Lipopolysaccharide Enhances The Protective Effect Of Bone Marrow Mesenchymal Stem Cells On Hypoxia-reoxygenation Injury Of H9c2 Cardiomyocytes Through HMGB1/P-STAT3 Signal Pathway

Background and Objective Acute myocardial infarction(AMI)is a serious threat to human health.Percutaneous coronary artery surgery(PCI)is the main treatment at present,but the myocardial injury seems to be further aggravated after the recovery of blood flow.This process is called ischemia-reperfusion injury(I/R),Bone marrow-derived mesenchymal stem cells(BM-MSCs in this paper all refer to BM-MSCs)have the functions of cell differentiation,tissue repair,anti-inflammation and immunosuppressive.Their strong paracrine can reduce myocardial Imax R injury and promote cardiac repair.High mobility group protein B1(HMGB1),which mainly exists in eukaryotic nucleus,is a highly conserved chromosome binding protein,it participates in gene replication and transcription,and can be actively secreted or passively released outside the cell as a damage-related molecular model to participate in inflammatory response,while appropriate concentration of HMGB1 can protect cells from apoptosis.Signal transducer and transcriptional activator 3(P-STAT3)is a signal transcriptional protein that affects cell growth and apoptosis.Previous studies in our group have shown that paracrine is an important mediator of the protective effect of MSCs on cardiomyocytes.P-STAT3 participates in the paracrine effect of MSCs.It is not clear whether MSCs can exert paracrine effect through HMGB1/P-STAT3 signal pathway.In this study,rat H9c2 cardiomyocytes were selected to establish a model of hypoxia-reoxygenation(H/R)to study the regulation of HMGB1/P-STAT3 signal pathway in the paracrine effect of MSCs,so as to provide a theoretical basis for elucidating the protective effect of MSCs on cardiomyocytes.Methods1.Apoptosis of H9c2 cardiomyocytes was induced by hypoxia for 4 h,6 h,10 h and 12 h,followed by reoxygenation for 6 h,12 h and 24 h.The apoptosis rate of H9c2 cardiomyocytes was detected by flow cytometry,the concentration of myocardial injury factors LDH and c-Tn was detected by ELISA,changes in the morphology and number of cells were observed under a microscope.2.H9c2 cardiomyocytes were co-cultured with MSCs conditioned medium and MSCs conditioned medium pretreated with lipopolysaccharide for 24 hours,then treated for hypoxia and reoxygenation.The apoptosis rate,the concentration of myocardial injury factors LDH and c-Tn and the viability of H9c2 cardiomyocytes were detected by flow cytometry,ELISA and CCK8,the degree of apoptosis was observed by TUNEL fluorescence staining.3.The expression of HMGB1 and P-STAT3 in MSCs pretreated with LPS was observed by Western Blot technique,and the concentration of paracrine factors VEGF,HGF and IGF in the culture supernatant was detected by ELISA.4.Si RNA technique was used to silence HMGB1 and P-STAT3,ELISA in MSCs to detect the concentration of VEGF,HGF and IGF in the supernatant.Then the silent MSCs conditioned medium was co-cultured with H9c2 cardiomyocytes for 24 hours,and then treated with hypoxia-reoxygenation.The apoptosis rate of H9c2 cardiomyocytes was detected by flow cytometry,the contents of myocardial injury factors LDH and c-Tn were detected by ELISA,the cell viability was detected by CCK8,and apoptosis was detected by TUNEL fluorescence staining.Results1.The apoptosis rate of H9c2 cardiomyocytes and the content of myocardial injury factors LDH and c-Tn increased gradually after hypoxia-reoxygenation for different time,and reached the ideal apoptosis state after hypoxia for 10 h and reoxygenation for6 h.2.MSCs conditioned medium and LPS preconditioned MSCs conditioned medium,the apoptosis rate,myocardial injury factor content and TUNEL fluorescence intensity of H9c2 cardiomyocytes decreased significantly,especially in MSCs group of LPS preconditioning group,indicating that MSCs conditioned medium protected H9c2 cardiomyocytes from hypoxia-reoxygenation injury,while MSCs conditioned medium preconditioned by LPS could play a more significant protective role.3.After pretreatment of LPS,the content of paracrine factors in the culture supernatant of MSCs was significantly higher than that in the untreated group,and the expression of HMGB1 and P-STAT3 in MSCs was up-regulated.4.After silencing HMGB1 in MSCs,the expression of P-STAT3 also decreased,and the content of paracrine factor was significantly lower than that in untreated group.After co-culture with H9c2 cardiomyocytes,the activity of cardiomyocytes decreased significantly,which weakened the protective effect of MSCs on H9c2 cardiomyocytes.5.After silencing P-STAT3 in MSCs,the content of MSCs paracrine factor decreased.After co-culture with H9c2 cardiomyocytes,the apoptosis rate of H9c2 cardiomyocytes increased,the content of myocardial injury factor increased,cell viability was decreased,the fluorescence intensity of TUNEL was enhanced.Conclusion1.The hypoxia-reoxygenation model of H9c2 cardiomyocytes was established by Anaero Pack System,and reached a stable state of apoptosis after hypoxia for 10 hours and reoxygenation for 6 hours.2.Co-culture of MSCs with H9c2 cardiomyocytes could significantly improve the hypoxia-reoxygenation injury of H9c2 cardiomyocytes,and MSCs pretreated with LPS showed a stronger protective effect.3.LPS exerts its protective effect on H9c2 cardiomyocytes through HMGB1/P-STAT3 signal pathway.