The Study On Mechanism Of MiR-122-5p In Regulating GIT1 In The Pathogenesis Of Septic Myocardial Depression

Objective: Sepsis is a life-threatening organ dysfunction syndrome caused by altered host response to infection.The morbidity and fatality rate remain high,and it is one of the common diseases in pediatric intensive care units.As a dynamic organ of circulation,heart is the most easily affected target organ in sepsis and septic shock,and septic myocardial depression is the key factor for prognosis.The pathogenesis of septic myocardial depression is complicated.Studies have shown that cardiomyocyte apoptosis may play an important role in the pathogenesis of septic myocardial depression.Intervention of myocardial cell apoptosis may improve the prognosis of septic myocardial depression.Micro RNAs(mi RNA)are a class of endogenous non-coding small RNA molecules with length of about 18 to 25 nucleotides,and widespread in eukaryotes.Mi RNAs can participate in important physiological processes such as cell growth,proliferation and apoptosis by inhibiting target genes m RNA translation process or promoting the degradation of the target m RNA.Up to now,mi RNAs as a biomarker for septic myocardial depression and target has become the study hotspot.Mi R-122-5p is considered as a new marker for acute myocardial infarction,and overexpression of mi R-122-5p can aggravate the apoptosis of myocardial cells.Previous sequencing results of our research group showed that the expression of mi R-122-5p was significantly increased in the myocardium of rats with septic shock,but the effect of mi R-122-5p on myocardial apoptosis in septic myocardial depression remains unclear.Bioinformatics software predicted that there was a binding site between GIT1 and mi R-122-5p,and GIT1 was reported to be involved in the process of cardiomyocyte apoptosis.Therefore,we speculated that mi R-122-5p may target GIT1 to affect cardiomyocyte apoptosis and participate in the pathogenesis of myocardial depression in sepsis.In this study,LPS was used to construct animal and cell models of septic myocardial depression,in order to verify myocardial apoptosis and the expression of mi R-122-5p in myocardial tissues and cells,with the aim to clarify the role and regulatory mechanism of mi R-122-5p in septic myocardial depression,and to provide a new intervention target for the treatment of septic myocardial depression.Methods: In the first part,animal experiments were carried out to create septic shock models: SPF male Wistar rats were randomly divided into control group and experimental group,with 10 rats in each group.After intraperitoneal injection of 20% urethane 6 ml/kg to induce anesthesia,septic shock rats model was established by intraperitoneal injection of LPS,and the control group rats received equal dose of normal saline.The mean arterial pressure(MAP)was monitored 12 h through femoral artery catheter by connecting to Biopac multi-conducting physiological recorder pressure transducer.HE staining method was applied to observe the pathological changes of myocardial tissue,and TUNEL staining method was used to detect myocardial cell apoptosis.Real-time PCR was used to detect the m RNA levels of Bax,Bcl-2 and Caspase-3 in myocardial tissue,and Western blot method was used to detect the protein expression of the above genes.Real-time PCR was used to detect the expression of mi R-122-5p in myocardial tissue.Bioinformatics software Targetscan was used to predict the target genes that could bind to mi R-122-5p.Real-time PCR and Western blot were used to detect the m RNA and protein expression of GIT1 in myocardium respectively.The targeted binding relationship between mi R-122-5p and GIT1 was verified by dual luciferase reporter genes.In the second part,animal experiments were conducted to study the effect and mechanism of mi R-122-5p on myocardial depression in rats with septic shock: mi R-122-5p antagomir and NC were injected into the tail vein to inhibit the expression of mi R-122-5p in the myocardium of rats,24 h later the rats were intraperitoneally injected with LPS to establish the septic shock model.The experimental groups were: Control group,LPS group,LPS+NC antagomir group,LPS+mi R-122-5p antagomir group,with 6 rats in each group.The mean arterial pressure was continuously monitored 12 h through femoral artery catheter by connecting to Biopac multi-conducting physiological recorder pressure transducer.Real-time PCR was used to detect the expression of mi R-122-5p in the myocardial tissues of the four groups of rats.HE staining method was applied to observe the pathological changes of myocardial tissue,and TUNEL staining method was used to detect myocardial cell apoptosis.Real-time PCR was used to detect the m RNA levels of Bax and Bcl-2,and Western blot was used to detect the protein expression of Bax,Bcl-2,pro and cleaved-Caspase 3 in myocardial tissues respectively.Inflammatory factors(TNFa,IL-6,IL-1?),oxidative stress indexes(ROS,SOD,CAT,GSH-Px)and myocardial enzymes(c Tn I and LDH)in myocardium of each group were detected by Elisa and biochemistry kits respectively.Real-time PCR and Western blot were used to detect the m RNA and protein expression of GIT1 in myocardial tissue.In the third part,cell experiment: H9c2 rat cardiomyocytes were cultured in vitro.The optimal intervention concentration and intervention time of LPS were determined by CCK-8 method,and the model of septic myocardial depression was constructed at the cell level,meanwhile the morphological changes of cardiomyocytes after LPS intervention at different times were observed.Cell experiment(I): Effects of mi R-122-5p on H9c2 cardiomyocytes after LPS intervention: mi R-122-5p mimics,inhibitor and corresponding NC were synthesized,and the expression of mi R-122-5p under mimics and inhibitor transfection as well as before and after LPS intervention was detected by Real-time PCR to verify the transfection efficiency of mi R-122-5p.Then the cells were divided into Control group,LPS group,LPS+NC mimics group,LPS+mi R-122-5p mimics group,LPS+NC inhibitor group,and LPS+mi R-122-5p inhibitor group.The apoptosis of H9c2 cardiomyocytes was detected by TUNEL staining and flow cytometry.Real-time PCR was used to detect the m RNA levels of apoptosis-related proteins Bax and Bcl-2 in H9c2 cardiomyocytes,and the protein expressions of Bax,Bcl-2,pro and cleaved-Caspase 3were detected by Western blot.Inflammatory factors(TNF-a,IL-6,IL-1?),oxidative stress indexes(ROS,SOD,CAT,GSH-Px)and myocardial enzymes(c Tn I and LDH)in H9c2 cardiomyocytes were detected by Elisa and biochemistry kits respectively.Real-time PCR was used to detect the m RNA expression of GIT1 in H9c2 cardiomyocytes,and Western blot was used to detect the protein expression level of GIT1.Cell experiment(?): To verify the role of GIT1 in cardiomyocytes and the targeted regulation of GIT1 by mi R-122-5p.The cells were divided into Control group,LPS group,LPS+NC si RNA group,LPS+GIT1si RNA group,LPS+mi R-122-5p inhibitor+NC si RNA group,and LPS+mi R-122-5p inhibitor+GIT1 si RNA group.Real-time PCR and Western blot were used to detect m RNA and protein expression of GIT1 in H9c2 cardiomyocytes respectively,and evaluate the transfection efficiency of GIT1 si RNA.Myocardial cell apoptosis was detected by TUNEL staining and flow cytometry.Real-time PCR was used to detect the m RNA levels of apoptosis-related genes Bax and Bcl-2 in H9c2 cardiomyocytes,and Western blot was used to detect the protein expressions of Bax,Bcl-2,pro and cleaved-Caspase 3.The contents of inflammatory factors(TNF-a,IL-6,IL-1?),oxidative stress indexes(ROS,SOD,CAT,GSH-Px)and myocardial enzymes(c Tn I and LDH)in H9C2 cardiomyocytes were detected by Elisa and biochemistry kits respectively.Results: The first part: About 2 hours after intraperitoneal injection of LPS,blood pressure began to drop,indicating septic shock,and blood pressure did not recover or improve within 12 hours.HE staining results showed significant myocardial injury and inflammatory cell infiltration in left ventricular muscle of rats with sepsic shock.TUNEL staining confirmed increased myocardial cell apoptosis in sepstic myocardial depression.Real-time PCR and Western blot method respectively showed increased m RNA and protein levels of Bax and cleaved-Caspase 3,whereas decreased Bcl-2 expression.Moreover,the increased expression of mi R-122-5p in the myocardium of rats with septic shock was confirmed by Real-time PCR with expanded sample size.Targetscan predicted that GIT1 had binding sites with mi R-122-5p,and the m RNA and protein levels of GIT1 decreased in the myocardium of rats with septic shock,contrary to the trend of mi R-122-5p.Finally,dual luciferase assay was used to show that mi R-122-5p mimics significantly downregulated GIT1-3'-UTR luciferase activity in the wild group,but not in the mutant group,compared with the mimics negative control group,which proved that GIT1 could be directly bound to mi R-122-5p.The second part: Antagomir could effectively inhibit the expression of mi R-122-5p in the myocardium of rats with septic shock.HE staining showed that myocardial injury and inflammatory factor infiltration were reduced,with TUNEL staining showing decreased cell apoptosis.PCR and Western blot results showed decreased expression levels of Bax and cleavd-Caspase 3 whereas increased expression level of Bcl-2.In septic shock rats,myocardial inflammatory factors(TNF-a,IL-6,IL-1?)content increased significantly,with increased ROS levels,contrast with decreased antioxidants(SOD,CAT and GSH-Px)level.Besides,elevated level of myocardial enzymes(c Tn I and LDH)indicated myocardial injury obviously,and inhibition of mi R-122-5p can effectively reduce the myocardial inflammatory factors(TNF-a,IL-6,IL-1?)content,lower level of ROS,increase the antioxidants(SOD,CAT and GSH-Px)level,and decrease the level of myocardial enzymes(c Tn I and LDH).PCR and Western blot showed inhibition of mi R-122-5p increased the expression of GIT1 from aspects of m RNA and protein level.The third part: CCK8 method determined the optimal LPS intervention concentration of10?g/ml,the optimal time 24 h.After 10?g/ml LPS intervention,with the extension of time,the activity of cells gradually decreased,and cell death even occured.Mi R122-5p inhibitor could effectively inhibit the expression of mi R-122-5p in H9c2 cardiomyocytes after LPS intervention,reduce myocardial cell apoptosis,reduce Bax and cleaved-Caspase 3expression,increase Bcl-2 expression,reduce myocardial inflammatory factors(TNF-a,IL-6,IL-1?)content,lower levels of ROS,increase the antioxidants(SOD,CAT and GSHPx)level,lower the levels of myocardial enzymes(c Tn I and LDH),as well as increase the expression of GIT1 from aspects of m RNA and protein level by PCR and Western bolt method respectively.On the contrary,after the application of mi R-122-5p mimics,the apoptosis of myocardial cells was increased,the expression of Bax and cleaved-Caspase 3was increased,the expression of Bcl-2 was decreased,the content of myocardial inflammatory factors(TNF-a,IL-6,IL-1?)was increased,the ROS level was increased,the level of antioxidants(SOD,CAT,GSH-Px)was decreased,the level of myocardial enzymes(c Tn I and LDH)was increased,and m RNA and protein expression of GIT1 was decreased.Through GIT1 si RNA interference and co-transfection with mi R-122-5p inhibitor,we found that the lack of GIT1 can aggregate apoptosis of H9c2 cardiomyocytes,increase Bax and cleaved-Caspase 3 expression,reduce the expression of Bcl-2,increase myocardial inflammatory factors(TNF-a,IL-6,IL-1?)content,increase the level of ROS,reduce antioxidants(SOD,CAT and GSH-Px)level,and increase the level of myocardial enzymes(c Tn I and LDH).Moreover,the effect of inhibiting mi R-122-5p on the antiinflammatory,anti-oxidative and anti-apoptotic protection of H9c2 cardiomyocytes after LPS intervention was partially offset by GIT1 deficiency.Conclusions: 1.Apoptosis of cardiomyocytes may play an important role in septic myocardial depression.2.Inhibition of mi R-122-5p could increase the expression of GIT1,thus reducing myocardial inflammation,oxidative stress and apoptosis in rats with septic shock,then alleviate myocardial injury.3.Mi R-122-5p can inhibit expression of GIT1 to aggravate the inflammation,oxidative stress and apoptosis of H9c2 cardiomyocytes induced by LPS,thereby aggravating apoptosis and myocardial injury.