Mechanism Of Yishen Huayu Formula In The Intervention Of Renal Interstitial Fibrosis Based On The Regulation And Control Of The Signaling Pathway Of The TGF-?1/smad

Purpose1.Through the establishment of UUO model and TGF-?1 of induction NRK52E differentiated cells turn,clear TGF-?1/Smad way signaling proteins activation and related cell factors change and TEMT express process and renal fibrosis connection.2.By inhibiting epithelial cells differentiation and controlling TGF-?1/Smad signal protein,it can intervene the abnormal activation RIF effect,the papes explores and benefiting kidney to remove stasis antagonist RIF role and the molecular mechanism of possible targets.Methods1.By using the unilateral ureter ligation method to establish the rat renal fibrosis(UUO)model,random rats were divided into control group,the model group,benefiting kidney high dose group,remove party benefiting kidney to remove the party dose group of benefiting kidney,low dose group,to remove party Ang ? receptor antagonist losartan 6 groups of group.UUO model in the 7 d.14 d,28 d executed rats,Dynamic observation of YiShenHuaYu Fang on UUO rat renal function and renal histopathologic effects,using immunohistochemistry methods were used to remove party simulating test and benefiting kidney differentiation mark protein alpha SMA and E-cad heri expression,and the influence of TGF-?1,Smad7 signal-the regulation effects of protein expression.2.Use the in vitro culture rat proximal tubular epithelial cells(NRK52E)as the research object,choose the currently accepted the fibrosis strong effect factor TGF-?1 for stimulating factor,observation NRK52E cell type change and benefiting kidney to remove the serum containing medicine party repair function,the cell immunohistochemical method observe different concentration of benefiting kidney medicine serum containing remove party of renal tubular epithelial cells turn in the process of differentiation TGF-?1/Smad way signal protein p-Smad2/3,Smad7 express the influence,and attempts to observe this party genetic level of TGF-1 mRNA,BMP7mRNA expression of controlling effect.Results1.To make an intervention study on the influence of the prescription on TGF ?1/Smad signal-the way.(1)The 7 d group rats that significantly impaired kidney function,and with the obstruction the extension of time serum BUN,Scr level gradually raised,tip UUO made model success.After treatment removing stasis and benefiting kidney dose group of each party and losartan rats,BUN,Scr level than that of a decreasing model group(P<0.01 or P<0.05),benefiting kidney high dose group and remove party losartan curative effect of quite.14 when d benefiting kidney to remove the party dose group of Scr levels dropped the most significant,and group compared with model with significant difference(P<0.01).Tip in early to mid to good curative effect of Chinese medicine.(2)The results show that the prescription can remove different degree UUO,reduce rat kidney tissues pathological damage,decreased the interstitial inflammatory cell,expand or shrink renal tubular,the treatment group different time points renal interstitial injury score and model group dropped significantly by(P<0.01);the 14d Yishen Huayu in high dose group repair renal interstitial injury the most significant,and valsartan group efficacy equal.(3)Immunohistochemical,according to the results of the model rats after obstruction 7d kidney tissues of muscle fiber cell phenotype sign into protein alpha SMA is strong positive expression,and sign the epithelial cells of the protein E-cadherin expression instead.Tip in renal fibrosis of early muscle fibroblasts has started in interstitial increased,TEMT has started to happen.The prescription can significantly inhibited the expression of alpha SMA,its high dose group and losartan renal tissues in the group E-cad herin expression to have obvious enhancement.Alpha SMA expression OD value shows,the treatment group in different time points of alpha with SMA express a model group all have reduce(P<0.01 or P<0.05).but E-cadherin expression was significantly enhanced.(4)Immunohistochemical results show,after the model of 7D model rats renal tissue f'ound in the TGF-?1 expression,and as the obstruction time in renal tubular epithelial cell cytoplasm was persistent cluster of' high expression,but Smad7 expression decreased.Remove party benefiting kidney can inhibit the rats in the kidney tissues TGF-?1 early high expression,improved Smad7 express activity,in each observation points are significantly better than model group(P<0.01 or P<0.05).Benefiting kidney high dose group and remove party losartan Smad7 group in kidney mesenchymal express the most positive elevated,TGF-?1 protein expression of positive weakened gradually,both a curative effect.2.To remove party control benefiting kidney TGF-?1/Smad signal way NRK52E intervention mechanism study of differentiation turn.(1)Experiments have f'ound that give TGF-(31 10 ng/ml concentration NRK52E cells stimulate 48h,renal tubular epithelial cells form change,spin increase with a spindle shape,intercellular broadens,have fiber appearance characteristics.And benefiting kidney and remove party medicated joint action of serum,the cells form as benefiting kidney medicine to remove with serum concentrations of the party in a certain extent increase cell gap narrowed,cells form intends to to normal.(2)The results show that,remove party including benefiting kidney medicine and TGF serum-?1 NRK52E joint action.Cell 48 h,but different degree cut p-Smad2/3 expression and nuclear inversion,and raised Smad7 expression,and this eff'ect is dose dependent.Bivariate analysis results show that p-Smad2/3 of the expression and Smad7 expression is a significant negative correlation,the correlation coefficient for r = 0.809,P<0.01.(3)RT-PCR results showed that the NRK52E stimulated by TGF-?1 48 h,BMP-7 mRNA in cell cytoplasm expression decreases,and the external TGF-?1 mRNA expression then rises.And the prescription medicated joint action of serum,BMP-7 mRNA express significant rise and exogenous TGF-?1 mRNA expression then decreased significantly,and has the dose dependent.And losartan serum containing medicine to BMP-7 and TGF-?1 mutual control consistently.Reflect BMP-7 TGF-?1 to the fibrosis effect has antagonist role.Conclusion1.The prescription can to a certain extent to improve the general condition of UUO rats,reduce rat renal heavy index,reduce UUO rats histopathologic kidney damage and lower renal interstitial injury degree.And it can reduce UUO rats serum BUN,Scr level.It is confirmed that the prescription has obvious improvement in renal function,delays renal interstitial fibrosis.2.To remove stasis through benefiting kidney groups was statistically the expression of alpha SMA,upward E-cad herin expression level,restrain rats in the kidney tissues TGF-?1 early high expression,raised Smad7 activity,and reflects the inhibition renal tubular epithelial cells turn differentiation effect and regulation TGF-?1/Smad7 signal protein function.3.Prescription of benefiting kidney to remove stasis of serum TGF-?1 exciting NRK52E cells has repair function,and at the same time cuts endogenous TGF-?1 mRNA expression,increases resistance inhibitory factor BMP-7 mRNA fibrosis expression.And in Smad paths through the cut p-Smad2/3 expression and nuclear inversion,the expression of the Smad7 raised,and regulation TGF ?1/Smad way signal activation and protein expression.And this effect is dose dependent.In short,prescription of benefiting kidney to remove stasis have certain renal protection effect on UUO rats,can slow down the process of renal fibrosis.The control mechanism and relieving renal pathology organization may damage,the suppression turn differentiation marks the expression of protein alpha SMA,raised the epithelial cells mark protein E-cadherin closely related to the expression,and in the kidney tissues transmembrane signal way control TGF-?1/Smad signal protein expression and abnormal activation.And at the same time through to TGF-?1 NRK52E turn differentiation process of induction of antimicrobial resistance function,genes and protein levels in raised Smad7,BMP-7 mRNA expression,restrain exogenous TGF-?1 mRNA high expression and p-Smad2/3 activity,and reflects the antagonist TGF-?1 cause of fibrosis effect and antimicrobial resistance epithelial cells turn disintegration.Embodied in the tissue and cellular levels of TEMT process and RIF inhibition effect,this is the prescription of clinical application to provide scientific theory and new ideas for reference.