Effects Of Tanshinone ⅡA Combined With Dapt On Hes-1 Protein And Snail Protein In The Process Of Renal Fibrosis

Objective: To observe the effects of TanshinoneⅡA(TanⅡA)combined with DAPT on Hes-1 and Snail proteins in the process of UUO renal interstitial fibrosis,so as to provide experimental basis for preventing and treating kidney diseases by the combination of traditional Chinese and Western medicine.Methods: Sixty healthy Sprague-Dawley rats were randomly divided with computer grouping software into the normal group(n=12),UUO group(n=12),DAPT group(n=12),TanⅡA group(n=12)and DAPT + TanⅡ A compatibility group(compatibility group,n=12).Each group was half male and half female.UUO model was established in the rats of UUO,DAPT,TanⅡA and compatibility group.DAPT solution,TanⅡA solution and DAPT+TanⅡA mixed solution were intragastrically administered daily to the rats in the DAPT,Tan Ⅱ A and compatibility groups for 14 days,respectively.Serum cystatinC(CYS-C),kidney tubular injury molecule-1(KIM-1),24 hour urinary protein(24h UPr)and urinary alpha-1 microglobulin(α1-MG)were detected using ELISA.Left kidneys were dissected along the longitudinal axis,1/2 kidneys were harvested for pathological observation using HE,Masson staining and immunohistochemistry(IHC-P).The other 1/2kidneys were used to detected the relative expression of Hes-1,Snail1 and Slug molecule in rat kidneys with western blotting(WB)and reverse transcriptase-polymerase chain reaction(RT-PCR).Results:1 HE staining There were no pathological changes in renal interstitium of normal rats.In UUO rats,the cortex renis became thinner;the boundary between cortex and medulla was blurred;the arrangement gap of renal tubules was loose;the renal tubule lumen was compensatory hypertrophy,rupture even atrophied.All administration groups were improved to varying degrees.The HE scores of Tan Ⅱ A group were significantly lower than those of DAPT group(P < 0.05).2 Masson staining Renal pathological positive staining was not obvious in normal group.In UUO group,a large number of banded or flaky blue-stained collagen deposition were found in the rats interstitium.Collagen staining was deepened,accompanied by dilatation and atrophy of renal tubules.Necrotic epithelial cells could be seen in the lumen,and brush border cells of renal tubules were lost.Collagen deposition in renal tissue of each administration group was significantly reduced compared with UUO group(P < 0.05).The Masson scores of TanⅡA group were significantly lower than those of DAPT group(P <0.05).3 Detection of rat serum by ELISA The levels of CYS-C and KIM-1 in TanⅡA group were significantly lower than DAPT group(P < 0.05);the levels of CYS-C and KIM-1 in compatibility group were significantly lower than single administration groups(P < 0.05).4 Detection of rat urine by ELISA The 24 hUPr and α1-MG levels in TanⅡA group were significantly lower than those in DAPT group(P < 0.05),while the the level of 24 hUPr in the compatibility group were lower than single administration group(P < 0.05).5 Detection of renal tissue by IHC-P The MOD values of Snail1 and Slug proteins in DAPT group were significantly lower than those in Tan Ⅱ A group(P < 0.05),and the MOD values of Snail1 protein and Slug protein in compatibility group were significantly lower than single administration groups(P < 0.05).6 Detection of renal tissue by WB The relative expression of Hes-1 protein in TanⅡA group was significantly lower than that in DAPT group(P < 0.05);the relative expression of Snail1 protein in DAPT group was significantly lower than that in TanⅡA group(P <0.05);the relative expression of Hes-1 and Slug proteins in compatibility group were significantly lower than single administration group(P < 0.05).7 Detection of renal tissue by RT-PCR The relative expression of Hes-1mRNA in TanⅡ A group was lower than that in DAPT group(P < 0.05);the relative expressions of Snail1 mRNA and SlugmRNA in DAPT group were significantly lower than that in TanⅡA group(P < 0.05);the expression level of Hes-1mRNA,Snail1 mRNA and SlugmRNA in compatibility group were relatively lower than that in single administration group(P <0.05).Conclusion: Tan Ⅱ A,DAPT and their compatibility can improve the renal general structure,tubular function and pathology to a certain extent,and have certain protective effect on UUO rats with renal interstitial fibrosis.Its mechanism may be related with effectively reducing of Hes-1,Snail1 and Slug molecules in Notch pathway of UUO kidney tissue and delay of tubular epithelial-mesenchymal transition.Among them,the compatibility administration has the better significant effect,which indicates that TanⅡA and DAPT may play a synergistic role in down-regulating the expression of Hes-1 and Snail molecules in the kidney of UUO rats.