Lipopolysaccharide-induced Endothelial Cell-derived Exosomes Mediate Myocardial Cell Injury

objective: Sepsis is a clinical syndrome of infection-induced host dysregulation and life-threatening organ dysfunction.It usually occurs after infection,ischemia-reperfusion injury,trauma,shock,and other severe disease states.Endotoxin is the main pathogenic factor of bacterial sepsis.It is released by various pathogenic bacteria after invading the host,especially Gram-negative bacteria.The main component of endotoxin is lipopolysaccharide(LPS),which promotes the release of multiple inflammatory factors by inflammatory cells,leads to a waterfall outbreak of systemic inflammatory response and causes multiple organ dysfunction.Sepsis progresses rapidly and the mortality rate reaches 20%.Sepsis combined with cardiac dysfunction is called sepsis-induced cardiomyopathy(SIC).SIC is a severe condition and with a mortality rate of 70%.Although the research on SIC has been going on for decades,its pathogenesis has not yet been fully elucidated.Endothelial cells(EC)are distributed on the inner side of the blood vessels.They form a smooth interface to maintain normal blood flow and are also an important natural barrier of the vasculature.ECs are the first to be violated when the body is subjected to various stimuli such as infection,ischemia-reperfusion,trauma,shock,and so on.After breaking through the vascular intima,the pathogens cause damage to the tissue and organs of the whole body.Being stimulated by pathogens,ECs synthesis and releaseextracellular vesicles(EVs)which containing biologically active substances such as proteins,lipids,and nucleic acids.EVs can mediate crosstalk between cells.Exosomes are a type of EVs that have received increasing attention in recent years.They are about 40-100 nm in diameter and can carry microRNAs(miRNAs).It has been found that miRNAs regulate the function of cardiomyocytes and their mechanisms have been gradually reported.In this study,we aimed to explore the association and mechanism of miRNAs carried by endothelial cell-derived exosomes and myocardial cell dysfunction in sepsis.Methods: We cultured the thoracic aorta endothelial cells of Sprague-Dawley rats by enzymatic digestion method.ECs were stimulated with LPS in the experimental group for 6 h.PBS was used to treat ECs in the control group.The total RNA of the two groups were collected respectively and subjected to a miRNA sequencing to identify the differentially expressed miRNAs(fold change>1.5,p<0.05).It was found that the expression of miR-155-5p was significantly increased in LPS challenging group.Endothelial cell-derived exosomes were harvested by differential centrifugation in low-temperature and polyethylene glycol precipitation.The expression of miR-155-5p was also increased in exosomes from LPS challenging ECs.MiR-155-5p mimics were transfected into cultured cardiomyocytes(CMCs).The content of malondialdehyde(MDA)and the relative expression of BCL-2/BAX protein in CMCs were detected.The cell viability was detected by CCK-8 method.The level of oxidative stress and the degree of cellular damage were assessed.Results: 1.The maturity,activity,and purity of the ECs isolated and cultured from blood vessels by collagenase digestion method met the experimentalrequirements.Reliable CMCs with high purity can be obtained by trypsin combined with type II collagenase digestion method.2.The exosomes extracted by differential centrifugation in low-temperature and polyethylene glycol precipitation method have high purity and activity.By detecting by electron microscopy,they had typical characteristics of the exosome.3.ECs expressed a variety of microRNAs under LPS stimulation.Among them,miR-155-5p can be released to the outside of the cell by exosomes.4.In vitro,miR-155-5p mimics induced oxidative stress and promoted apoptosis in neonatal rat cardiomyocytes.Conclusions: Vascular ECs produce exosomes containing miR-155-5p when stimulated by LPS.Endothelial cell-derived exosomes may act on cardiomyocytes through paracrine or peripheral circulation.They mediate oxidative stress and promote apoptosis of cardiomyocytes during sepsis by miR-55-5p.It may be an essential pathogenic mechanism of sepsis-induced cardiomyopathy.