Study On Protective Effect And Mechanism Of Tangshen Formula On Diabetic Nephropathy Rats

ObjectiveDiabetic nephropathy(DN) is a common microvascular complication of diabetes mellitus.About 20 to 40%patients develop end stage renal disease.This disease do harm to human health.The pathogenesy of DN is very complicated,ACEI and angiotensin receptor to control developing of DN.traditional chinese medicine have evident special feature and superiority in treating DN.One of the hot spot of studying DN is seeking for safe and high-performance chinese medicine.this study set up two DN model.Type 1 DN rat model was induced streptozotocin.Spontaneous type 2 DN is the other model.In the base of prove the drug action of Tangshen Formula,we study the mechanism of action of Tangshen formula on DN in TGF-β1 and unbalance of synthesis and degradation of extracellular matrix which is important factor in inducing DN,and provide theory evidence for clinical application.Methods:1.Type 1 DN rat model was induced by right-kidney nephrectomy plus peritoneal injection of low-dosage streptozotocin(40 mg/kg) a week later,rat were randomly divided into five groups:normal,model,high-dose TSF,low-dose TSF and monopril,10 rats in each group.general states,weigh,blood glucose,Urine protein/creatinine were observed in the 0^th, 4^th,8^th,12^th,16^th,20th week.The rats were sacrificed at the end of the 20th week.Whole Blood viscosity and blood plasma viscosity were detected by blood rheometer.Blood fat total serum protein,albumin and function of kidneys were detected by blood biochemistrymeter. Pathological change of glomerular and renal tubularinterstitium were observed by semiquantitative assessment.CollagenⅣand TGF-β1 of kidneys were detected by immunohistochemistry.the mRNA expression of TGF-β1,MMP-2 and MMP-9 were mesured by RT-PCR.2.Spontaneous animal model of type 2 DN--OLETF rats were introduced abroad. The rats were randomly divided into model group,TSF group and Monopril group,LETO rats with same genetic background but not developing diabetic symdromes,same week age and sex were used as control group.General states,weight,blood glucose,Urine protein in 24 hours of the rats are observed in every 4 weeks.And successive administration was made for 24 weeks and 44 weeks.The rats were sacrificed at 36 and 56 weeks of age respectively. Blood viscosity and plasma viscosity were detected by blood rheometer.Serum total serum protein,albumin,lipid and function of kidneys were detected by blood biochemistrymeter. Pathological change of glomerular and renal tubularinterstitium were observed by semiquantitative assessment.Result:1 Effects of Tangshen fomula on type 1 DN rats induced by streptozotocin:1) General state and weight of model group was significantly worse than that of control group,and the difference was more significant followed by the progress of the experiment, and the differece was peaking in 20^th week.The general state of rats of every medication administration group was better than that of model group.Body weight of low-dose TSF group was increased from 4^th week and continued until the end of the experiment.Urine protein/creatinine of the rats of model group were signicantly increased from 12^th week.Urine protein / creatinine of rats of TSF group and Monopril group was signicantly lower than that of model group from 16^th week.Blood glucose,cholesterol,triglyceride,plasma viscosity, Blood urea nitrogen,Glomerulosclerosis index(GSI) and renal tubularinterstitial fibrosis index(RIFI) of model group were higher significantly than that of control group.Plasma viscosity,cholesterol,triglyceride,GSI and RIFI of high dose TSF group was lower than that of model group.Blood glucose,urea nitrogen,whole blood viscosity,total serum protein, albumin,creatinine of rats of TSF group were the same as that of model group.2) The result of immunohistochemistry shows that the expression of renal tissue of model group was higher significantly than that of control group.TGF-βlandⅣcollagen of low dose TSF and high dose TSF group were lower than that of mdel group.Experiment of RT-PCR shows that expression of mRNA of TGFβ1 of renal tissue of model group was higher significantly than that of control group.Expression of mRNA of MMP-9 of renal tissue of model group was lower significantly than that of control group.Expression of mRNA of TGFβ1 of renal tissue of high dose TSF group was lower significantly than that of model group.Expression of mRNA of MMP-9 of renal tissue of high dose TSF group was higher significantly than that of model group.Expressions of renal tissue of MMP-2 among the groups were no different.2.The effect of TSF on OLETF rats--a spontaneous animal model of type 2 DNBody weight of model group of rats was significantly lower than control group from 6 weeks old.The difference was most significant at 40 weeks of age,and then the difference becomes less.And at 52 weeks of age,there was no difference between two group.Body weight of TSF group was significantly lower than model group in 40,44 and 48 week of age, Blood glucose and urine protein of 24 hour of model group was significantly higher than those of control group from 6 week of age,and followed by the progression of the experiment, and the difference was biggest at 56 week of age.Urine protein of 24 hour of TSF group was significantly lower than that of model group from 36 week of age;blood glucose of TSF group was significantly lower than that of model group from 52 weeks of age.Plasma viscosity,triglyeride,GSI,RIFI of model group was significantly higher than those of control group in 36 week of age.Plasma viscosity of TSF group was significantly lower than that of model group,the effect is better than that of monopril.Triglyceride of monopril group was significantly lower than that of model group.The effect is better than that of TSF group.GSI and RIFI in TSF group and monopri group was significantly lower than that of model group. Cholesterol,triglyceride,whole blood viscosity,plasma viscosity,urea nitrogen,GSI,RIFI of model group were higher than those of control group in 56 week of age.Level of total serum protein and albumin of model group were lower than those of control group.Blood viscosity, plasma viscosity,cholesterol,GSI and RIFI of high dose TSF gorup were significantly lower than those of model group.The level of total serum protein and albumin of TSF group was significantly higher than those of model group.There was no difference between every group in the level of urea nitrogen.Conclusion:1 TSF can reduce urinary protein excretion,improve the impairment of kidney,the mechanism is related to improve unbalance of lipid metabolism and abnormal haemorheology,but it is not related with blood glucose.2 Accumulation of extracellular matrix is an key cause in the pathogenesis progression of DN.TSF can inhibit the expression of TGF-β1 mRNA and protein of type 1 DN,and increase the expression of MMP-9 mRNA,reduce the expression of collageⅣwhich is the main component of excellular matrix,this is the important mechanism of TSF of protect effect on rat with type 1 DN.3 TSF can reduce 24 hour urine protein of OLETF rats in early stage and advanced stage,and could improve pathological changes of glomerular and renal tubularinterstitium. Correcting disorder of lipid and glucose metabolism,improving blood rheology are renoprective mechanism of TSF on type 2 DN.4 Correcting disorder of lipid metabolism and abnormal haemorheology share common mechanism of treating effect of TSF on animal model of type 1 and type 2 DN.