The Effects And Mechanism Of MicroRNA-21 On Cardiac Fibrosis And Cardioprotective Effects Of Celastrol

Part1: The effects and mechanism of miR-21 on the regulation of cardiac fibroblasts[Objective] Cardiac fibroblasts(CFs)are the effector cells of myocardial fibrosis(MF).The functional status of CFs plays a core role in the pathological process of MF.Study found that miR-21 is involved in the regulation of fibrotic diseases.This part of the study will discuss the role and mechanism of miR-21 to regulate the function and state of CFs.[Methods] The primary CFs was isolated and cultured,and a cell growth promoting fibrosis model(TGF-beta 1)was constructed,and transfected with miR-21 mimic and miR-21 inhibiter to inhibit or overexpress miR-21.They were randomly divided into control group,TGF-beta 1 group,miR-21-mimic group and TGF-beta 1+ miR-21-inhibiter group.The expression of miR-21 was detected by real-time PCR,and the expression of p ERK/ERK and alpha-SMA was detected by Western blot,and the proliferation of cells was detected by MTT.[Results] Compared with control group,group TGF-beta 1 CFs proliferation was significantly increased;the difference was statistically significant(P < 0.05).Compared with control group,collagen synthesis and the expression of alpha-SMA increased significantly in TGF-beta 1 group,the difference was statistically significant(P < 0.05);TGF-beta 1 can increase the level of miR-21 in CFs.Compared with control group,the proliferation of CFs was significantly increased in miR-21 mimic group(P < 0.05),collagen synthesis and the expression of alpha-SMA was significantly increased.TGF-beta 1 +miR-21-inhibiter group compared with the TGF-beta 1 group,collagen expression decreased(P < 0.05),alpha-SMA expression level has been decreased(P < 0.05);miR-21 mimic group compared to the control group of p ERK/ERK cells increased and the difference was significant(P < 0.05),while the TGF-beta 1+miR-21-inhibitergroup,the expression of p ERK/ERK was significantly lower than in TGF-beta 1 group.(P < 0.05)[Conclusion] This study indicates that miR-21 may be a downstream regulator of TGFbeta 1,which has a fibrotic effect on CFs,promotes CFs proliferation and activation,and increases collagen synthesis and alpha-SMA expression.MiR-21 may regulate p ERK/ERK mediated TGF-beta 1 effect.Part 2: The effect and mechanism of miR-21 on endothelial-mesenchymal transition of endothelial cells[Objective] Endothelial cell endothelium transformation(End MT)is one of the sources of myofibroblast.End MT is derived from the loss of inherent characteristics of endothelial cells and the acquired phenotype of mesenchymal cells.The human umbilical vein endothelial cells(HUVECs)model was established in vitro,and discussed the effect and mechanism of miR-21 on End MT.[Methods] HUVECs was isolated and cultured,and End MT were induced by TGF-beta 1 in HUVECs,and miR-21 mimic and miR-21 inhibite were transfected to inhibit or over express miR-21.The cells were divided into 4 groups: the control group,the TGF-beta 1 group,the miR-21-mimic group and the TGF-beta 1+ miR-21-inhibiter group.The real-time PCR was used to detect the expression of miR-21.Immunofluorescence and immunoblotting were used to detect the expression of FSP-1,a marker of endothelial cell marker CD-31 and interstitial cells.[Results] TGF-beta 1 up-regulated HUVECs miR-21 level,there was significant difference compared with control group(P < 0.05);TGF-beta 1/miR-21 can induce HUVECs End MT expression for endothelial cell marker CD31 expression decreased(P < 0.05),and mesenchymal cell marker FSP-1 expression increased(P < 0.05);the inhibition of miR-21 can inhibit TGF-beta 1 induced by End MT.[Conclusion] TGF-beta 1 can regulate End MT induced by miR-21.Inhibition of miR-21 partially inhibits End MT induced by TGF-beta 1.Part 3: The regulate effect and mechanism of celastrol on cardiac fibroblasts[Objective] Celastrol is an effective natural bioactive compound extracted from the roots of Tripterygium wilfordii.It has been proved to be of great value in anti-inflammatory,anticancer,anti rheumatic and autoimmune diseases.The study found that Tripterygium wilfordii has a protective effect on the heart.MiR-21 participates in the regulation of the proliferation and activation of CFs.In this part,the purpose of this study is to explore the effect of celastrol on the functional status of CFs and the ability to regulate miR-21 and to explore the mechanism.[Methods] The primary CFs was isolated and cultured,and the TGF-beta 1 fibrotic model was constructed,and celastrole was intervened.HUVECs were randomly divided into control group,TGF-beta 1 group and celastrol group.The expression of miR-21 was detected by real-time PCR method and the expression of alpha-SMA and p ERK/ERK was detected by Western blot;MTT method was used to detect the proliferation of cells;El ASA method was used to detect the cell proliferation.[Results] The proliferation of CFs was detected by MTT method.The analysis data showed that the inhibitory effect of celastrol on CFs was dependent on concentration and time.With different concentrations of celastrol(0.2,1,5 M)intervention could inhibit TGF-beta 1 fibroblasts proliferation level induced increased,which was statistically significant in 1 M and 5 M concentration(P < 0.05).Compared with group TGF-beta 1,celastrol group CFs culture supernatant of collagen type III collagen levels decreased(P < 0.05,P < 0.05),the expression level of alpha-SMA also decreased(P < 0.05);celastrol can inhibit TGF-beta 1 induced increasing of miR-21;TGF-beta 1 compared with control group cells increased the level of p ERK/ERK,the difference was statistically significant(P < 0.05),while the celastrol group compared with the TGF-beta 1 group,the expression of p ERK/ERK was significantly lower(P < 0.05).[Conclusion] This study indicates that celastrol can inhibit the proliferation and activation of CFs and collagen synthesis,and the effect of celastrol may be related to down regulating the miR-21 and p ERK/ERK signaling pathways of CFs.Part 4: The effects and mechanism of celastrol on End MT of HUVECs[Objective] This study investigates the effect and mechanism of celastrol on End MT in HUVECs.[Methods] HUVEC was isolated and cultured,and TGF-beta 1 was established to induce the End MT model of HUVECs,and celastrol was intervened.HUVECs were randomly divided into three groups: control group,TGF-beta group,and celastrol group.real-time PCR method was used to detect the expression of miR-21.Immunofluorescence and Western blotting were used to detect the expression of FSP-1 and CD-31 in endothelial cell markers.The expression of inflammatory factors(ICAM-1,VCAM-1 and TNF-a)was detected by Western blotting.[Results] TGF-beta 1 induced End MT in HUVECs by up regulating the level of miR-21,down regulating the expression of CD31,and upregulate the expression of FSP-1,a specific marker of interstitial cells.Celastrol can inhibit the increase of miR-21 expression induced by TGF-beta 1 and inhibit the occurrence of End MT.It shows that the expression of CD31,a specific marker of endothelial cells,is increased,while the expression of FSP-1 in interstitial cells is decreased.Celastrol inhibits the End MT effect of TGF-beta 1 by inhibiting the level of miR-21 and the expression of inflammatory factors in HUVECs.[Conclusion] TGF-beta 1 can induce EndMT by regulating miR-21 induced HUVECs.Celastrol can improve End MT induced by TGF-beta 1 by inhibiting miR-21 and inhibiting the expression of inflammatory related factors.Part 5: The effect and mechanism of celastrol on cardiac fibrosis in pressure overload animals[Objective] Celastrol has a protective effect on CFs and HUVECs in vitro.In this study,we observed the effects of celastrol on cardiac fibrosis and cardiac function in pressure overload animals model,and discussed the mechanism.[Methods] C57BL/6 mice were selected and the experimental animals were randomly divided into sham operation group,TAC group and celastrol group(TAC+ celastrol).The transverse aortic constriction(TAC)method was used to establish an animal model of myocardial fibrosis induced by pressure overload.Administered 1 time daily(1mg/kg,intraperitoneal injection),12 weeks after the test.Heart tissue samples were collected,HE staining,Masson staining and picrosirius red staining observe changes of myocardial tissue.The expression of miR-21 was detected by real-time PCR method and the expression of a-SMA was detected by Western blot.Western blotting and immunofluorescence were used to detect the expression of endothelial cell markers(CD31)and fibroblast markers(FSP-1)in myocardium.To detect the expression of p ERK/ERK in each experimental animal,inflammatory factors(ICAM-1,VCAM-1,TNF-a);using echocardiography in detecting left ventricular diastolic anterior wall thickness(LVAWd),left ventricular systolic anterior wall thickness(LVAWs),left ventricular diastolic posterior wall thickness(LVPWd),left ventricular systolic posterior wall thickness(LVPWd),left ventricular end diastolic intemal diamension(LVEDd),left ventricular end systolic internal diamension(LVEDs),ejection fraction(EF),left ventricular fractional shortening(FS).[Results] Compared with the sham group,HE staining,Masson staining and Sirius red staining showed significant myocardial fibrosis in the TAC group.Compared with the sham group,the expression of collagen expression was increased in the TAC group.The expression of protein expression of alpha-SMA in the TAC group was increased than sham group,and the expression of miR-21 in TAC group was increased compared with sham operation group.Compared with the sham group,the p ERK/ERK of myocardial tissue in the TAC group was increased.The expression of CD31 in endothelial cells was decreased in TAC group than in sham group,while the expression of FSP-1 in was increased.Compared with the sham group,the expression of inflammatory factor TNF-a,proinflammatory factor VCAM-1 and ICAM-1 in the model group of mice was significantly increased.Compared with sham group,TAC group of animal cardiac ventricular wall thickening significantly: left ventricular diastolic anterior wall thickness(LVAWd),left ventricular systolic anterior wall thickness(LVAWs),left ventricular diastolic posterior wall thickness(LVPWd),left ventricular systolic posterior wall thickness(LVPWs)increased.In the TAC group,there was significant decrease in cardiac function: a significant decrease in ejection fraction(EF)and a significant reduction in the short axis shortening(FS)of the left ventricle.The treatment of celastrol can significantly ameliorate the pathological change of myocardial fibrosis and reduce the collagen deposition in myocardial tissue.The expression of type I collagen and type III collagen in celastrol group was lower than that in TAC group,and the expression of alpha-SMA in celastrol group was lower than that in TAC group,and miR-21 expression in celastrol group was lower than that in TAC group.p ERK/ERK in celastrol group was lower than that in TAC group,and celastrol could inhibit the changes of End MT in the myocardium of pressure overload mice.The intervention of celastrol inhibited the inflammatory response of myocardium in mice induced by pressure overload,and increased the expression of TNF-a,VCAM-1 and ICAM-1 compared with the TAC group.The intervention of celastrol significantly improved the ventricular wall thickening / dilatation of the heart cavity and the decrease of cardiac function in the experimental mice.Compared with TAC group,left ventricular diastolic anterior wall thickness(LVAWd),left ventricular systolic anterior wall thickness(LVAWs),left ventricular diastolic posterior wall thickness(LVPWd)and left ventricular systolic posterior wall thickness(LVPWs)decreased.The ejection fraction(EF)was increased compared with that of the TAC group,and the short axis shortening rate(FS) of the left ventricle was higher than that of the TAC.[Conclusion] Celastrol can improve the myocardial fibrosis,End MT and impaired heart function induced by pressure overload.The effect of celastrol on cardiac fibrosis and End MT may be related to inhibition of miR-21,p ERK/ERK and inhibition of inflammatory factor TNF-a,proinflammatory cytokines VCAM-1 and ICAM-1.