The Effect Of Antiapoptotic Protein HAX-1 On Myocardial Injury Induced By Lipopolysaccharide (LPS) And Its Underlying Mechanism

Cardiovascular disease is a common type of disease in the clinic,many of it is accompanied by inflammatory reactions such as myocarditis,dilated cardiomyopathy,acute myocardial infarction,heart failure and so on.The common pathogen that induces inflammation is Gram-negative bacteria(G-),such as E.coli,which produces endotoxin--LPS(lipopolysaccharide)is the main pathogenic component that induces inflammation in the body,which forms endotoxemia when accumulated in a large amount in the blood,eventually induce the failure of multiple organ function.In recent years,endotoxin damage to the heart has gradually attracted the attention of many researchers.HAX-1(HS1-associated protein X-1,HS1 associated protein X-1)is a novel apoptosis regulatory protein,some studies have found that during the inflammatory response of certain diseases(such as actinic keratosis),the synthesis and expression of intracellular inflammatory factors are increased,while the anti-apoptotic protein HAX-1is down-regulated.However,there is no relevant research report on the possible role of HAX-1 and its mechanism of action in the process of inflammatory injury of cardiomyocytes.As a novel apoptosis regulatory protein,HAX-1 is widely expressed in cardiomyocytes and is one of the anti-apoptotic proteins that play a role in cardioprotection.Therefore,we hypothesized that HAX-1 may play a role in LPSinduced myocardial inflammatory injury.This topic takes NRCMs(Neonatal Rat Cardiac Myocytes)obtained by laboratory independent separation as the research object,the cardiomyocyte inflammatory injury model was established by treating lipopolysaccharide with NRCMs,and transfection of NRCMs with HAX-1 adenovirus to regulate the expression of HAX-1 in cells,to explore the effect of HAX-1 on inflammatory injury of cardiomyocytes and the possible mechanism of action,in order to obtain new ideas for the treatment of myocardial inflammatory injury.Objective: To study the effects of anti-apoptotic protein HAX-1 on inflammatory injury of neonatal rat cardiomyocytes.Method: 1.Select the birth 1-3 days SD rat neonatal rats,isolated and cultured NRCMs in vitro,and treated with an anti-α-SMA antibodies by immunofluorescence method identified NRCMS;2.LPS treatment of neonatal rat cardiomyocytes,MTT and Q-PCR method was used to design and establish a model of myocardial cell inflammatory injury in vitro,and the expression of HAX-1 protein in NRCMs was detected by Western Blot;3.The HAX-1 adenoviral vector which can be expressed in cardiomyocytes was constructed and transfected into neonatal rat cardiomyocytes to up-or down-regulate the expression level of HAX-1 protein in the cells,and then the NRCMs were treated with LPS to establish an in vitro inflammatory injury model.The effects of HAX-1 on inflammatory injury of cardiomyocytes induced by LPS and its possible mechanism were explored by Q-PCR and Western Blot.Result: 1.Successfully isolate,culture and identify primary cardiomyocytes of NRCMs.In this experiment,an effective and practical method for isolation and culture of NRCMs was used to obtain cardiomyocytes with good growth state and separation purity of more than 90%,which can ensure the scientificity,rigor and accuracy of experimental research;2.In the LPS-induced inflammatory injury model of NRCMs,the NF-κB inflammatory signaling pathway was activated,and the expression level of intracellular inflammatory factors is increased by 5-fold(IL-1β)and 9-fold(IL-6)compared with the normal group,and the expression level of HAX-1 protein was significantly decreased.The results showed that HAX-1 may play a role in inflammatory injury of NRCMs;3.When HAX-1 is overexpressed,compared with the GFP model group,the synthesis and expression of related inflammatory factor m RNA levels in NRCMs was reduced by about 30%.In the NF-κB inflammatory signaling pathway,the expression of IκBα protein was increased by about 30%,and the expression of phosphorylated protein(P-p65,P-IκBα)was down-regulated by about 25%.The results showed that HAX-1 may inhibit the activation of NF-κB inflammatory signaling pathway by inhibiting the phosphorylation degradation of IκBα protein,reduce the expression of downstream protein,and down-regulate the expression of inflammatory cytokine m RNA in cardiomyocytes.HAX-1 plays a protective role in inflammatory damage of NRCMs.Conclusion: In inflammatory injury of neonatal rat cardiomyocytes,HAX-1 inhibits LPSinduced activation of NF-κB inflammatory signaling pathway by inhibiting the downregulation of IκBα expression and decreasing the phosphorylation level of p65 and IκBα protein,and then down-regulating the transcriptional synthesis and expression of intracellular inflammatory factors,thereby reducing myocardial cell inflammatory damage,play a protective role.