Research Of Autophagy In LPS Preconditioning In Cardiac Myocytes Of Neonatal Rats

Background and objective:Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.Still it remains a major challenge to pediatric intensive care clinicians and is one of the leading causes of pediatric death worldwide.The exact mechanism of sepsis-induced cardiomyopathy is not fully understood.Several studies suggest that proinflammatory mediators,released during the host immune response,act as "myocardial depressant factors.”Endotoxemia refers to the presence of Lipopolysaccharides in the systemic circulation,which often exists with sepsis.In addition,Lipopolysaccharides is also likely to play a signifcant role in the structural modifcations in the myocardium and alterations in myocardial bioenergetics.Autophagy is one of the innate immune defense mechanisms against microbial challenges.It is activated initially in sepsis,and is thought to play a protective role against multiple organ injuries.The phenomenon of ischemic preconditioning refers to the resistance to prolonged ischemia induced by brief episodes of ischemia in cardiac myocytes.And it has been demonstrated that pretreatment with LPS can also induce protective effect in cardiac myocytes against ischemia.However it is unknown if the LPS preconditioning could induce the resistance to lethal dose LPS.Based on the information above,we hypothesized that low dose LPS preconditioning to could induce the resistance to lethal dose LPS in cardiac myocytes,in which the changes of autophagy play a role in this study,we attempted to experimentally prove the protective role of low dose LPS preconditioning and investigate the mechanism involved.MethodsPart 1:Neonatal rat ventricular cardiac myocytes were isolated and cultured.After cultured for 24h,cardiac myocytes were randomly divided into 6 groups:(1)Control group:DEME treatment for 20h.(2)PRE group:1?g/ml Lipopolysaccharides(LPS,dissolved in DMEM)for 4h,after the preconditioning the LPS was replaced by normal DMEM for 16h.(3)PRE+High dose 5?g/ml group:after the preconditioning by 1?g/ml LPS for 4h,LPS was removed for 12h,and then 5?g/ml LPS was added again and stimulation for 4h.(4)High dose 5?g/ml group:5?g/ml LPS treatment for 4h.(5)PRE+High dose 50?g/ml group:after the preconditioning by 1?g/ml LPS for 4h,LPS was removed for 12h,and then 50p,g/ml LPS was added again and stimulation for 4h.(6)High dose 50?g/ml group:50?g/ml LPS treatment for 4h.Part 2:Cardiac myocytes were randomly divided into 8 groups:(1)Control group:DEME treatment for 20h.(2)Rapamycin group:0.1?M/ml rapamycin treatment for 4h,and then replaced by normal DMEM for 16h.(3)PRE group:1?g/ml LPS for 4h,after the preconditioning the LPS was replaced by normal DMEM for 16h.(4)PRE and 3-MA group;1?g/ml LPS with 5mM/ml 3-MA for 4h,followed by replacement of normal DMEM for 16h.(5)Rapamycin+ High dose LPS group:after the pretreatment by 0.1?M/ml rapamycin for 4h,rapamycin was removed for 12h,and then 5?g/ml LPS was added again and stimulation for 4h.(6)PRE+High dose LPS group:after the preconditioning by 1?g/ml LPS for 4h,LPS was removed for 12h,and then 5 ?g/ml LPS was added again and stimulation for 4h.(7)PRE and 3-MA+High dose LPS group:after the preconditioning by l?g/ml LPS and 5mM/ml 3-MA for 4h,LPS was removed for 12h,and then 5?g/ml LPS was added again and stimulation for 4h.(8)High dose LPS group:5?g/ml LPS treatment for 4h.Part 3:Cardiac myocytes were randomly divided into 6 groups:(1)Control group:DEME treatment for 20h.(2)PRE +4h group:1?g/ml LPS stimulation for 4h,after the preconditioning the LPS was replaced by normal DMEM for 4h.(3)PRE+High dose LPS 4h group:after the preconditioning by 1?g/ml LPS for 4h,then replaced by 5?g/ml LPS and stimulating for another 4h.(4)PRE group:1?g/ml LPS for 4h,after the preconditioning the LPS was replaced by normal DMEM for 16h.(5)PRE+High dose LPS group:after the preconditioning by 1?g/ml LPS for 4h,LPS was removed for 12h,and then 5?g/ml LPS was added again and stimulation for 4h.(6)High dose LPS group:5?g/ml LPS treatment for 4h.Cardiac myocytes were harvested and cell culture medium was collected after the treatment.These samples were analyzed for the mRNA expression of IL-1? and TNF-? by RT-PCR,the autophagy related protein LC3-? by western-blot.Also cell culture mediums were detected for the level of lactate dehydrogenase(LDH).Results1.LPS preconditioning reduced the mRNA expression of IL-1? and TNF-a as well as the level of LDH in cell culture medium after treated with high dose LPS.Autophagy activity was enhanced by LPS preconditioning.2.Early increased autophagy by rapamycin also reduced the mRNA expression of IL-1? and TNF-a as well as LDH.Interestingly 3-MA decreasing a little the autophagy induced by LPS preconditioning and PMH group got the best protection.3.When high dose LPS was given immediately after the preconditioning,the PRE+ High dose LPS 4h group still got the decreasing mRNA expression of IL-1?and TNF-a as well as the level of LDH.The autophagy was activated again.Conclusion1.Preconditioning by low dose LPS renders the cardiac myocytes resistance to subsequent lethal dose LPS,indicating the presence of "LPS preconditioning in cardiac myocyte".2.Early upregulated autophagy induced by low dose LPS preconditioning play a role in the resistance to LPS injury.However,greater the autophagy,may not come with the better resistance.3.Even if lethal dose LPS was given immediately after the preconditioning without the resting phase,the cardiac myocytes may still get the protection.