Oxymatrine Inhibits TGF-β1 Induced Cardiac Fibroblast To Myofibroblast Transformation (FMT) By Mediating The Notch Signaling Pathway In Vitro

Objective: To elucidate the effects of Notch signaling and oxymatrine(OMT)on the cardiac fibroblast(CFs)to myofibroblast transformation(FMT)induced by transforming growth factor β1(TGF-β1);Confirmed oxymatrine regulated Notch signaling inhibition of TGF-β1 induced FMT.Methods: The primary CFs were obtained from the hearts of SD rat neonatal rats aged 1-3 days by trypsin digestion and differential adherent method.The purity of CFs was identified by vimentin immunocytochemistry.The passage 2 CFs were used in the experiment.The dose and time effect of TGF-β1-induced CFs proliferation and the inhibitory effect of OMT and Notch signaling pathway inhibitor DAPT on the proliferation of CFs induced by TGF-β1 were observed by MTT assay.The does-effect of TGF-β1-induced CFs proliferation was divided into control group(Control,Serum-free DMEM)and model group(TGF-β1,1 ng/mL,2.5ng/mL,10 ng/mL,20 ng/mL,30 ng/mL).The time-effect of TGF-β1-induced CFs proliferation were divided into control group,model group(TGF-β1,10ng/mL),with 2 designing time points 12 h and 24 h.The inhibitory effects of OMT on the proliferation of CFs induced by TGF-β1 were divided as follow group : control group,TGF-β1 group(10ng/mL),OMT group(0.001mg/mL,0.005mg/mL,0.01mg/mL,0.04 mg/mL,0.05 mg/mL).The inhibitory effect of Notch signaling pathway inhibitor DAPT on the proliferation of CFs induced by TGF-β1 was divided into control group,TGF-β1 group(10ng/mL),DAPTgroup(1μmol/L,5μmol/L and 10μmol/L).The experiment was divided into five groups: control group,TGF-β1 group(10ng/mL),DAPT group(1μmol/L),OMT low dose group(OMT-L,0.005mg/mL),OMT high dose group(OMT-H,0.01mg/mL).The morphological changes of CFs in each group were observed by HE staining.Collagen fibers were observed by picric acidsirius red staining.Immunofluorescence staining was used to detect the expression of α-SMA.The migration of CFs in each group was observed by scratch test.The changes of hydroxyproline in collagen secretion in each group were detected by hydroxyproline content assay.Flow cytometry was used to determine the proportion of CFs in each cell cycle.The experiment was divided into control group and TGF-β1 group(10ng/mL),DAPT group(1μmol/L),OMT-L group(0.005mg/mL),OMT-H group(0.01mg/mL),OMT-L + DAPT group and OMT-H + DAPT group,and used western blot to detect the protein expression of Notch-1,Jagged-1,DLL-4 and Hes-1.Results: MTT results showed that TGF-β1(10ng/mL)stimulated the CFs proliferation and the optimal treatment duration effect time was 24 h(p<0.01).After OMT and DAPT pretreatment,can significantly inhibit the proliferation of CFs(p<0.01).HE staining showed that the nucleus were stained blue-purple and the cytoplasm was stained pink of CFs.Compared with the control group,the number of cells in the TGF-β1 group was significantly increased,especially the number of binuclear cells was increased.Compared with the TGF-β1 group,the cells number in OMT group and DAPT group were decreased obviously and the intercellular space increased.The results of picric acid sirius red staining showed that pretreatment with OMT and DAPT can significantly decreased the cells number and collagen fibers experssion induces by TGF-β1.Immunofluorescence staining showed that α-SMA was expressed in green fluorescence and blue fluorescence of nucleus of CFs.The green fluorescence in TGF-β1 group was significantly increased compared with control group,and the treatment drug could significantly decreased the green fluorescence expression.Scratch test results showed that the migration ability of TGF-β1 group was significantly increased compared with control group,and the migration ability of CFs were inhibited by OMT and DAPT.Compare with control group,TGF-β1 could significantly promote the content of hydroxyproline in the supernatant,OMT and DAPT treatment can attenuates the effects.Flow cytometry showed that the ratio of cells in G1 phase was significantly decrease and the percentage of cells in S phase wassignificantly increased in TGF-β1 group.Compared with TGF-β1 group,OMT and DAPT can siginificantly inhibits the distribution of cells in S phase induced by TGF-β1.Western blot results showed that TGF-β1 induced the protein expression of Notch-1,Jagged-1,DLL-4 and Hes-1 were significantly increased.When OMT and DAPT joint application,there was no significant difference in expression of each protein between DAPT group and OMT + DAPT group.Conclusion: The results confirm that OMT inhibits Notch signaling can improve the CFs proliferation,differentiation and collagen synthesis in FMT induced by TGF-β1.Further in vivo studies will provide it more of a role in cardiovascular disease.