The Effect And Mechanism Of The Tripterygium Glycosides On Diabetic Renal Tubulointerstitial Lesions

Objective:Diabetic nephropathy(DN)is one of the most important microvascular complications of diabetes mellitus,and the lesion not only affects the glomerulus but also affects the renal tubulointerstitium.Renal tubulointerstitial lesion is characterized by infiltration of inflammatory cells,disorder of cytokine expression and deposition of extracellular matrix.Tripterygium glycosides is a traditional Chinese medicine that has been used for many years in the treatment of chronic kidney disease due to its immunosuppressive and anti-inflammatory effects.However,nearly all of the previous researches on tripterygium glycosides' nephroprotective effect mainly focused on glomerulus structure and function,while the effect of tripterygium glycosides on renal tubuloinsterstitial injury in DN remains to be identified.The aim of the present study was to investigate the effect of tripterygium glycosides on diabetic renal tubulointerstitial lesions and its potential mechanism.Methods:1.Clinical studyA total of 138 patients were included in the study and randomly divided into conventional treatment group and tripterygium glycosides group.Conventional group was treated with routine dose of angiotensin converting enzyme inhibitors(ACEI)or angiotensin II type 1 receptor blockers(ARBs)treatment.In the tripterygium glycosides group,we added tripterygium glycosides(10 mg 3 times per day)on the basis of conventional group.The patients were treated and followed up for 12 weeks.Blood glucose,blood lipid,blood pressure,24-hour urinary protein,urinary retinol binding protein(U-RBP),urinary beta 2-Microglobulin(?2-MG),urinary retinol binding protein(U-RBP)were assessed at end of treatment.2.Animal experiment75 rats were randomly assigned to normal control group(NC group,n = 15),which were fed with standard diet,and HFD group(n = 60),which were allowed free access to the high-fat diet to induce dyslipidemia for 8 weeks.Then,the HFD rats were administered a single tail intravenous injection of 30 mg/kg STZ.About 72 h after STZ injection,the diabetic model was considered to be successful when the random blood glucose was >16.7 mmol/L.The diabetic rats were then randomly divided into four groups: diabetic rats without drug treatment(DM group,n = 15),and diabetic rats treated with tripterygium glycosides at a low dose group(1 mg/kg,n = 15),medium dose group(3 mg/kg,n = 15)and high dose group(6 mg/kg,n = 15),respectively.Tripterygium glycosides was given by daily gastric gavage for 8 weeks.The rats in NC group and DM group received equal volumes of saline every day.At the end of this study,blood and urine samples were collected for biochemical examination,and the renal tubulointerstitial lesions was quantified by HE,PAS and Masson staining.The m RNA and protein levels of the TLR4,My D88,NF-?B,IL-1?,TGF?1,TNF-? and VCAM-1,?-SMA,vimentin,type IV collagen and E-cadherin were detected by immunohistochemistry,real-time PCR and western blotting analysis,respectively.Results:1.Clinical studyEight patients in the tripterygium glycosides group and 6 in the conventional group did not complete the study.In overall,124 patients complete the study.After 12 weeks of follow-up,no significant differences were found between the two groups in changes of systolic blood pressure,diastolic blood pressure,fasting plasma glucose,Hb A1 c,triglycerides,total cholesterol and aspartate aminotransferase.Compared with conventional group,UMA,NAG,U-RBP and ?2-MG were significantly reduced by tripterygium glycosides treatment.2.Animal experiment1)Compared with NC group,the level of blood lipids,insulin,HOMA-IR and HOMA-? were significantly increased in HFD group after high-fat diet;blood glucose was significantly higher accompanied by polydipsia,polyuria and polyphagia after injection of STZ,which indicating that the model of type 2 diabetic rats was induced successfully.2)General indicators:(1)Blood glucose and blood lipids: The level of blood glucose,TG and TC in DM group were significantly increased compared with NC group(P<0.05);however,no significant effect was observed after intervention of tripterygium glycosides compared with DM group(P>0.05);(2)KW/BW: Compared with NC group,the level of KW/BW was increased significantly in DM group(P<0.05),and decreased after intervention of tripterygium glycosides;(3)Liver,renal function and blood routine: The parameterof liver,renal function and blood routine did not differ among the five groups(P>0.05).3)Compared with NC group,both UMA and NAG were significantly increased in diabetic groups(P<0.05);compared with DM group,UMA and NAG were significantly decreased by intervention of tripterygium glycosides(P<0.05).4)The structure of renal tubulointerstitial area is normal in rats of NC group,however,some of the renal tubular epithelial cell degeneration,loss,tubulointerstitial area increased stromal matrix between the endoplasmic,inflammatory cell infiltration and renal interstitial fibrosis could be seen in DM group,while those were ameliorated by treatment with tripterygium glycosides.5)Compared with the NC group,m RNA and protein expression of ?-SMA,vimentin and type IV collagen were all significantly increased,and the expression of E-cadherin was decreased in DM group by immunohistochemisty,real-time PCR and western blotting analysis(P< 0.05).Compared with the DM group,m RNA and protein expression of ?-SMA,vimentin and type IV collagen were significantly decreased dose-dependently,and the expression of E-cadherin was increased in group of tripterygium glycosides.6)Compared with NC group,m RNA and protein expression of TLR4,My D88,NF-?B,IL-1?,TGF?1,TNF-? and VCAM-1 were all significantly increased in DM group by immunohistochemisty,real-time PCR and western blotting analysis(P< 0.05);all of which were reduced in the tripterygium glycosides treated diabetic rats dose-dependently,but still higher than those in NC group(P < 0.05).7)Compared with NC group,serum NF-?B,IL-1?,TGF?1,TNF-? and VCAM-1 level were significantly increased in DM group by ELISA;compared with DM group,serum NF-?B,IL-1?,TGF?1,TNF-? and VCAM-1 levels in different dose intervention group of tripterygium glycosides were significantly decreased(P < 0.05).Conclusion: Tripterygium glycosides could ameliorate diabetic renal tubulointerstitial lesion,the mechanism of which may be at least partly associated with the suppression of EMT through blocking TLR4/My D88/NF-?B signaling pathway and reducing the inflammatory reaction.