Protective Effects And Mechansism Of Shenshuaining On The Kidney Injury Of Diabetic Rats Induced By STZ

Objective:In this study, we established streptozotocia(STZ)-induced diabetic rats model to observe the effects of Shenshuaining on renal function, nephropathology, extracellular matrix(ECM) and the expressions of renal transforming growth factor-beta 1(TGF-β1), plasminogen activator inhibitor-1(PAI-1) and tissue inhibitor of metalloproteinase-1(TIMP-1) of diabetic rats. At last we discussed the protection and mechanism of Shenshuaining used independently and combining with Benazepril on the early kidney injury of diabetic rats, and we hope it may provide a theoretical basis for clinic using.Methods:40 healthy male Wistar rats were fed for a week. And then picking up 8 rats randomly as the normal control group(N group), the other 32 rats were induced to the diabetes models by a single intraperitoneal streptozotocia injection with the dose of 65mg/kg, and the rats in N group were injected with equivalent sodium citrate buffer solution. And then the rats injected with STZ were divided randomly into diabetes model group(M group), Benazepril treating group(B group), Shenshuaining treating group(S group) and Benazepril united Shenshuaining treating group(BS group),8 rats each group. After the injection, the rats in B group, S group and BS group were given drugs by gavage once a day for 8 weeks, according to the dose:Shenshuaining-0.7g/kg, Benazepril-4mg/kg, while the rats in N group and M group were given normal saline with equal volume by gavage at the same time. Blood glucose was tested with the blood from tail vein at the end of the fourth and eighth week respectively and at the end of the eighth week serum creatinine(SCr), blood urea introgen(BUN),24h urine protein excretion quantity and kidney-to-body weight ratio were detected. And then renal pathological examination was observed to contrast the pathological changes of nephridial tissue in each group by HE, PAS and MASSON staining and the testing of glomerular area(GA), extracellular matrix(ECM) and the ratio of the two indexes(ECM/GA). And the expression of TGF-β1, laminin(LN), and collagen IV(ColⅣ) were detected by immunohistochemistry. In addition, the expression of the molecular biological parameters in nephridial tissue, TGF-β1, PAI-1 and TIMP-1, were measured by real-time fluorescent quantitation RT-PCR.Results:(1) Compared with N group, the levels of blood glucose, SCr, BUN,24h urine protein excretion quantity and kidney-to-body weight ratio in M group and 3 treating groups were significantly raised(P<0.01). And compared with M group, all the parameters above in 3 treating groups were significantly decresed(P<0.01), except the level of blood glucose(P>0.05). And among treating groups, except the levels of blood glucose and BUN(P>0.05), there were significant differences between the rest parameters in BS group and that in B group, S group(P<0.01). Besides, compared with B group, the levels of SCr and 24h urine protein excretion quantity in S group were significantly declined statistically(P<0.01) and the body weight of rats in S group was significantly increased(P<0.05), while there were not significant differences between other parameters of the two groups(P>0.05).(2) Pathological changes of nephridial tissue in each group:The kidney structure of N group was clear, and there were not significant abnormalities in glomerulus, tubule and interstitial area. While in M group there were early pathological changes of diabetic kidney injury, the glomerular volume enlarged, basement membrane thickened, mesangial cells and matrix proliferated, part of tubular epithelial cells showed vacuolar degeneration and there were a quantity of inflammatory cells infiltrating in the interstitial area. And compared with M group, the pathological changes of nephridial tissue in 3 treating groups were lightened.Changes of pathological parameters:Compared with N group, glomerular area(GA), extracellular matrix(ECM) and the ratio of the two indexes(ECM/GA) in M group and 3 treating groups were significantly increased(P<0.01). Compared with M group, all the parameters above in 3 treating groups were significantly decresed(P<0.01). And among treating groups, there were also significant differences between the parameters above in BS group and that in B group, S group(P<0.01), that means the levels of all the parameters above decresed more significantly in BS group, while there were not significant differences between the parameters above in S group and that in B group(P>0.05).(3) Immunohistochemistry results:Compared with N group, the expression of TGF-β1, LN and ColⅣin M group and 3 treating groups were significantly increased(P<0.01). Compared with M group, all the parameters above in 3 treating groups were significantly decresed(P<0.01). And among treating groups, there were also significant differences between the parameters above in BS group and that in B group, S group(P<.05 or P<0.01), that means the expression of all the parameters above decresed more significantly in BS group, while there were not significant differences between the parameters above in S group and that in B group(P>0.05).(4) RT-PCR results:Compared with N group, the relative expression quantity of TGF-β1, PAI-1 and TIMP-1 in nephridial tissue in M group and 3 treating groups were significantly increased(P<0.01). Compared with M group, all the parameters above in 3 treating groups were significantly decresed(P<0.01). And among treating groups, there were also significant differences between the parameters above in BS group and that in B group, S group(P<0.01), that means the relative expression quantity of all the parameters above decresed more significantly in BS group. And there were not significant differences between the expression of TGF-β1 and TIMP-1 in S group and that in B group(P>0.05), but the expression of PAI-1 in S group was declined more significantly(P<0.05).Conclusion: Used independently, Shenshuaining has a protective effect on the early kidney injury of diabetic rats, similarly to Benazepril. It can reduce urinary protein, improve renal function and lighten the pathologic injury of kidney. And compared with Benazepril, Shenshuaining can play a more effective role in reducing creatinine and urinary protein excretion and increasing the body weight of diabetic rats. The key mechanism of its protective effect may be it down-regulates the expression of TGF-β1, PAI-1 and TIMP-1 in nephridial tissue and suppresses the accumulation of ECM to slow down the process of renal fibrosis. In addition, Shenshuaining also can be used combining with Benazepril to protect kidney from injury and improve the efficacy of treating early diabetic kidney injury.