| Background:Sepsis is a syndrome of host dysregulation and organ dysfunction caused by infection.Severe septic shock is a common cause of death in patients in intensive care unit(ICU).Sepsis occurs in 48.9 million patients worldwide every year and causes 11 million deaths,accounting for nearly one fifth of the world’s deaths.Different countries and regions have different morbidity,and the morbidity and mortality rate of sepsis are higher in developing and underdeveloped countries.Sepsis-induced myocardial injury,also known as septic cardiomyopathy or sepsis-related myocardial depression,is one of the most common organ dysfunction in sepsis,with an incidence of more than 50% and a high mortality rate.SIC is an acute cardiac dysfunction caused by non-myocardial ischemia in patients with sepsis.It has been found that the cardiac dysfunction caused by sepsis can be mainly attributed to the direct damage of myocardial cells caused by inflammation,mitochondrial dysfunction,the release of inducible nitric oxide(i NO),the dysfunction of autonomic nervous system,the disorder of calcium circulation and the apoptosis of myocardial cells.However,the pathogenesis and molecular mechanisms of cardiac dysfunction in sepsis have not been fully elucidated.Therefore,it is of great significance to carry out in-depth study of relevant signaling pathways to understand the mechanism of cardiac dysfunction in sepsis.Micro RNA(mi RNA)is a kind of non-coding RNA with the length of 21-25 nucleotides,which widely exists in animals and plants.Mi RNAs can regulate cell proliferation,differentiation and apoptosis,and affect the occurrence and development of diseases.Circulating mi RNAs are present in human blood,sweat,urine,and milk,and some mi RNAs in the plasma of septic patients have been identified as diagnostic and prognostic markers of sepsis.At present,the relationship between mi R-195 and sepsis has been studied.The mi R-195 gene is located at17p13.1,and two mi RNAs,mi R-195-5p and mi R-195-3p,can be transcribed and expressed,but their functions are different in myocardial tissues and cells.Mi R-195-5p regulates cell function by targeting multiple targets,and in order to fully elucidate the role of mi R-195 in myocardial dysfunction in sepsis,it is necessary to further and comprehensively explore the downstream target genes of mi R-195-5p and their functions.ATF6 is a major stress sensor protein in the ERS signal transduction pathway.In the process of endoplasmic reticulum stress,ATF6 cooperates with related response elements to regulate the synthesis and degradation of cellular proteins.Studies have shown that the endoplasmic reticulum stress pathway is highly activated in sepsis,and the inhibition of ATF6 can significantly improve the inflammatory response of tissues and cells,suggesting that ATF6 plays an important role in the occurrence and development of sepsis.This study was intended to the following three aspects.Firstly,we determined the expression characteristics of mi R-195-5p in the cardiomyocyte injury model induced by LPS,and explored the regulatory effect of mi R-195-5p on the cell model.Secondly,this study confirmed that ATF6 was the target of mi R-195-5p,and the role of mi R-195-5p/ATF6 in cardiomyocyte injury model.Finally,the effect of mi R-195-5p on myocardial injury in sepsis was verified in vivo.In conclusion,this study aims to confirm that mi R-195-5p mediates cardiomyocyte apoptosis,inflammatory response and oxidative stress through inhibiting of ATF6 expression in myocardial dysfunction in sepsis.This study provides a new idea for improving the mechanism of myocardial injury in sepsis and early diagnosis and treatment of the disease.Part one: Expression of mi R-195-5p in cardiomyocytes induced by LPSObjective: This part was to determine the expression characteristics of mi R-195-5p in cardiomyocyte injury model induced by LPS,and to explore the potential effect of mi R-195-5p on the LPS-induced cells.Methods: 1)Neonatal rat cardiomyocytes were isolated and treated with LPS.The expression of mi R-195-5p was detected by q RT-PCR;2)mi R-195-5p mimics were transfected in to LPS-induced cells and then the expression of mi R-195-5p is detected by q RT-PCR.inflammatory response was determined by ELISA.The expression of Cleaved caspase 3,CHOP,ATF6 and IRE1 were measured by Western blot,the cell apoptosis and ROS(oxygen free radicals)were detected by flow cytometry.SOD and MDA were detected.Immunofluorescence was used to detect the expression and localization of ATF6 in the cells.3)One-way analysis of variance was used to perform the statistical analysis.Results: 1)Mi R-195-5p expression was down-regulated in LPS-induced cardiomyocytes;2)mi R-195-5p mimics significantly attenuated the levels of IL-1β,IL-6,TNF-α and MCP1 in LPS-treated cells,and significantly down-regulated the cardiomyocyte apoptosis induced by LPS.Besides,mi R-195-5p alleviated endoplasmic reticulum stress and alleviated oxidative stress and reduced cardiomyocyte injury.Conclusion: The expression of mi R-195-5p is down-regulated in LPS-induced cardiomyocyte,and the overexpression of mi R-195-5p is helpful to reduce cell injury.Part two: The effect of miR-195-5p/ATF6 axis in cardiomyocytes induced by LPSObjective: 1)This part was to confirm that mi R-195-5p protected LPS-induced cardiomyocytes by inhibiting.Methods: 1)The relationship between mi R-195-5p and ATF6 was verified by dual-luciferase assay and Western blot.2)mi R-195-5p mimics were transfected and ATF6 was overexpressed in LPS-induced cardiomyocytes.Afterwards,the expression of mi R-195-5p was detected by q RT-PCR,and inflammatory response,cell apoptosis,endoplasmic reticulum stress related proteins was detected.Besides,ROS,SOD and MDA was determinated.3)One-way analysis of variance was used to perform the statistical analysis.Results: 1)mi R-195-5p inhibited expression of ATF6 by targeting 3’UTR of ATF6;2)mi R-195-5p protected LPS-induced cardiomyocytes.While,ATF6 reversed the protective role of mi R-195-5p by enhancing inflammatory response,oxidative stress,cell apoptosis and endoplasmic reticulum stress.Conclusion:1)mi R-195-5p inhibited ATF6 expression in LPS-induced cell injury models.2)mi R-195-5p can play a key role in apoptosis,oxidative stress and endoplasmic reticulum stress by targeting ATF6 in injured cardiomyocytes.Part three: Effect of mi R-195-5p on septic cardiomyopathy animal modelObjective: This part of experiments was to explore the effect of mi R-195-5p on myocardial injury in sepsis in vivo.Methods: Mouse sepsis model was established by cecal ligation punctue(CLP),then the sepsis mouse model was treated with mi R-195-5p.1)±dp/dtmax was detected by hemodynamics.2)Serum inflammatory factors and oxidative stress were detected by ELISA,and endoplasmic reticulum was detected by electron microscopy.The expression of endoplasmic reticulum associated protein and ATF6 was detected by Western blot.Results: 1)In the sepsis model group,the cardiac hemodynamics results showed that dp/dtmax was significantly weakened,but restored after mi R-195-5p treatment.The survival of rats was recovered by mi R-195-5p delivery.2)Inflammatory cytokines,IL-1β,IL-6,TNF-α and MCP1 in the serum of the animal model group were significantly up-regulated,and they were significantly down-regulated after mi R-195-5p treatment.Meanwhile,SOD and MDA were significantly recovered after mi R-195-5p treatment.electron microscopy results showed that the endoplasmic reticulum in the model group appeared different degrees of swelling,and the morphology of endoplasmic reticulum was significantly improved after mi R-195-5p treatment.After mi R-195-5p treatment,the expression level of ER stress was significantly reversed compared with the model group.Conclusion: mi R-195-5p can improve inflammatory response,endoplasmic reticulum stress level and myocardial injury in sepsis model established by CLP.Conclusions: mi R-195-5p can inhibit inflammatory response,apoptosis and endoplasmic reticulum stress in cardiomyocyte injury model induced by LPS,which is mainly achieved by targeting ATF6,inhibiting ATF6 expression,and then regulating endoplasmic reticulum stress. |
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