A Study Of Tanshinone ⅡA Induction Of Human Placental Mesenchymal Stem Cell Differentiation Into Cardiomyocytes,the Enhanced Homing Of Pre-differentiated Cells And The Related Mechanisms

Objective To evaluate the effect of tanshinone ⅡA in the differentiation of human placenta-derived mesenchymal stem cells(h PDMSCs)into cardiomyocytes,the homing of pre-differentiated cells,and to investgate the related underlying mechanisms.Methods1.The h PDMSCs were isolated using the explant culture method,and the relevant antigens were identified by the flow cytometry.2.The forth to the sixth generation h PDMSCs were treated with different concentrations of tanshinone ⅡA.The MTS method was used to measure cell proliferation every four days,and select the non-toxic doses which had no effect on cell proliferation(no critical concentration affected)for experiments.3.The h PDMSCs were treated with tanshinoneⅡA or tanshinoneⅡA combined with the β-catenin agonist WAY-262611,and a control group was established.The MTS method was used to measure cell proliferation every four days.4.The h PDMSCs were treated with tanshinoneⅡA or tanshinoneⅡA combined with the β-catenin agonist WAY-262611,and a control group was established.The scratch test was used to analyze the migration and movement of cells.5.The h PDMSCs were treated with tanshinoneⅡA or tanshinoneⅡA combined with the β-catenin agonist WAY-262611,and a control group was established.After 20 days,the collected cells were detected by Western-blot assay for the following related proteins:(1)The expression of cell cycle regulatory proteins(CDK4,CDK6,Cyclin D1),cell proliferation related antigens(Ki67,PCNA)and apoptosis regulatory proteins(Caspase-3,Cleaved Caspase-3);(2)The expression of myocardial-specific proteins(GATA-4,ANP,α-SCA,and c Tn I);(3)The expression of canonical Wnt signaling pathway related proteins(APC,Gsk-3β,β-catenin)and non-canonical Wnt signaling pathway related proteins(Wnt5a,Wnt11);(4)The expression of homing-associated chemokines(CXCR-4,CCR-2).Results1.The h PDMSCs were successfully isolated by the explant culture method.2.Compared with the control group,the proliferation of cells treated with tanshinone IIA decreased significantly,but the decrease was not accompanied by changes in apoptosis.The expression of apoptosis regulatory proteins(Caspase-3,Cleaved Caspase-3)were not change significantly(P>0.05),but the expression of cell proliferation related antigens(Ki67,PCNA)and cell cycle regulatory proteins(CDK4,CDK6,Cyclin D1)were significantly decreased(P<0.05).3.Compared with the control group,the expression of myocardial-specific proteins(GATA-4,ANP,α-SCA,and c Tn I)in cells treated with tanshinoneⅡA were significantly increased(P<0.05),and the ability of cells to migrate to the injured area were significantly enhanced(P<0.05).4.Compared with the control group,the expression of APC and Gsk-3β in the canonical Wnt signaling pathway in cells treated with tanshinoneⅡA were significantly increased(P<0.05),while the expression of β-catenin significantly decreased(P<0.05),and the expression of Wnt5 a and Wnt11 in the non-canonical Wnt signaling pathway also significantly increased(P<0.05).At the same time,the expression of homing-associated chemokines CXCR-4 and CCR-2 were also significantly increased(P <0.05).5.After adding the β-catenin agonist WAY-262611,the cell proliferation were significantly increased,the expression of cell proliferation related antigens(Ki67,PCNA)and cell cycle regulatory proteins(CDK4,CDK6),Cyclin D1)were significantly increased(P<0.05),the expression of myocardial-specific proteins(GATA-4,ANP,α-SCA,and c Tn I)were significantly decreased(P<0.05).At the same time,the ability of cell to migrate to the injured area were significantly decreased(P<0.05),and the expression of homing-associated chemokines CXCR-4 and CCR-2 were also significantly decreased(P< 0.05).Conclusions Tanshinone ⅡA can induce differentiation of h PDMSCs into cardiomyocytes and enhance the homing ability of pre-differentiated cells by regulating the Wnt/β-catenin signaling pathway.