The Renal Protective Effect Of Hedysarum Polybotys Saccharide On Diabetic Nephropathy In Db/db Mice And Effect On Tgf-b1/smads Signal Pathway

Objective:Through the observation of effect of Hedysarum Polybotys Sac char ide?HPS?on renal pathology and TGF-?1?CTGF?BMP-7 gene and protein expression level in renal tissue and TGF-?1/Smads signaling pathway on diabetic nephropathy in db/db mice,to objectively evaluate the renal protective effect of HPS on diabetic nephropathy in db/db mice,and further reveal the mechanism and provide theoretical basis for further clinical application of HPS in prevention and treatment of diabetic nephropathy.Methods:50 SPF grade 12 weeks old male BKS db/db mice were randomly divided into 5 groups:HPS large dose group?middle dose group?low dose group?enalapril group?model control group,with 10 mice in each group;another 10 syngeneic non diabetic db mice as normal control group.HPS of high dose group were given HPS 400mg.kg^-1.d^-1;middle dose group were given HPS 200mg.kg^-1.d^-1;low dose group were given HPS 100mg.kg^-1.d^-1;enalapril group with 1 Omg.kg^-1.d^-1;The model control group and normal control group were given the same dose of normal saline.once a day by intragastric administration for 8 weeks.During the experiment,body weight was measured every week;measuring the blood glucose every 2 weeks;at the end of the experiment,the eyeballs were removed to get blood,blood samples were collected for biochemical analysis;After the mice were sacrificed,the kidneys were removed immediately.the right kidney was fixed in 4%paraformaldehyde,embedded in paraffin,sliced for pathological and immunohisto chemical detection.Pathology underwent HE and Masson staining,changes of the morphology and ultrastructure of renal pathology were observed under the light microscope and electron microscope,then evaluate the degree of renal fibrosis.Left kidney was preserved for RT-PCR and Western-blot detection in-70? refrigerator after frozen in liquid nitrogen.mRNA expression were detected by RT-PCR method of TGF-?1?CTGF?BMP-7?Smad2?Smad3?Smad7 in renal tissue,Immunohistochemical and Western Blot method detected the target proteins.Results:? The effect on body weight:body weight in diabetic db/db mice was significantly higher than that of normal control group?P<0.01?;after 8 weeks of treatment,in addition to the body weight of normal group did not change significantly,other groups showed varying degrees of reduction,compared with the model group,the body weight of treatment groups decreased more obviously,differences were statisticaly significant?P<0.05orP<0.0 1?,especially in HPS high dose group.?blood glucose:blood glucose of diabetic mice was significantly higher than that in non diabetic mice?P<0.01?;during the experiment,except normal control group,blood glucose of other groups increased in varying degrees,especially the model group increased most significantly;after 8 weeks,there was significant difference between the HPS of high dose group and model group?P<0.05?,but no statistical significance between the other treatment groups and model group?P>0.05?.?Serum lipid:compared with the normal group,the TG and TC levels of model group were significantly higher than that in normal control group?P<0.01?;after treatment of 8 weeks,serum TG and TC levels of HPS high?medium dose groups are significantly lower than the model group,the difference was statistically significant?P<0.01?,especially the difference of HPS high dose group was more obvious.? Kidney function:compared with the normal control group,the serum BUN and SCr in DN mice are significantly higher?P<0.01?;compared with the model group,BUN and SCr decreased significantly in the HPS high dose group and the enalapril group,the difference is statistically significant?P<0.01?.?the pathological changes:under the light microscope and electron microscope,nephric morphology in the normal control group mice are normal,podocyte structures are complete,arranged orderly and clear,without basement membrane thickening,no proliferation of mesangial matrix and renal interstitial fibrosis and inflammatory cell infiltration;In model group mice,a significant increase in glomerular volume,diffuse thickening of glomerular basement membrane in different degrees,diffuse proliferation of mesangial matrix,mesangial region widen,fusion of foot processes,endothelial cells arranged in disorder?shedding,part of tubular epithelial cells vacuolar degeneration,tubular atrophy,protein cast,renal capsule adhesion,visible mesangial fibrosis hyperplasia and renal fibrosis;compared with the model group,foot process fusion?deposition of ECM and GBM thickness were improved in the treatment groups in different degrees?P<0.05 or P<0.01?,HPS high dose group and enalapril group are the most obvious.?Gene expression:re suits of RT-PCR suggest that:compared with normal control group,TGF-?1?CTGF?Smad2?Smad3 mRNA expression increased significantly in model group?P<0.01?,BMP-7?Smad7 mRNA expression obviously decreased?P<0.01?;compared with the model group,except HPS low dose group showed no significant difference?P>0.05?,TGF-?1?CTGF?Smad2?Smad3 mRNA expression in other treatment groups decreased compared with model group?P<0.01?,BMP-7?Smad7 mRNA expression are higher than the model group?P<0.01?,especially HPS high dose group and enalapril group.?The protein expression:immunohi st o chemistry and Western Blot results showed that:compared with the normal control group,there were significantly increased expression of TGF-?1,CTGF,Smad2,Smad3 protein in renal cortex of mice in model group?P<0.01??and the protein expression of BMP-7?Smad7 decreased significantly?P<0.01?;after HPS treatment,the renal TGF-?1?CTGF?Smad2?Smad3 protein expression decreased compared with the model group?P<0.01?,but the expression of BMP-7,Smad7 increased significantly?P<0.01?,especially in the high dose of HPS group.Conclusions:HPS could inhibit the elevated blood glucose in db/db mice with diabetic nephropathy,lower the blood lipids,protective the renal function in a certain extent,and improve diabetic renal damage.The mechanism may be through upregulation of BMP7?Smad7 expression in renal tissue,downregulation of TGF-?1?CTGF?Smad2?Smad3,thus affecting the transduction of TGF-?1/Smads signal pathway.