Study On The Effect And Mechanism Of Shenqi Pills Against Renal Interstitial Fibrosis In Rats

Part 1 Study the function and molecular mechanism of Shenqiwan on Renal Interstitial FibrosisObjective:In this project,we focus on EMT to explore the effects and mechanisms of Shenqiwan on tubulointerstitial fibrosis,furthermore,providing new ideas and new methods for better carry out Chinese medieine against renal tubulointerstitial fibrosis.Method:The SD rats were randomly divided into the control group,adenine model group and Shenqiwan group(n=10).The model group rats were treated with adenine(150mg/kg)once per day for 35 days.The rats of Shenqiwan group were intragastrieally administrated with Shenqiwan 3g/(kg·d)per day for 21 days,The sham group were intragastrieally with identieal volume of normal saline.On the 35th,rats were anesthetized,and blood was collected by aorta abdominalis and separated for serum to detect the contents of BUN、Scr、24hUTP and TGF-β1.HE staining,PAS staining and Masson staining were used to observe the pathological changes of each group.Vimentin and E-cadherin were observed by immunohistoehemical method.E-cadherin and Vimentin protein were detected by western blot and E-cadherin mRNA、Vimentin mRNA、β-catenin mRNA、LEF1 mRNA and Wnt4 mRNA expression were detected by real-time quantitative PCRResults:1.In adenine group,renal interstitial became widening,inflammatory cells infiltrating serious,renal artery intimal thickening and luminal narrowing.Regional distribution of fibrosis was spotty.Glomerulus have gathered in serious regional phenomenon.Tubulointerstitium interstitial fibrosis feature was apparented.All the pathological changes above show that the experiment model would were successful.These morphological changes were relieved,tubulointerstitium damage degree was notably reduced after treated with Shenqiwan.The results indicate that Shenqiwan can relieve renal pathologieal changes in rats and protect the kidney.2.TGF-β1 expression of model group were increased compared with those in sham group,after treatment with Shenqiwan,TGF-β1 expression was deceased compared with those in model group.There results indicate that Shenqiwan inhibit the expression of TGF-β1 in renal tissue.3.Vimentin expression of model group was increased compared with this in sham group,Vimentin expression was decreased after treatment with Shenqiwan compared with this in model group.E-cadherin expression of model group was decreased compared with this in sham group after treated with Shenqiwan,E-cadherin was markedly increased compared with those in model group.The results indicate that Shenqiwan can inhibit or retard the development of EMT.4.TGF-β1 mRNA、Snail mRNA、Twist mRNA、LEF1 mRNA and Wnt4 mRNA expression of model group were increased compared with those in sham group,treatment with Shenqiwan could significantly decrease the above indexes compared with those in model group.The results indicate that Shenqiwan can inhibit or retard the occurrence or development of tubulointerstitium fibrosis by prevent EMT.Conclusion:Pathological process of EMT has happened with tubulointerstitial fibrosis model rats.Shenqiwan can significantly relieve main renal pathopogical damage caused by adenine,inhibit or retard the occurrence or development of tubulointerstitium fibrosis.Part 2 Study the function and molecular mechanism of Shenqiwan on TGF-β1-induced EMT in renal epithelial cellsObjective:To observe the influence of Shenqiwan on TGF-β1-induced epithelial mesenchymal transition(EMT)in renal tubular epithelial and explore the mechanism of preventing renal fibrosis by Shenqiwan.Method:Renal tubular epithelial cells were divided into six groups as follows:control group,TGF-β1 group,blank serum group+TGF-β1(10ng/ml),Shenqiwan 1.5g/(kg·d)+TGF-β1(10ng/ml),Shenqiwan 3.0g/(kg·d)+TGF-β1(10ng/ml),Shenqiwan 6.0g/(kg-d)+TGF-β1(10ng/ml).Using inverted microscope to observe the morphological changes of cells in every group;The content of collagen I and FN was measured by ELISA method;Expression of E-cadherin and Vimentin protein were detected by immunofluorescence method;Expression of E-cadherin、Vimentin、smad2/3、p-smad2/3、ILK、GSK-3β、p-GSK-3β and β-catenin protein was detected by westernblot method;Expression of E-cadherin mRNA、Vimentin mRNA、β-catenin mRNA、LEF1 mRNA and Wnt4 mRNA were detected by RT-PCRResults:1.the cells morphology were changed into fibroblast by TGF-β1(10ng/ml)under inverted microscope.2.Elisa results showed that the content of collagen I and FN were significantly raised in cells stimulated by TGF-β1(10ng/ml)compared with the blank control group(p<0.01),but when joined with Shenqiwan,the content of collagen I and FN were obviously decreased(p<0.01).3 Immunofluorescence results showed that the expression of E-cadherin was decreased and the expression of Vimentin was raised in TGF-β1(10ng/ml)incubation,when joined with Shenqiwan,the expression of E-cadherin was raised and the expression of Vimentin was decreased.4.Smad2/3 was phosphorylated as early as 15 mins after TGF-β1(10ng/ml)incubation,peaked at 30 mins in cells.Western blot results proved that the expression of p-smad2/3、ILK、p-GSK-3β in TGF-β1(10ng/ml)group were significantly higher than the blank control group,when joined with Shenqiwan,the expression of p-smad2/3、ILK、p-GSK-3β protein were obvious decreased.RT-PCR results proved that the expression of Vimentin mRNA、β-catenin mRNA、LEF1 mRNA and Wnt4 mRNA in TGF-β1(10ng/ml)group were significantly higher than the blank control group,the expression of E-cadherin mRNA was decreased,when joined with Shenqiwan,the expression of Vimentin mRNA、β-catenin mRNA、LEF1 mRNA and Wnt4 mRNA were obvious decreased,the expression of E-cadherin mRNA was raised.Conclusion:Shenqiwan can maintain the morphological stability of HK-2 cell and NRK-52E cell,prevent cells from fibrosis and inhibit cell proliferation and secretion of collagen I and FN induced by TGF-β1.It can also regulate the renal tubular epithelial cell signaling pathway that mediated by TGF-β1.Shenqiwan can inhibit the renal interstitial fibrosis to a certain extent.