾组织自噬有关.
Abstract
Objective:To observe the effect of Yiqi Huoxue Yangyin Qingre Formula(益气活血养阴清热方)on renal tissue damage and the AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/Unc-51-like kinase 1(ULK1)signaling pathway in dia-betic nephropathy(DN)rats,and explore its underlying mechanism for the treatment of DN,aiming to provide an objective theoretical basis for clinical application of Yiqi Huoxue Yangyin Qingre Formula in the treatment of DN.Methods:Ninety SPF-grade male SD rats were ran-domly divided into a normal control group and an experimental group according to the random number table method,and they were fed on a regular diet and a high-sugar high-fat diet,respectively.After 6 weeks,the DN model was induced by intraperitoneal injection of streptozotocin(STZ)in rats of the experimental group.After successful modeling,the model rats were further randomized into a model control group,low-,medium-,and high-dose Yiqi Huoxue Yangyin Qingre Formula groups(7.7,15.3,and 30.6 g/kg),and an irbesartan group(0.017 g/kg).Following administration,fasting blood glucose(FBG),24-hour urine protein(24 h-UTP),creatinine(Scr),and blood urea nitrogen(BUN)levels were measured.Histological changes in renal tissues were observed using hematoxylin-eosin(HE)staining,Masson's staining,and pe-riodic acid-silver methenamine(PASM)staining.Ultrastructural changes in renal tissues were examined by transmission electron microsco-py.Immunohistochemistry(IHC)was used to determine the expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ)and Beclin 1 in rat renal tissues.Western blot was employed to assess the protein expression of AMPK,p-AMPK,mTOR,p-mTOR,ULK1,and p-ULK1 in rat renal tissues.Real-time polymerase chain reaction(PCR)was performed to measure the mRNA expression of Ampk,mtor,and Ulk1 in rat renal tissues.Results:Compared with the normal control group,the model control group exhibited significantly increased FBG,Scr,BUN,and 24 h-UTP levels(P＜0.01),enlarged renal glomeruli,thickened basal membranes,mesangial ma-trix proliferation,significant capillary dilation,podocyte fusion,decreased expression levels of LC3Ⅱ and Beclin 1(P＜0.01),downregulated AMPK,p-AMPK,ULK1,and p-ULK1 expression(P＜0.05 or P＜0.01),upregulated mTOR and p-mTOR expression(P＜0.01),downregu-lated Ampk and Ulk1 mRNA expression(P＜0.01),and upregulated mtor mRNA expression(P＜0.01).Compared with the model control group,the high-dose Yiqi Huoxue Yangyin Qingre Formula group showed significantly reduced FBG,Scr,BUN,and 24 h-UTP levels(P＜0.01),mitigated enlargement of renal glomeruli,improvement in renal tissue pathological changes,clear autophagic lysosomes,increased ex-pression levels of LC3Ⅱ and Beclin 1(P＜0.01),upregulated AMPK,p-AMPK,ULK1,and p-ULK1 expression(P＜0.01),downregulated mTOR and p-mTOR expression(P＜0.01),upregulated Ampk and Ulk1 mRNA expression(P＜0.01),and downregulated mtor mRNA ex-pression(P＜0.01).Conclusion:Yiqi Huoxue Yangyin Qingre Formula can alleviate renal tissue damage in DN rats,and its mechanism may be related to the regulation of renal tissue autophagy mediated by the AMPK/mTOR/ULK1 signaling pathway.
基金项目
河北省自然科学基金(H2022423367)
政府资助临床医学优秀人才培养项目(13000022P00DE34100247)
河北省中医药局科研项目(2021167)
河北中医学院研究生创新能力培养资助项目(XCXZZBS2022018)
国家科技期刊平台
NSTL
万方数据