Cytoprotection And Mechanism Of M₃-mAChR Against On Hypoxia Injury Induced By CoCl₂ In Rat Cardiomyocyte Line H9c2

Objective To investigate the cytoprotection and mechanism of carbachol（CCH）stimulate M₃-m ACh R against on hypoxia injury induced by Co Cl₂ in rat cardiomyocyte line H9c2.Methods Select the normal rat cardiomyocyte line H9c2 as the control group,rat cardiomyocyte line H9c2 were managed with Co Cl₂ to establish hypoxia injury model,M₃-m ACh R specific agonist CCH and antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide（4-DAMP）were used for intervention.The cell viability was tested by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT）;The apoptosis in cardiomyocyte was detected by flow cytomery（FCM）;The intracellular levels of reactive oxygen species（ROS）weremeasuredby2’,7’-Dichlorodihydrofluorescein diacetate（DCFH-DA）staining;Using hoechst 33258 nuclear staining observe changes of the morphology and quantity of apoptotic cells by fluorescence microscope;The expression levels of caspase-3,hypoxia inducible factor-1（HIF-1）and heme oxygenase-1（HO-1）proteins were measured by Western blot assay.Results In hypoxia group,apoptosis rate and ROS levels were significantlyincreased（P<0.01）,cellproliferationwasdistinctly decreased（P<0.01）,and the expression of HIF-1α,caspase-3 and HO-1 proteins were up-regulated obviously（P<0.01）;After treated with CCH,apoptosis rate and ROS levels of cardiomyocytes was significantly decreased（P<0.01）,while the proliferation of cells was significantly increased（P<0.01）,and the expression of HIF-1αand HO-1 proteins were increased（P<0.01）,and the expression of caspase-3 protein was significantly decreased（P<0.01）.Moreover,when applying 4-DAMP for intervention,these effects that mediated by CCH was abolished.Conclusions CCH stimulate M3-m ACh R against on hypoxia injury induced by Co Cl₂ in rat cardiomyocyte line H9c2,and the mechanism may be related to down-regulation of caspase-3 expression and up-regulation of HIF-1αand HO-1 protein expression.