Mechanism Of Yiqihuoxuejiangzhuo Formula’s Effect On Renal Tubular Epithelial Cell Transdifferentiation Induced By TGF-β1

1research backgroundRenal insterstitial fibrosis is the common feature and main pathological basis from various kidney disease to end stage renal failure.its main pathological basis is the atrophy of renal tubular,infiltration of inflammatory cells and the sedimentary of excessive fiber components on renal tubular interstitial.Its main feature is excessive external cells matris such as collagen I,III,IV fibronectin and laminin accumulating on renal tubular interstitial and the fibroblast proliferation on renal interstitium.Its characteric feature is connective tissue deposit on renal parenchyma and do damage to kidney.Its core mechanism is epithelial-mesenchymal transition(EMT).Tubular EMT is a process in which the tubular renal cells lose their phenotypical epithelial characteristics and acquire new ones, which are characteristics of the mesenchymal cells. So these cells exhibit a transformation capacity, after which they can produce extracellular matrix.The key link of RIF is the activation and proliferation of mass myofibroblast.Of the progression of RIF the EMT exert a significant role so inhibiting EMT play an important role on reversing and delaying renal fabrosis.But there is no effective treatment aiming at this key pathological link.The traditional Chinese medicine has advantage of better efficacy and lower side effect.Therefore,it is of important significance to research the YIQIHUOXUEJIANGZHUO herbal prescription’effect on preventing and treating renal fibrosis.On aspect of TCM,the pathogenesis of renal fibrosis is oriented spleen-kidney deficiency and standarded wet-stasis mutual resistance.The therapy should strengthen healthy energy to eliminate pathogenic factors and seek both temporary and permanent solutions. Threapy should be supplementing QI and activating blood,grooming humidity and descending turbid.The eldly kidney professor headed by professor Dai Xiwen study a large of CRF cases and establish the YISHENJIANGYA formula dominated in supplementing QI,activating blood and descending turbid.Prophase clinical study show that this formula can reduce the accurrence of endpoint events.Basis of research show that this formula can reduce the expression of TGF-β1,increase the expression of smad6、7,recover the equilibrium of metalloproteinase enzyme and inhibitory factor reduce the abnormal accumulation of ECM,so as to alleviate or inhibit the development of renal fabrosis. 2resaerch purposeWith the ambition of illustrating the mechanism of YIQIHUOXUEJIANGZHUO formula preventing renal fabrosis,we will study the signal transduction pathway of TGF-β1/smads,p38MAPK,rho-ROCK by which the YIQIHUOXUEJIANGZHUO formula inhibit EMT.We will also find the concrete target and therapy difference of variety dosage,find the most proper therapy scheme and lay solid theory basis for therapy of TCM to renal fabrosis and chronic renal failure.3research subject and methodThis experiment will use method of whole level,cell level,gene level and signal transduction pathway,use test in vitro of cell culture and use various experiment method such as electron microscopic,immunohistochemistry,rt-PCR,western Blot to investigate the concrete target and mechanism of YIQIHUOXUEJIANGZHUO formula preventing the renal fabrosis.This research provide scientific basis for the TCM’s therapy and prevention of renal fabrosis and chronic renal failure.4result4.1the aspect of cell morphologyThe TCM formula drug serum has the trend of reversing EMT of HK-2stimulated by TGF-β1.The large dose group drug containing serum show the most significant effect.4.2the aspect of regulation protein expressionFirst,YIQIHUOXUEJIANGZHUO formula can inhibit the expression of a-SMA which is the marker protein of myofibroblast,recover expression of E-cadherin which is marker protein of epithelial cells.Second,this formula can inhibit the key molecular such as smad2/3,smad6,smad7of signal transduction pathway TGF/smad so as to inhibit the progression of EMT.4.3the aspect of cytokinesThe formula can regulate the cycytokines which have relations to EMT,such as TGF-β1、HGF、BMP-7、ILK、CTGF、collagen I in order to delay the progression of EMT.4.4the aspect of regulate expression of DNAThere is no obvious change in the expression of Rho-A and p38MAPK,but the middle dose group drug containing serum may inhibit the expression of p38MAPK. 5conclusionThe formula YIQIHUOXUEJIANGZHUO can inhibit the expression of a-SMA which is the marker protein of myofibroblast, inhibit the key molecular such as smad2/3,smad6,smad7of signal transduction pathway TGF/smad,inhibit the cycytokines which have relations to EMT,such as TGF-β1、HGF、BMP-7、ILK、CTGF、collagen I,recover expression of E-cadherin and increase the cycytokines HGF、BMP-7which is related to inhibiting renal EMT.