Mechanisms Responsible For Inhibition Of NE-induced Apoptosis By Berberine In Neonatal Rat Cardiomyocytes

ObjectiveTo observe the effect of berberine(Ber) on norepinephrine(NE) - induced apoptosis in neonatal rat cardiomyocytes,and further investigate the underlying mechanisms.MethodsFirstly,neonatal rat cardiomyocytes were isolated,cultured,and identified by immunofluorescence staining with monoclonal antibodies directed against troponin I observed under a confocal laser-scanning microscope.Secondly,the cultured neonatal rat cardiomyocytes were stimulated with various concentrations of NE or/and various concentrations of Ber in serum-free conditions for 48 h or 72 h.The activity of lactate dehydrogenase(LDH) in the supernatant was examined.Thirdly,the neonatal rat cardiomyocytes were exposed to various concentrations of NE or/and various concentrations of Ber.After 48h-incubation,hoechst33258,isothiocyanate (FITC)-conjugated annexin-Ⅴand propidine iodide(PI) staining were used to detect apoptosis of neonatal rat cardiomyocytes,and the DNA fragmentation was also confirmed by ladder formation in agarose gels.Lastly,production of intracellular reactive oxygen species(ROS) and activation of caspase 2 and caspase 3 were measured by using the flow cytometry.Furthermore,the level of secreted tumor necrosis factor a(TNFa) was measured by ELISA.ResultsTroponin I was found to express in the cultured cells.NE at concentrations of 10,50,100μmol/L induced an increase in activity of LDH in the supematant,which were inhibited by treatment with 0.5,1.0,2.0μmol/L Ber,respectively.Hoechst33258,FITC-conjugated Annexin-Ⅴ,PI staining and agarose gel electrophoresis demonstrated that 50μmol/L of NE induced apoptosis of cardiomyocytes and Ber significantly attenuated NE-induced apoptosis in a dose - dependent manner.Moreover,Ber at dose of 2μmol/L markedly decreased the ROS and TNFαproduction,inhibited the activation of caspase-2,3 induced by NE.ConclusionBer inhibits NE-induced apoptosis in neonatal rat cardiomyocytes through blocking a ROS-TNFα-Caspase signaling pathway.