Baicalin Alleviates Cardiomyocyte Oxidative Injury Through Suppressing Wnt/?-catenin Signal Pathway

Objective:To evaluate the effect of baicalin on cardiac oxidative injury and investigate the underlying molecular mechanism base on Wnt/?-catenin signal pathway.Methods:We first used TOPFLASH/Renilla reporter gene assay to evaluate the effect of baicalin on the activation of Wnt/?-catenin pathway.Then,we used H2O2-treated H9c2 cells as an in vitro model for evaluating the effects of baicalin on cardiac oxidative injury.MTT assay was used for evaluating the effct of baicalin on the cell viability in H2O2-treated H9c2 cells.Western Blot was used for determining the effct of baicalin on Cleaved caspase-3,Bcl-2,Bax,P-catenin and its downstream targets Axin-2 and c-Myc,following treatment with different concentrations of baicalin in H2O2-stimulated H9c2 cells.Moreover,we used siRNA for downregulating p-catenin expression to verify the role of p-catenin in H2O2-treated H9c2 cells.Finally,we used LiCl,a GSK-3? inhibitor which is commonly used to stabilize ?-catenin,to verify above-observed results.Furthermore,we used H2O2-treated SD neonatal rat cardiomyocytes as an in vitro model for verifying above conclusion.Results:The results of TOPFLASH/Renilla reporter gene assay indicated that Wnt/?-catenin signaling could be significantly activated upon stimulation with different agents,including Wnt3a,LiCl,BIO and ?-catenin?N.However,baicalin exhibited a specific and significant inhibition effect on TOPFLASH activation with a dose-dependent manner.MTT assay showed that baicalin could significantly inhibit H2O2-induced loss of H9c2 cell viability.Western Blot assay showed that baicalin profoundly attenuated H2O2-induced cell apoptosis,as evidenced by the down-regulation of Cleaved caspase-3 and the increase of anti-apoptotic Bcl-2/Bax ratio after baicalin treatment in H2O2-stimulated H9c2 cells.Furthermore,baicalin remarkably decreased the expression of ?-catenin and its downstream Axin-2 and c-Myc in H2O2-treated H9c2 cells;and knockdown of ?-catenin expression also inhibited H2O2-induced cell apoptosis.Finally,LiCl(a ?-catenin stabilizer)induced H9c2 cell apoptosis by increasing P-catenin expression,which however could be significantly neutralized by baicalin treatment.We also found that baicalin significantly inhibited H2O2-induced loss of cell viability,cell apoptosis,as well as the expression of nuclear ?-catenin in SD neonatal rat cardiomyocytes.