| ObjectiveDiabetic nephropathy(DN)is one of the main microvascular complications of diabetes mellitus(DM).Current researches on the pathogenesis of DN involve numerous aspects,but the damage of glomerular barrier function is the direct cause of proteinuria.Glomerular endothelial cell is an important part of glomerular filtration membrane.In this study,we established the animal model of type 2 diabetes mellitus and diabetic nephropathy using sugary and fatty feedstuff and low dose streptozotocin(STZ)injection through caudal vein as the research objects,and conducted the intervention by tripterygium wilfordii glycosides(TWP)and berberine(BBR)in normal albuminuria phase and microalbuminuria phase,in order to investigate the effect on glomerular endothelial cell related factors such as monocyte chemotactic protein-1(MCP-1)and vascular cell adhesion molecule-1(VCAM-1).Methods260 clean grade male SD rats were raised,20 of which were randomly selected as prevention normal control(PNC.n=10)and treatment normal control(TNC,n=10)group,respectively,fed with regular feedstuff.Others were fed with sugary and fatty feedstuff and STZ(30mg/kg)was injected through caudal vein to induce T2DM animal model after 8 weeks.The successfully induced rats were randomly divided into prevention(P)groups and treatment(T)groups,n=120.respectively.P groups were divided into diabetes model(PDM)group,single TWP groups,single BBR(PB,BBR 150mg/kg·d)group and groups combined with TWP and BBR.Single TWP groups were further divided into TWP low-dose(TWP lmg/kg·d),TWP middle-dose(TWP 3mg/kg·d),TWP high-dose(TWP 6mg/kg·d)(PTL,PTM,PTH)group according to the application of dose of TWP;groups combined with TWP and BBR were further divided into TWP low-dose and BBR(PTBL,TWP lmg/kg-d+BBR 150mg/kg·d)group,TWP middle-dose and BBR(PTBM,TWP 3mg/kg·d+ BBR 150mg/kg·d)group,TWP high-dose and BBR(PTBH,TWP 6mg/kg·d+ BBR 150mg/kg·d)group according to the application of dose of TWP,n=15,respectively.experimental rats were started TWP and BBR administration by gavage after successfully induced,PNC and PDM groups were treated with equal amount of distilled water(2ml),and P groups were executed and removed organs after 8 weeks.T groups were divided into diabetes model(TDM)group,single TWP groups,single BBR(TB,BBR 150mg/kg·d)group and groups combined with TWP and BBR.Single TWP groups were further divided into TWP low-dose(TWP 3mg/kg·d),TWP middle-dose(TWP 6mg/kg·d),TWP high-dose(TWP 9mg/kg·d)(TTL,TTM,TTH)group according to the application of dose of TWP;groups combined with TWP and BBR were further divided into TWP low-dose and BBR(TTBL,TWP 3mg/kg-d+BBR 150mg/kg·d)group,TWP middle-dose and BBR(TTBM,TWP 6mg/kg·d+ BBR 150mg/kg·d)group,TWP high-dose and BBR(TTBH,TWP 9mg/kg·d+ BBR 150mg/kg·d)group according to the application of dose of TWP,n=15,respectively,experimental rats were started TWP and BBR administration by gavage 6 weeks after successfully induced,TNC and TDM groups were treated with equal amount of distilled water(2ml),and T groups were executed and removed organs after 8 weeks.Changes of body weight(BW),blood glucose,24h urine microalbumin(UMA)of rats were recorded in the process of experiment;BW,kidney weight(KW)were recorded when executed;blood samples were kept to detect blood glucose,hepaticand renal function,blood lipids and blood routine;ELISA was used to detect serum MCP-1 and VCAM-1 level;renal tissues were kept,electron microscope was used to observe the changes of ultrastructure of glomerular filtration membrane,RT-PCR,Western Blotting and immunohistochemical methods were used respectively to detect mRNA and protein expression of MCP-1 and VCAM-1.Results1.The level of blood lipids,insulin,HOMA-IR and HOMA-β in sugary and fatty feedstuff group were significantly higher(P<0.05);after injection of STZ,blood glucose was significantly higher in rats of sugary and fatty feedstuff group and body weight was significantly lower,accompanied by polydipsia,polyuria and polyphagia,UMA of diabetic rats increased significantly 6 weeks after successfully induced.2.General indicators:①BW and blood glucose:compared with PNC and TNC group,the blood glucose in P and T groups increased significantly(P<0.05)while the body weight decreased significantly after successfully induced(P<0.05),no significant effects were observed after intervention of TWP and BBR(P>0.05);②KW/BW:compared with PNC and TNC group,the level of KW/BW increased significantly in PDM and TDM group(P<0.05),the level of KW/BW decreased in P groups after intervention of TWP and BBR(P<0.05),especially in PTH group;after intervention of TWP and BBR in T groups,the level of KW/BW decreased in TTH.TB,TTBL,TTBM and TTBH groups(P<0.05);③Blood lipids:compared with PNC and TNC group,the level of TG and TC in P and T groups increased significantly(,P<0.05),no significant effects were observed after intervention of TWP and BBR(P>0.05);④Hepatic and renal function and blood routine:no significant differences of hepatic and renal function and blood routine were seen among groups(P>0.05).no significant effects were observed after intervention of TWP and BBR(P>0.05).3.UMA:①P groups:compared with PNC group.UMA was significantly higher in diabetic groups(P<0.05);compared with PDM group,UMA declined significantly in single TWP groups,single BBR group and groups combined with TWP and BBR(P<0.05).and the effect was dose-dependent;②T groups:compared with TNC group,UMA was significantly higher in diabetic groups(P<0.05);compared with TDM group,UMA declined significantly in single TWP groups and groups combined with TWP and BBR(P<0.05),the effect was dose-dependent.4.Electron microscopic observation of glomerular filtration membrane:There was not obvious abnormity of the shape and size of the capillary lumens and content of glomerular endothelial cell in rats of normal control group.But in rats of diabetic group,there were ultrastructure changes of glomerular endothelial cell just like in early DN,such as visible hyperplasia in glomerular endothelial cell accompanied by swelling congestion,protruding into the capillary lumen,it was much serious in T group than that in P group.The pathological ultrastructure were improved in single TWP groups,single BBR group and groups combined with TWP and BBR.5.Use ELISA method to detect the level of MCP-1 and VCAM-1 in serum of rats:①P groups:compared with PNC group,level of MCP-1 and VCAM-1 in serum of rats were significantly higher in diabetic groups(P<0.05);compared with PDM group,level of MCP-1 and VCAM-1 in serum declined significantly in group PTH,PTBM and PTBH(P<0.05);②T groups:compared with TNC group,level of MCP-1 and VCAM-1 in serum of rats were significantly higher in diabetic groups(P<0.05);compared with TDM group,level of MCP-1 in serum declined significantly in group TTH,TTBL,TTBM and TTBH(P<0.05),level of VCAM-1 in serum declined significantly in TTM,TTH,TTBL,TTBM and TTBH(P<0.05).6.Use RT-PCR method to detect the mRNA expression of MCP-1 and VCAM-1 in kidney of rats:①P groups:compared with PNC group,the mRNA expression of MCP-1 and VCAM-1 in kidney of rats were significantly higher in diabetic groups(P<0.05);compared with PDM group,the mRNA expression of MCP-1 and VCAM-1 in kidney of rats declined significantly in single TWP groups,single BBR group and groups combined with TWP and BBR(P<0.05),and the effect was dose-dependent;②T groups:compared with TNC group,the mRNA expression of MCP-1 and VCAM-1 in kidney of rats were significantly higher in diabetic groups(P<0.05);compared with TDM group,the mRNA expression of MCP-1 and VCAM-1 in kidney of rats declined significantly in single TWP groups and groups combined with TWP and BBR(P<0.05),the effect was dose-dependent.7.Use Western Blotting method to detect the protein expression of MCP-1 and VCAM-1 in kidney of rats:①P groups:compared with PNC group,the protein expression of MCP-1 and VCAM-1 in kidney of rats were significantly higher in diabetic groups(P<0.05);compared with PDM group,the protein expression of MCP-1 and VCAM-1 in kidney of rats declined significantly in single TWP groups,single BBR group and groups combined with TWP and BBR(P<0.05),and the effect was dose-dependent;②T groups:compared with TNC group,the protein expression of MCP-1 and VCAM-1 in kidney of rats were significantly higher in diabetic groups(P<0.05);compared with TDM group,the protein expression of MCP-1 and VCAM-1 in kidney of rats declined significantly in single TWP groups and groups combined with TWP and BBR(P<0.05).the effect was dose-dependent.8.Use immunohistochemistry method to detect the protein expression of MCP-1 and VCAM-1 in kidney of rats:compared with PNC and TNC group,there were marked cellular infiltration of MCP-1 and VCAM-1 in glomerular in diabetic groups(P<0.05);after intervention of TWP individually,the expression of MCP-1 and VCAM-1 declined significantly in kidney of rats(P<0.05),group PTM,PTH.TTM and TTH produced the most obvious;when administrated with BBR,the effect can be observed in P groups(P<0.05),but was not obvious in T groups after the intervention of BBR individually(P>0.05);in groups combined with TWP and BBR,the expression of MCP-1 and VCAM-1 declined significantly in kidney(P<0.05),group PTBM and PTBH,TTBM and TTBH produced the most obvious decline.Conclusions1.We successfully established the animal model of T2DM and DN by sugary and fatty feedstuff and low dose STZ.2.Through electron microscopic observation,we can see that there were ultrastructure changes of glomerular endothelial cell.The mRNA and protein expression of MCP-1 and VCAM-1 in kidney of DN rats increased significantly,indicated that there may be correlation between changes of glomerular endothelial cell related cytokines and adhesion molecules and the development of DN.3.The intervention of single TWP,single BBR and the combination of them can decline the level of UMA of rats in P groups and postpone the onset of DN.the intervention of single TWP and the combination of TWP and BBR can decline the level of UMA in T groups and retard the development of DN,the intervention of BBR individually didn’t have effect on T group.No obvious damage of hepatic and kidney function and loss of cells in peripheral blood were observed in experimental rats after using each dose of TWP and berberine.indicating a good security.4.TWP and berberine intervention can postpone the onset of DN and retard the development of DN by reducing the expression of glomerular endothelial cell related cytokines and adhesion molecules,represented by MCP-1 and VCAM-1,alleviate the inflammatory reaction of endothelial cell and repaire the structure of glomerular filtration membrane. |
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