Study On The Mechanism Of Shenkang Injection In Regulating Renal Fibrosis And HUVEC Angiogenesis In UUO Mice

Objective:To investigate the effects of Shenkang Injection(SKI)on renal fibrosis of UUO mice and angiogenesis of HUVEC,by regulating VEGF activityMethods:48 male C57BL/6 mice,8 weeks old,were randomly divided into sham group(sham),unilateral ureteral obstruction group(UUO),losartan-administrated group(ARB),and Shenkang-administrated groups with high,medium and low doses(SKI-H,SKI-M,SKI-L).Mice except the sham ones were subjected to UUO operation.ARB mice were administrated with losartan at a dose of 13 mg/kg per day,while SKI-H,SKI-M,SKI-L mice were administrated with SKI at doses of 7.8 g/kg,3.9 g/kg and 1.95 g/kg per day,respectively.Mice were sacrificed 14 days after administration.Levels of creatinine,urea nitrogen and cystatin C in serum were determined.Renal vasculature was observed by micro CT.Formalin-fixed paraffin-embedded renal sections were stained with hematoxylin and eosin to show morphological changes,and stained with sirius red to show fibrotic changes.Levels of VEGF and VEGFR-2 were determined by immunohistochemistry.Human umbilical vein endothelial cells(HUVEC)were cultured and Medicated with SKI at doses of 8 mg/ml and 2 mg/ml respectively for 24 hours.Transfection was performed using RNAi(Si-VEGF)to knock down VEGF genes in HUVEC,and medicated with SKI as mentioned above.Angiogenesis of HUVEC in each group was observed and determined by in vitro angiogenesis kit.Gene expressions and protein expressions of VEGF,VEGFR-2,AKT and eNOS were determined by real-time PCR and Western blot respectivelyResultsBody weights were significantly decreased in UUO mice than in sham mice(P<0.05 or P<0.01),and significantly increased in ARB,SKI-H and SKI-M mice than in UUO mice(P<0.05).Levels of creatinine,urea nitrogen and cystatin C were significantly higher in UUO mice than in sham mice(P<0.05 or P<0.01),and significantly lower in ARB and SKI-H mice than in UUO mice(P<0.05 or P<0.01).Seldom abnormal vasculature and pathologic changes were observed in sham mice.Vasculature damage and pathologic changes were obviously observed in UUO mice,with small vessels lost,vessel tortuosity and vessel branching points decreased,hyperplasia of interstitium,and infiltration of inflammatory cells.Vasculature damage and pathologic changes were alleviated in ARB and SKI mice compared with UUO mice.Levels of VEGF and VEGFR-2 were significantly lower in UUO mice than in sham mice(P<0.01),and significantly higher in ARB and SKI mice than in UUO mice(P<0.01).Gene expression of VEGF significantly decreased under condition of transfection with Si-VEGF(P<0.01).SKI promotes angiogenesis,with paraments of angiogenesis higher and small vessels denser in SKI-medicated HUVEC than in the control HUVEC.SKI at high dose(8 mg/ml)upregulated gene expressions of VEGF,VEGFR-2 and eNOS(P<0.05 or P<0.01).SKI at both doses upregulated these gene expressions under condition of transfection(P<0.05 or P<0.01).SKI upregulated protein expressions of VEGF,VEGFR-2,eNOS,p-eNOS(P<0.05 or P<0.01)Conclusions:SKI alleviated pathologic changes and fibrotic lesions in UUO mice.SKI upregulated expressions of VEGF and VEGFR-2,and alleviated vasculature damages of the obstructive kidney in UUO mice.SKI promoted angiogenesis of HUVEC and upregulated expressions of VEGF,VEGFR-2 and eNOS.In all,SKI might inhibit renal fibrosis and promote angiogenesis by regulating VEGF pathway.