Inhibitory Effect And Mechanism Of Qidan Dihuang Decoction To Renal Fibrosis Of Diabetic Nephropathy

Intended To identify the effect of Qidan Dihuang decoction(QDD)on renal function in DM rats;To identify the effect of QDD on pathological structure of kidney in DN rats.To study the effect of QDD on renal fibrosis in DN rats.To study the effect of QDD on mesangial cell and myofibroblast induced by high glucose.To identify the effect of QDD on renal RAS in DN rats.Methods Male Sprague-Dawley(SD)rats were reandomly divided into normal group and diabetes group and intraperitoneally injected with sodium citrate buffer and sodium citrate STZ buffer.After 1 week,rats whose fasting blood glucose was measured and included fasting blood glucose>16.67were subjected to follow-up experiments.Rats with successful modeling were randomly assigned to the QDD group,Losartan group,and model group.Blood glucose and body weight were measured at 0,2,4,6 and 8 weeks after administration,respectively.24 hours urine of rats were collected and the urine volume(U-vol)were recorded at 0,4 and 8 week after administration.24h urine protein(24hU-Pro)and urea nitrogen(BUN)were detected.Serum creatinine and 24h urinary microalbuninnuria(MAU)were measured at 8 week after administration.Kidneys were weighted and the kidney-to-body-weight ration(KW/BW)was calculated.Some kidney tissues were taken for HE,PAS and Masson pathological staining.The expression of a-SMA and TGF-β1 in the kidney was analyzed by immunohistochemistry;The proteins expression of a-smooth muscle actin(α-SMA),transforming growth factor β1(TGF-β1),tumor necrosis factor a(TNF-α),renin,angiotensin Ⅱ receptor 1(AT1)were detected by western blot.Preparation of drug serum:QDD was decocted,filtered,concentrated and preserved;30 SD rats were randomly divided into normal group,losartan group and QDD group,and 10 rats in each group.Menstruum,losartan and QDD were given by gavage for three days and twice a day respectively.Blood was taken from the inferior vena cava and serum was separated by centrifuge,inactivated,filtered,and stored at-80℃.Culture,passage and intervention of mesangial cells and fibroblasts:After starvation for 12 h,the mesangial cells and NRK-49F cells were divided into control group,high glucose group,high glucose +losartan serum group,high sugar + QDD serum group.After 48 hours of drug stimulation,cell proliferation was measured by MTT assay.The expressions of FN,α-SMA,TGF-β1,TNF-α and renin,angiotensin Ⅱ receptor 1(AT1)and prorenin receptor(PRR)were detected by Western blot.The expressions of renin,PRR and AT1 in DN rats were detected by western blot.Results After treatment for 8 weeks,there was no significant difference in the blood glucose among the QDD group,the diabete group and the Losartan group;QDD did not affect the body weight and U-vol of DN rats,nor did it significantly improve the results of BUN and KW/BW.However,QDD could significantly reduce 24h MAU,24hU-Pro and Scr in DN rats.After treatment for 8 weeks,HE,PAS and Masson staining showed that the pathological structure of renal tissue of the QDD and Losartan groups was improved in the DN rats,and collagen and glycogen deposition were reduced as well.Immunohistochemical analysis showed that QDD can inhibit the protein expression of a-SMA and TGF-β1 in glomeruli.Western blot results showed that the expression of TGF-Pi,a-SMA,COL-Ⅰ,TNF-α in kidney of DM rats in the QDD group was decreased.Drug serum containing QDD inhibits the proliferation rate of mesangial cells and NRK-49F cells,and can also reduce the expressions of FN,α-SMA,TGF-β1,TNF-α in mesangial cells and NRK-49F cells,the expressions of renin,PRR,and AT,in both cells were reduced as well;After 8 weeks of administration,QDD can inhibit the expression of renin,PRR and AT1 in the renal RAS cascade of DN rats.Conclusion QDD can improve the renal function and tissue structure of DN,inhibit its fibrosis,and these positive effects may be related to the inhibition of RAS.