Effect of Yiqi Huoxue Yangyin Qingre Formula(益气活血养阴清热方)on AMPK/mTOR/ULK1 Autophagy Signaling Pathway in Renal Tissue of Diabetic Nephropathy Rats
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.
Yiqi Huoxue Yangyin Qingre Formula(益气活血养阴清热方)Diabetic nephropathyAMP-activated protein kinaseMamma-lian target of rapamycinUNC-51 like kinase 1
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