Effects Of Matrine On The Proliferation And Collagen Synthesis Of Rat Cardiac Fibroblasts Induced By Aldosterone

AIM: To investigate the effects of matrine on the proliferation and collagen synthesis of rat cardiac fibroblasts(CFs) induced by aldosterone(Ald),to research the mechanism of regression of heart fibrosis.METHODS: The neonatal rat cardiac fibroblasts were extracted by enzymatic digestion and anchorage velocity-dependent separation method. Through the technique of cell culture in vitro , establish a model which Aid induced CFs fibrosis, then add disposal factors.groups were divided into 7 groups, group A: Normal central group; group B: Ald (1.0xl0-7mol/L ) group; group C: Ald (1.0xl0-7mol/L) + Spi (l.0xl0-6moI/L) group; group D: Ald (1.0xl0-7mol/L)+losartan (l.0xl0-6mol/L) group; group E: Ald (1.0xl0-7mol/L) + Mat (0.125mmol/L) group; group F: Ald (1.0xl0-7mol/L)+Mat (0.25 mmol/L) group; group G: Ald (1.0xl0-7mol/L)4-Mat (0.5 mmol/L) group . After 24 hours' operation, prolifertion was measured by thiazolyl blue(MTT) assay; cell total protein content was measured by coomasie brilliant blue; Cellcycle distribution was dertermined with flow cytometer(FCM); the expression of proliferation cell nuclear antigen(PCNA) was measured by immunofluorescence staining method and FCM. Collagen synthesis was defermined by means of hydroxyproline concentration staining; the expression of fibronectin(FN) was measured by immunofluorescence staining method; collagen type I / III ratio was analyzed by pepsin extraction and SDS-PAGE; enzyme activity was evaluated by gelatinzymography and densitometric analysis; the tissue inhibitor metalloproteinase (TIMP-1) mRNA expression was measured by semi-quantitatative RT-PCR analysis, the expression of transforming growth factor beta(TGFβ-l) was dertermined by immunofluorescence staining method, FCM,and western-blot; smad7 mRNA expression was measured by semi-quantitatative RT-PCR analysis.RESULTS:(1) Aid's effects: Compared with Normal central group , Aid groups markly enhanced CFs MTT results, cell total protein content, and the expression of PCNA (p< 0.01= , Cellcycle distribution analysis indicate that Aid groups' cell number were decreased in Go-Gi period, markedly increased in S and Ga-M period (p<0.01=; Aid greatly enhanced Collagen synthesis content, enhanced the expression of FN,and enhanced the ratio of collagen type I and type IE (p<0.01) .(2) Aid effects' mechanism: Compared with Aid groups, Ald+Spi groups markly decreased CFs MTT results, cell total protein content, and the expression of PCNA (p <0.01) , Cellcycle distribution analysis indicate that Ald+Spi groups' cell number were increased in Go-Gi period (p<0.01) , markedly decreased in S and Ga-M period (p< 0.01) ; Ald+Spi groups greatly decreased Collagen synthesis content, the expression of FN,and the ratio of collagen type I and type III (p<0.05) , all this indicate that Spi inhibits voluminous of Aid's action. Compared with Aid groups, Ald+losartan groups inhibits only part of Aid's action, Ald+losartan groups decreased CFs MTT results, cell total protein content, Collagen synthesis content, the ratio of collagen type I and type III(p<0.05) , had no obvious in the expression of PCNA,total cell number in S period,and the expression of FN (p>0.05) . All this indicate that Aid promote CFs proliferation not only through membrane-receptor signal,but also through nucleus-receptor signal,but the latter is leading.(3) Aid 's effects and its mechanism: Compared with Aid groups, Ald+Mat groups decreased CFs MTT results, cell total protein content, and the expression of PCNA, Collagen synthesis content, the expression of FN, the ratio of collagen type I and type III (p<0.01) , Cellcycle distribution analysis indicate that Ald+ Mat groups' cell number were markedly decreased in S and G2-M period (p<0.01) ,Ald+Mat groups markedly decreased the expression of TGF8-1 (p<0.01), enhanced the enzyme activity and the expression of smad7 (p<0.01) ,all of this indicate that Mat can inhibits CFs proliferation and secretion,and the effect was of dose dependent-manner.When Matconcentration was above 0. 5mmol/L, Mat's effects was t...