Tanshinone ?A Promotes Myocardial Regeneration By Regulating Wnt Signaling Pathway

ObjectiveTo evaluate the effect of tanshinone ?A on the differentiation of rat embryonic myocardium H9c2 cells into cardiomyocytes,and to explore the role of tanshinone ?A in myocardial regeneration and its related mechanisms.Methods1.Treat H9c2 cells with different concentrations of tanshinone ?A(0,0.01,0.02,0.04,0.06,0.08,0.1,0.2,0.4,0.6,0.8,1,2,4,6,8,10,20 mg/L),after 4days,MTS assay was used to detect cell proliferation,and Western-blot assay was used to detect expressions of cell cycle regulatory proteins(CDK4,CDK6,CyclinD1),cell proliferation-associated antigen(Ki67,PCNA),and apoptosis regulatory proteins(Cleaved Caspase-3).A suitable concentration of tanshinone ?A was screened according to the above criteria for the following experiment.2.H9c2 cells were treated with tanshinone ?A,tanshinone ?A+?-catenin agonist WAY-262611,simultaneously setting a control group without drugs.after 7 days,the experiment was terminated and the following related tests were performed:(1)Western-blot assay was used to detect the expression of canonical Wnt signaling pathway-related proteins(GSK-3?,APC,?-catenin)and non-canonical Wnt signaling pathway-related proteins(Wnt11,Wnt5a);(2)Western-blot assay was used to detect the expression of cell cycle regulatory proteins(CDK4,CDK6,CyclinD1)and cell proliferation-associated antigens(Ki67,PCNA);(3)Immunohistochemistry and Western-blot assay were used to detect theexpression of cardiac troponin I(cTnI)and cardiac troponin T(cTnT).Results1.When the concentration of tanshinone ?A reached 0.4 mg/L,the proliferation rate of H9c2 cells decreased significantly,when the concentration reached 0.6 mg/L~2 mg/L,the cell proliferation rate slowed down but remained in a proliferative plateau.We choose 0.6 mg/L tanshinone ?A to treat cells,the expression of cell cycle regulatory proteins(CDK4,CDK6,CyclinD1)and cell proliferation-associated antigens(Ki67,PCNA)were significantly decreased(P<0.05),but there was no significant change in the expression of apoptosis-regulated proteins(Cleaved Caspase-3)(P>0.05).In view of the inhibition of cell proliferation during proliferative plateau but no apoptosis,we used 0.6mg/L tanshinone ?A for the study.2.Compared with the control group,expressions of cTnI and cTnT in H9c2 cells treated with tanshinone ?A were significantly increased(P<0.05),expressions of GSK-3? and APC in the canonical Wnt signaling pathway were significantly increased(P<0.05),and the expression of ?-catenin was significantly decreased(P<0.05),expressions of Wnt11 and Wnt5 a were significantly increased in the non-canonical Wnt signaling pathway(P<0.05).3.Compared with the tanshinone ?A treatment group,expressions of?-catenin,CDK4,CDK6,CyclinD1,Ki67 and PCNA in cells treated with tanshinone ?A+?-catenin agonist WAY-262611 were significantly increased(P<0.05).while expressions of cTnI and cTnT were significantly decreased(P<0.05).ConclusionsTanshinone ?A can promote the differentiation of H9c2 cells into cardiomyocytes,and its mechanism is related to its ability to regulate Wnt signaling pathway.Tanshinone ?A can promote myocardial regeneration to treat ischemic heart disease.