| Objective Diabetic nephropathy(DN)is a idiopathic systemic microvascular complications of diabetes mellitus and it has become the major cause of end stage renal disease(ESRD).Studies found that thickening of the glomerular basement membrane in diabetic nephropathy is a major cause of proteinuria leaking.Using type 2 diabetes and diabetic nephropathies SD rats animal model which were built by high-sugar and high-fat diet combined with low dose STZ tail vein injection as our research objects and taking Tripterygium Wilfordii Glycosides(TWP)and Berberine(BBR)as the therapeutic drugs.We investigated the intervention of different doses of TWP combined with BBR on the occurrence and development of DN and the repair mechanisms that may be existed on glomerular basement membrane.Methods 20 of 260 clean grade male SD rats were randomly assigned to the prevention normal control group(PNC groud,n=10)and treatment normal group(TNC groud.n=10)respectively that were fed with regular rat chow.Other 240 rats were fed with high-sugar and high-fat diet.After 8 weeks on their respective diets,STZ was injected into caudal vein of the 240 high fat diet-fed rats to develop type 2 diabetes mellitus.Successful model rats were randomly divided into prevention group(P)and treatment group(T).These two groups were continue divided into 9 groups respectively:including diabetes model group(PDM,TDM).TWP low dose group(PTL,lmg/kg·d;TTL,3mg/kg·d),TWP middle dose group(PTM,3mg/kg·d;TTM,6mg/kg·d).TWP high dose group(PTH,6mg/kg·d;TTH,9mg/kg·d),BBR group(PB,150mg/kg·d;TB,150mg/kg·d).TWP low dose and BBR group(PTBL,lmg/kg·d +150mg/kg·d;TTBL,3mg/kg·d +150mg/kg·d),TWP middle dose and BBR group(PTBM,3mg/kg·d+15 0mg/kg·d;TTBM.6mg/kg·d+150mg/kg·d)and TWP high dose and BBR group(PTBH.6mg/kg·d +150mg/kg·d:TTBH,9mg/kg·d +150mg/kg·d).The rats of prevention group were began gavage 8 weeks after being DM model and was executed and removed organs in the 8th weekend.While the rats of treatment group were began gavage 8 weeks after being DN model in the 7th week and was executed and removed organs at the end of 14th weekend.Metabolic indicators were determined from the blood and urine samples during the different time of experiment.Glomerular morphology and glomerular basement membrane ultrasfructural changes were observed by light microscopy and electron microscopy.Col-? and LN in peripheral blood were observed by enzyme-linked immuno sorbent assay(ELISA).The expression of Col-? and LN cytokines were observed by RT-PCR and Western blotting personally from the genetic and protein levels in kidney tissues.At last,we use the method of immunohistochemical to locate the expression of Col-? and LN in kidney tissues.Results(1)After having been fed wth high sugar and fat diet for 8 weeks,the rats of this group has significantly higher triglyceride(TG).total cholesterol(TC),FINS,HOMA-IR and HOMA-? than normal control group(P<0.05).After STZ injection.the blood sugar,kidney weight/body weight and the body weight in this group are significantly higher and lower than normal control group respectively(P<0.05),meanwhile the 24-hour urinary UMA level of the rats continued to rise at the end of the sixth week.While the liver,kidney function and the blood routine examination had no significant difference(P>0.05).(2)The levels of 24-hour urinary UMA:?The prevention group:Compared with the PNC group and other intervention groups.the levels of 24-hour urinary UMA in PDM were significantly increased(P<0.05);Compared with the PDM group,the levels of 24-hour urinary UMA in TWP groups.BBR group and TWP and BBR joint intervention groups were in a dose-dependent reduction.That means PTM and PTH group produced the most obvious reduction of UMA than other intervention groups(P<0.05).?The treatment group:Compared with the TNC group and other treatment groups,the levels of 24-hour urinary UMA in TDM were significantly increased(P<0.05);Compared with the TDM group.the level of 24-hour urinary UMA in three dosage of TWP treatment groups were in a dose-dependent reduction(P<0.05).After application of BBR,comparing with the TDM group.the levels of 24-hour urinary UMA in three dosage of TWP and BBR joint treatment groups were decreased significantly.Meanwhile,PTBM and PTBH group produced the most obvious reduction than other treatment groups(P<0.05).(3)The result of light microscopy and electron microscopy:?The prevention group:The rats of PDM group had obvious renal pathological changes of early DN,such as glomerular atrophy and increased mesangial matrix mildly.At the same time.the glomerular basement membranes was thickened mainly within the subcutaneous additional thickening.Three layers of fine GBM structure was disappeared.The shape was slightly curled.Electron density was increasesand uniformly and narrowing of capillary lumen mildly.And so on.The renal pathological changes in three dosage of TWP intervention groups were improved to varying degrees.In the mean while,PTM and PTH group produced the most obvious improvement than other intervention groups.After application of BBR.The PTBM and PTBH groups produced the most obvious preventive effect than other groups.?The treatment group:the rats of TDM group had typical renal pathological changes of DN.TTM and TTH group produced the most obvious improvement than other treatment groups.After application of BBR,the renal pathological changes in.TTBL group was improved.While,TTBM and TTBH groups produced the most obvious improvement than other treatment groups.(4)Using ELISA to detect the levels of Col-??LN in serum:? The levels of serum Col-?:Compared with the normal control group,the levels of serum Col-?in diabetic model group,the intervention and treatment groups were significantly increased(P<0.05).Compared with diabetic model group,the levels of serum Col-?in three dosage of TWP intervention and treatment groups were in a dose-dependent reduction(P<0.05).After application of BBR,the levels of serum Col-? in three dosage of TWP,BBR joint intervention and treatment groups were in a dose-dependent reduction(P<0.05).? The levels of serum LN:Compared with the normal control group,the level of serum LN in diabetic model group,compared with diabetic model group,the levels of serum LN in three dosage of TWP intervention and treatment groups were decreased to varying degrees(P<0.05).After application of BBR,the levels of serum Col-? in PTBM group.PTBH group and three dosage of TWP and BBR joint treatment groups were declined obviously(P<0.05).(5)Using RT-PCR to detect the levels of Col-??LN mRNA in kidney:?The prevention group:The level of Col-??LN mRNA in kidney in PDM and other intervention groups were significantly increased than the PNC group(P<0.05);Compared with the PDM group,the level of Col-??LN mRNA in kidney in TWP groups,BBR group and TWP and BBR joint intervention groups were in a dose-dependent reduction.?The treatment group:Compared with TNC group,the level of Col-??LN mRNA in kidney in TDM and other treatment groups were significantly increased(P<0.05):Compared with the TDM group,the level of Col-??LN mRNA in kidney in three dosage of TWP treatment groups were in a dose-dependent reduction(P<0.05).After application of BBR,compared with the TDM group,the levels of Col-??LN mRNA in kidney in three dosage of TWP and BBR joint treatment groups were decreased significantly(P<0.05).(6)Using Western Blotting to detect the levels of kidney Col-??LN protein expression:?The prevention group:The level of kidney Col-??LN protein expression in PDM and other intervention groups were significantly increased than the PNC group(P<0.05);Compared with the PDM group,level of kidney Col-??LN protein expression in TWP groups,BBR group and TWP and BBR joint intervention groups were in a dose-dependent reduction.?The treatment group:Compared with TNC group,the level of kidney Col-??LN protein expression in TDM and other treatment groups were significantly increased(P<0.05);Compared with the TDM group,the levels of kidney Col-??LN protein expression in three dosage of TWP treatment groups were in a dose-dependent reduction(P<0.05).After application of BBR,Compared with the TDM group,the levels of kidney Col-??LN protein expression were decreased to varying degrees(P<0.05).In the mean while,the middle and high-dose of TWP treatment groups declined obviously(P<0.05).After application of BBR,compared with the TDM group,the levels of kidney Col-??LN protein expression in three dosage of TWP and BBR joint treatment groups were decreased significantly(P<0.05).(7)Using Immunohistochemistry staining to detect the levels of kidney Col-??LN protein expression:Col-? and LN are mainly expressed in glomerular,tubular basement membrane and mesangial zone.?The prevention group:The brown particles infiltration of Col-? and LN expressed in glomerular,tubular basement membrane and mesangial zone in PDM and other intervention groups were significantly increased than the PNC group(P<0.05);Compared with the PDM group,the level of kidney Col-??LN protein expression in TWP groups.BBR group,TWP and BBR joint intervention groups were decreased to varying degrees and in a dose-dependent(P<0.05).?The treatment group:Compared with TNC group,the brown particles infiltration of Col-? and LN expressed in glomerular,tubular basement membrane and mesangial zone in TDM and other treatment groups were significantly increased(P<0.05);Compared with the TDM group.the level of kidney Col-??LN protein expression in three dosage of TWP treatment groups,TWP and BBR joint treatment groups were in a dose-dependent reduction(P<0.05).Conclusions(1)We successfully prepared animal models of T2DM and DN.(2)The expression of Col-? and LN in kidney and the expression of Col-? and LN in serum in DN rats were significantly increased compared with the normal control group that indicate that the accumulation of collagen and non-collagenous glycoprotein in the glomerular basement membrane may involved in the development of DN.(3)The application of middle-dose,high-dose of TWP and BBR or joint intervention in T2DM rat model can effectively delay the occurrence of DN.The application of berberine can not effectively delay the development of renal disease in DN rat model.But the application of middle-dose,high-dose of TWP and BBR joint intervention can significantly lower the microalbuminuria level of the DN rats to delay and treat the development of type 2 diabetic nephropathies and has a high security.(4)TWP and BBR can repair the glomerular basement membrane,restore the selective permeability of glomerular filtration barrier and reduce the levels of UMA by regulating the imbalance of synthesis and degradation on Col-? and LN to delay the occurrence and development of type 2 diabetic nephropathies. |
PDF Full Text Request