The Role And Mechanism Of LncRNA-AABR07066529.3 In Sepsis-induced Myocardial Injury

Objective: sepsis is a life-threatening organ dysfunction caused by infection,which is one of the common diseases in intensive care unit.Septic shock,as a subtype of sepsis,has a higher mortality.Sepsis-induced myocardial injury is a common complication of septic shock,which promotes the progress and deterioration of septic shock and is an important cause of death.Therefore,clarifying the pathogenesis of sepsis-induced myocardial injury will help clinicians find new therapeutic targets to reduce the mortality of patients.The pathogenesis of sepsis-induced myocardial injury is complex,and there is no one or several theories that can be fully elucidated at present.The most widely accepted theory is that many factors interact with each other and participate in the pathogenesis of sepsis-induced myocardial injury,including inflammatory factors,apoptosis,mitochondrial dysfunction and so on.In recent years,studies have shown that pyroptosis may also be involved in the occurrence and development of myocardial injury in sepsis.Long non-coding RNA(long non-coding RNAs,lncRNAs)is a class of RNA found in eukaryotes with a length of more than 200 nt and no long open reading frame.In recent years,more and more studies have shown that lncRNAs can regulate gene expression at many levels,such as epigenetic,transcriptional and post-transcriptional,and then participate in a variety of important biological processes.Studies have found that a variety of lncRNA are involved in the pathogenesis of sepsis.However,there are few studies on lncRNA and sepsis-induced myocardial injury.Recently,our group conducted for the first time,whole genome sequencing of the heart tissue in septic shock adolescence rats and found that the pathways and genes related to inflammation and apoptosis were significantly enriched.At the same time,sequencing results showed that the expression of a new lncRNA-lncRNAAABR07066529.3 was significantly increased in the experimental group,and might be related to inflammation and apoptosis pathway.In addition,myeloid differentiation factor 88(myeloid differentiation factor 88 may be the target gene of lncRNAAABR07066529.3.MyD88 is a classical adaptor protein,which is involved in the regulation of inflammation and apoptosis in a variety of inflammation-related diseases.In addition,it has been reported that MyD88 can affect the expression of NLRP3 inflammasome,indicating that MyD88 is also involved in the regulation of process of pyrogenesis.Based on the above results,this study intends to study whether lncRNAAABR07066529.3 can affect cardiomyocyte injury by regulating inflammation,apoptosis and pyroptosis in sepsis-induced myocardial injury,and to explore the specific mechanism of the role of lncRNA-AABR07066529.3.Methods: Part ?: construction of sepsis-induced myocardial injury model and expression of lncRNA-AABR07066529.3 in rats with septic shock.1.Adolescent Wister rats were divided into control group and sepsis-induced myocardial injury group.The sepsis-induced myocardial injury model was established by intraperitoneal injection of LPS(20mg/kg),and the control group was given the same amount of normal saline.After 12 hours,the samples were taken and HE staining was used to observe the injury of myocardial tissue.2.H9c2 cardiomyocytes were treated with different concentrations of LPS for different time,and the myocardial viability was detected by CCK8 to determine the best intervention concentration and time of LPS intervention.3.Detection of inflammatory injury.Luminex multifactor technique was used to detect the contents of inflammatory cytokines TNF-? and IL-6 in abdominal aortic blood of rats in each group,and Western blot was used to detect the expression of TNF-? and IL-6 in H9c2 cardiomyocytes of each group.4.Detection of apoptosis.Tunel staining was used to detect the apoptosis in cardiac tissue of rats in each group,and flow cytometry was used to detect the changes of apoptosis rate in each group.Western blot was used to detect the expression of apoptosis-related proteins Bax,Bcl-2 and Cleaved-caspase3.5.Detection of pyroptosis.The contents of inflammatory cytokines IL-1? and IL-18 in abdominal aorta blood of rats in each group were detected by Luminex multifactor technique,and the myocardial injury was observed by electron microscope.Western blot was used to detect the expression of pyroptosis-related proteins: NLRP3,cleaved-caspase1 and GSDMD-NT.6.PCR was used to detect the expression of lncRNA-AABR07066529.3 in heart tissue and H9c2 cardiomyocytes of rats in each group.7.The cellular localization of lncRNA-AABR07066529.3 was predicted by website(http://www.csbio.sjtu.edu.cn/bioinf/lnc Locator/)and verified by fluorescence in situ hybridization(FISH).The size of lncRNA-AABR07066529.3 was observed by Northern blot experiment,and the full length of lncRNA-AABR07066529.3 was obtained by Rapid amplification of c DNA ends(RACE).Part ?: the effect of lncRNA-AABR07066529.3 on H9c2 cardiomyocytes induced by LPS.1.The lncRNA-AABR07066529.3 knockout vector was constructed by lentivirus.The transfection efficiency was observed by fluorescence microscope and the knockout efficiency was detected by PCR.The cells were divided into four groups:control group,LPS group,LPS+ NC-KD group and LPS+KD group.The changes of apoptosis in each group were detected by flow cytometry.Western blot was used to detect the expression of inflammatory cytokines TNF-? and IL-6,apoptosis-related proteins: Bax,Bcl-2 and cleaved-caspase3,and pyroptosis-related proteins: NLRP3,cleaved-caspase1 and GSDMD-NT in each group.2.Lnc RNA-AABR07066529.3 overexpression vector was constructed by adenovirus,and the overexpression efficiency was detected by PCR.The cells were divided into four groups: control group,LPS group,LPS+NC-OE group and LPS+OE group.The changes of inflammation,apoptosis and pyroptosis of cardiomyocytes were detected.Part ? : the possible mechanism of the role of lncRNA-AABR07066529.3 in myocardial injury in sepsis.1.Western blot was used to detect the expression of MyD88 in heart tissue and H9c2 cardiomyocytes in each group.2.The MyD88 knockdown vector was constructed by si-RNA,and the knockdown efficiency was detected by PCR and Western blot.Western blot assay was used to detect the changes of inflammation,apoptosis and the expression of pyroptosis related proteins in each group to determine the effect of MyD88 on LPS-induced injury of H9c2 cardiomyocytes.3.The expression of MyD88 was detected by Western blot after lncRNAAABR07066529.3 knockout or overexpression,and the regulatory effect of lncRNAAABR07066529.3 on MyD88 was observed.4.The rescue experiments of knocking down lncRNA-AABR07066529.3 and MyD88 at the same time were carried out to verify that lncRNA-AABR07066529.3 acted through MyD88.The cells were divided into LPS+KD+si-NC group and LPS+KD+si-MyD88 group to detect the expression of inflammatory cytokines,apoptosis and pyroptosis related proteins in each group by Western blot.5.RNA pulldown experiment was carried out to explore the regulation mechanism of lncRNA-AABR07066529.3 and MyD88.Results:Part ?: there were inflammation,apoptosis and pyroptosis injury in both in vivo and in vitro models of myocardial injury in rats with septic shock.The expression of lncRNA-AABR07066529.3 was increased in sepsis-induced myocardial injury model in vivo and in vitro,and mainly expressed in the cytoplasm.1.After 1-2 hours of LPS treatment,MAP and cardiac function decreased in septic shock group,and reached the shock level(MAP decreased more than 20%).HE staining showed that compared with the control group,the inflammatory infiltration in the septic shock group was increased and the myocardial fibers were broken,and when the concentration of LPS reached 10mg/ml(24 h),the viability of H9c2 cardiomyocytes decreased significantly,and the distribution of cells in LPS treated group was sparse and shrunk compared with the control group under light microscope.The results showed that the myocardial injury model of sepsis was successfully established in vivo and in vitro.2.Luminex multifactor experiment showed that the contents of TNF-?,IL-6,IL-1?and IL-18 in the abdominal aorta of the experimental group were higher than those of the control group(P<0.05).Tunel staining showed that the apoptosis was increased in the cardiac tissue of the sepsis-induced myocardial injury group(P<0.05),and the apoptotic bodies and inflammasome could be observed in the myocardial tissue of the experimental group under electron microscope.Compared with the control group,Western blot assay showed that the expression of TNF-? and IL-6,pro-apoptotic protein Bax and cleaved-caspase3,and pyroptosis-related proteins NLRP3,cleaved-caspase1 and GSDMD-NT increased while the expression of anti-apoptotic protein Bcl-2 decreased in LPS treated group.The results of flow cytometry also showed that cardiomyocyte apoptosis increased after LPS.The results showed that there were inflammation,apoptosis and pyroptosis injury in sepsis-induced myocardial injury model in vivo and in vitro.3.The results of PCR showed that the expression of lncRNA-AABR07066529.3increased in both in vivo and in vitro models of sepsis-induced myocardial injury induced by LPS,which was consistent with the results of sequencing.4.Website prediction and FISH experiments show that lncRNA-AABR07066529.3 is mainly located in the cytoplasm.In addition,Northern Blot and RACE experiments showed that the full length of lncRNA-AABR07066529.3 was 4786 nt.Part ?: lncRNA-AABR07066529.3 can inhibit inflammation,apoptosis and pyroptosis in H9c2 cells induced by LPS.1.PCR screened the lentiviral vector with the highest knockdown efficiency(p <0.005),and puromycin was successfully used to screen lncRNA-AABR07066529.3knockdown H9c2 stable cell line for follow-up experiments.2.Compared with LPS group and LPS+NC group,the expression of inflammatory cytokines TNF-? and IL-6,pro-apoptotic proteins Bax and cleaved-caspase3 and pyroptosis related proteins NLRP3,cleaved-caspase1 and GSDMD-NT increased,while the expression of anti-apoptotic protein Bcl-2 decreased in LPS+KD group.At the same time,flow cytometry also showed that apoptosis increased in LPS+KD group(p<0.005).These results showed that after inhibition of lncRNAAABR07066529.3,inflammation,apoptosis and pyroptosis increased,suggesting that lncRNA-AABR07066529.3 could inhibit inflammation,apoptosis and pyroptosis injury in H9c2 cardiomyocytes induced by LPS.3.PCR screened the adenovirus vector with the highest expression efficiency,and overexpressed lncRNA-AABR07066529.3 successfully.In contrast to knockout,overexpression of lncRNA-AABR07066529.3 decreased the expression of inflammatory cytokines TNF-? and IL-6,pro-apoptotic proteins Bax and cleavedcaspase3,and pyroptosis-related proteins NLRP3,cleaved-caspase1 and GSDMD-NT,while increased the expression of anti-apoptotic protein Bcl-2,which further indicated that lncRNA-AABR07066529.3 could inhibit inflammation,apoptosis and pyroptosis injury in H9c2 cardiomyocytes induced by LPS.Part ? : in H9c2 cardiomyocytes,lncRNA-AABR07066529.3 could inhibit LPS-induced inflammation,apoptosis and pyroptosis injury via inhibiting MyD88.1.The expression of MyD88 protein increased in sepsis-induced myocardial injury model in vivo and in vitro.2.PCR and Western blot selected the si-RNA,with the highest knockout efficiency and successfully knocked out MyD88(p<0.05).Compared with LPS group and LPS+si-NC group,the expression of TNF-?,IL-6,Bax,cleaved-caspase3,NLRP3,cleaved-caspase1 and GSDMD-NT decreased,while the expression of Bcl-2increased in LPS+si-MyD88 group,indicating that MyD88 could aggravate inflammation,apoptosis and pyrolysis injury in H9c2 cardiomyocytes induced by LPS.3.The expression of MyD88 protein increased after knockout of lncRNAAABR07066529.3,while the expression of MyD88 protein decreased after overexpression of lncRNA-AABR07066529.3,indicating that lncRNA-AABR07066529.3 could inhibit the expression of MyD88.4.Compared with knocking down lncRNA-AABR07066529.3 alone,the damage of inflammation,apoptosis and pyroptosis were alleviated when lncRNAAABR07066529.3 and MyD88 were knocked down at the same time(p < 0.05),suggesting that lncRNA-AABR07066529.3 might play its role through MyD88.5.The result of RNA pull-down results suggested that lncRNA-AABR07066529.3didn't play a role by directly combining with MyD88.Conclusions:1.In the sepsis-induced myocardial injury model induced by LPS in vivo and in vitro,there were inflammation,apoptosis and cell pyroptosis injury.The expression of lncRNA-AABR07066529.3 was increased in the in vivo and in vitro model of sepsis-induced myocardial injury induced by LPS,and the bioinformatics prediction is related to sepsis-induced myocardial injury.2.In H9c2 cardiomyocytes,lncRNA-AABR07066529.3 could inhibit LPS-induced inflammation,apoptosis and pyroptosis injury.3.In LPS-induced H9c2 cells,lncRNA-AABR07066529.3 could inhibit the expression of MyD88,which might be the mechanism of its role in inflammation,apoptosis and pyroptosis injury.