| Opening infarct-related vessels may lead to more severe acute injury than when the vessels are occluded in ischemic myocardium.This paradoxical aggravation of myocardial tissue injury after reperfusion is called myocardial ischemia-reperfusion injury(MIRI).Despite significant advances in interventional therapy strategies,diabetes is still associated with higher mortality after acute myocardial infarction.This disease increases the susceptibility of myocardium to ischemia-reperfusion injury,and changes the response of myocardium to ischemic preconditioning strategies by blocking or activating intracellular signaling pathways,thereby reducing cell resistance to death.The changes brought about by diabetes have become the basis of poor prognosis after acute myocardial infarction in diabetic patients.Autophagy is an evolutionary conservative mechanism in which intracellular proteins and organelles are isolated by double membrane vesicles called autophagosomes and delivered to lysosomes for degradation.Autophagy at the basic level is involved in maintaining cell homeostasis,but autophagy can also be further induced by stress,thus mediating protective or invasive functions in cells.HMGB1 widely exists in mammals,tH/Rough the active secretion and passive release of damaged,apoptotic,necrotic cells to regulate gene transcription,participate in DNA repair,mediate inflammatory response and other biological effects.Whether HMGB1 regulates autophagy mediated ischemia-reperfusion injury under high glucose tH/Rough related signaling pathways remains to be further studied.Human induced pluripotent stem cells derived cardiomyocytes(hiPSCsCMs),which can be induced by somatic cells,avoiding the damage and ethical problems caused by direct acquisition of human primary cardiomyocytes,better imitate the physiological state of human cardiomyocytes,get more objective experimental results.In this study,by constructing hiPSCs-CM hypoxia-reoxygenation model with high glucose preconditioning,the myocardial ischemia-reperfusion injury environment in diabetic patients was simulated,and the changes and characteristics of myocardial autophagy level after blood reperfusion were observed at the cellular and molecular levels.To investigate the internal relationship between myocardial injury and myocardial autophagy after ischemiareperfusion induced high glucose preconditioning,hypoxia and reoxygenation,and to reveal the physiological and pathological functions of HMGB1 and myocardial autophagy in myocardial injury induced by high glucose preconditioning and reoxygenation,and to reveal the physiological and pathological functions of myocardial autophagy and myocardial autophagy induced by ischemia-reperfusion.To clarify its specific effect on myocardial injury.In this study,hiPSCs-CM hypoxia-reoxygenation model with high glucose preconditioning was established to simulate myocardial ischemia-reperfusion injury environment in diabetic patients.To observe the changes and characteristics of myocardial autophagy level after hypoxia and reoxygenation at cellular and molecular levels,and to explore the internal relationship between myocardial injury and myocardial autophagy level after hypoxia and reoxygenation induced by hypoxia and reoxygenation.To reveal the physiological and pathological functions of HMGB1 and myocardial autophagy in myocardial injury under these conditions,and to clarify their specific effects on myocardial injury.Part 1 Establishment of hypoxia and reoxygenation model of hiPSCs-CM precultured with high glucoseObjective: Acquisition of hiPSCs after reprogramming with human urine cells.The model of hypoxia and reoxygenation hiPSCs-CM precultured with high glucose was established after differentiation of hiPSCs-CM.Methods: The urine of healthy male volunteer was collected and the primary urine cells were isolated and cultured.The primary urine cells were transfected with Sendai virus containing four reprogramming factors Oct,Sox2,Klf4 and cMyc to obtain hiPSCs.The obtained hiPSCs was identified by in vitro embryoid differentiation,in vivo teratoma tumorigenesis,immunofluorescence staining and other methods to identify the pluripotency of the obtained cell lines.The strategy of cardiac differentiation was induced by small molecules with clear chemical composition,using a culture medium containing only tH/Ree chemical components(basic medium RPMI1640,1-ascorbic acid 2-phosphate and rice derived recombinant human albumin)and small molecules.HiPSCs-CM was obtained by differentiation of hiPSCs.The cardiomyocytes were identified by immunofluorescence staining and flow cytometry.The obtained hiPSCs-CM was precultured with high glucose and modeled by hypoxia and reoxygenation,and the modeling was evaluated by CCK8 and other methods.Results:1.A multipotential human urine cell line hiPSCs was successfully established.2.The cardiomyocytes induced by small molecules without animal components have a complete sarcomere structure,and the purity can reach more than 95% after purification.3.The activity of hiPSCs-CM cells did not decrease when cultured with CDM3 at high glucose concentration of 25 mM,35mM and 45 mM for 24 hours(P > 0.05).4.After hiPSCs-CM was precultured with different concentrations of high glucose,the cells were cultured in tH/Ree-gas incubator for 2 hours and reoxygenated for 2 hours.The cell activity of all groups was significantly reduction than that of normal control group,especially in 35 mM group(all P < 0.05).Conclusion:The activity of cardiomyocytes decreased significantly after 2 hours of hypoxia and 2 hours of reoxygenation in 35 mM glucose concentration medium after 24 hours preculture.Part 2 The role of autophagy in hypoxia-reoxygenation injury of hiPSCs-CM precultured with high glucoseObjective:To investigate the relationship between autophagy level with mitochondrial function,apoptosis and other injury markers in hypoxiareoxygenation model of hiPSCs-CM precultured with high glucose.Methods: The strategy of cardiac differentiation was induced by small molecules with clear chemical composition,using a culture medium containing only tH/Ree chemical components(basic medium RPMI1640,1-ascorbic acid 2-phosphate and rice derived recombinant human albumin)and small molecules.The hypoxia-reoxygenation model of hiPSCs-CM precultured with high glucose was established after differentiated hiPSCs and cultured for 30 days according to the conditions obtained in the first part.Autophagy double standard adenovirus was used to detect autophagy flow,qPCR was used to detect the expression of autophagy related index mRNA,Western blot was used to detect the expression of autophagy related protein,JC-1 staining,mitochondrial DNA(mtDNA)staining,Mitotracker staining and Mitosox staining were used to detect the function of autophagy.QPCR was used to detect apoptosis-related genes mRNA and Western blot to detect the expression of apoptosis-related proteins.Results:1.Autophagy double label adenovirus detection of autophagy flow: the number of RFP spots in H/R group was higher than that in CON group,the number of RFP spots in 35H/R group was higher than that in 35 CON group,and the number of RFP spots in 35H/R group was significantly higher than that in H/R group(P < 0 05).2.The expression level of BECN1-mRNA in 35H/R group was significantly higher than that in 35 CON group(P < 0 05),and that in 35H/R group was significantly higher than that in H R group(P < 0 05).The expression level of SQSTM1-mRNA in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of LC3-mRNA in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.3.The expression level of BECN1 protein in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of SQSTM1 protein in 35H/R group was significantly lower than that in 35 CON group,and the expression level in 35H/R group was significantly lower than that in H/R group.The expression level of LC3 Ⅱ protein in 35H/R group was significantly higher than that in 35 CON group,and the expression level in 35H/R group was significantly higher than that in H/R group.4.JC-1 staining: the ratio of relative fluorescence intensity in 35H/R group was lower than that in 35 CON group(P < 0 01).The ratio of relative fluorescence intensity in 35H/R group was significantly lower than that in H/R group(P < 0 05).5.The average copy number of mtDNA in 35H/R group was significantly lower than that in 35 CON group(P < 0 05).The average copy number of mtDNA in 35H/R group was significantly lower than that in 35 CON group.The average copy number of mtDNA in 35H/R group was significantly lower than that in H/R group.6.Mitotracker staining flow cytometry showed that the average relative fluorescence intensity of single cell in 35H/R group was lower than that in 35 CON group(P < 0 01).The average relative fluorescence intensity of single cell in 35H/R group was significantly lower than that in H/R group(P < 0 05).7.Mitosox staining flow cytometry showed that the average relative fluorescence intensity of single cell in 35H/R group was lower than that in 35 CON group(P < 0 05).The average relative fluorescence intensity of single cell in 35H/R group was significantly lower than that in H/R group(P < 0 01).8.The expression of apoptosis-related gene mRNA in CASP3-mRNA: 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of BCL2-mRNA in 35H/R group was significantly higher than that in 35 CON group(P < 0 01),and the expression level in 35H/R group was significantly higher than that in H/R group(P < 0 01).The expression level of BAX-mRNA in 35H/R group was significantly higher than that in 35 CON group(P < 0 01),and the expression level in 35H/R group was significantly higher than that in H/R group(P < 0 01).9.The expression of apoptosis-related protein was detected by Western blot: the expression level of BAX protein in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of BCL2 protein in 35H/R group was significantly lower than that in 35 CON group,and that in 35H/R group was significantly lower than that in H/R group.10.Apoptosis was detected by flow cytometry: the number of Q3 quadrant cells in 35H/R group was significantly higher than that in 35 CON group(P < 0 05).The number of Q3 quadrant cells in 35H/R group was significantly higher than that in H/R group(P < 0 05).Conclusion:The autophagy level of hiPSCs-CM in high glucose precultured group was significantly higher than that in normal cultured cardiomyocytes after hypoxia and reoxygenation,and the damage and apoptosis of mitochondria in this group were the most serious.Part 3 HMGB1/TLR4/TRAF6/NF-κB signaling pathway regulates autophagy and participates in hypoxia-reoxygenation injury of hiPSCs-CM precultured with high glucoseObjective: To investigate the activation of HMGB1/TLR4/TRAF6/ NF-κB signaling pathway in hypoxia-reoxygenation model of hiPSCs-CM precultured with high glucose and its specific mechanism of mediating myocardial injury.Methods:The strategy of cardiac differentiation was induced by small molecules with clear chemical composition,using a culture medium containing only tH/Ree chemical components(basic medium RPMI1640,1-ascorbic acid 2-phosphate and rice derived recombinant human albumin)and small molecules.The hypoxia-reoxygenation model of hiPSCs-CM precultured with high glucose was established after differentiated hiPSCs and cultured for 30 days according to the conditions obtained in the first part.Glycyrrhizin,an inhibitor of HMGB1,and 3-MA,an autophagy inhibitor,were used to intervene hiPSCs-CM in hypoxia-reoxygenation group precultured with high glucose.CCK8 was used to detect the concentration and effect of inhibitor,immunofluorescence staining was used to detect autophagy flow,qPCR was used to detect the expression of mRNA,Western blot was used to detect the expression of signal pathway related proteins.The level of apoptosis was detected by flow cytometry.Results:1.The expression of HMGB1/TLR4/TRAF6/NF-kappa B signaling pathway related gene mRNA in HMGB1-mRNA: 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group(P < 0 05).The expression level of TLR4-mRNA in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of TRAF6-mRNA in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of NF-κ B-mRNA in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.2.Signaling pathway related gene protein expression: the expression level of HMGB1 protein in 35H/R group was significantly higher than that in 35 CON group,and the expression level in 35H/R group was significantly higher than that in H/R group.The expression level of TLR4 protein in 35H/R group was significantly higher than that in H/R group.The expression level of TRAF6 protein in 35H/R group was significantly higher than that in 35 CON group,and that in 35H/R group was significantly higher than that in H/R group.The expression level of NF-kappa B protein in 35H/R group was significantly higher than that in 35 CON group,and the expression level in 35H/R group was significantly higher than that in H/R group.3.The specific fluorescence of HMGB1 protein in the cytoplasm of HMGB1: H/R group and 35H/R cells was detected by immunofluorescence staining,and the expression of HMGB1 protein in 35H/R group was significantly higher than that in H/R group.4.CCK8 assay showed that: Gly(0.15 μ M group and 3-MA(10mM)group improved the cell injury induced by hypoxia and reoxygenation with high glucose,but there was no difference between Gly group and 3-MA group and H/R group.5.The expression of HMGB1 inhibitor in hypoxia-reoxygenation group was significantly lower than that in 35H/R group(P < 0.05).The expression of pathway-related genes and proteins in hypoxia-reoxygenation group was significantly lower than that in HAP group(P < 0.05).6.On the basis of the original grouping,HMGB1 inhibitor Glycyrrhizin intervention group(Gly group)and autophagy inhibitor 3-MA intervention group(3-MA group)were added to the original grouping.The expression of autophagyassociated proteins(BECN1,SQSTM1,BECN1)was detected at the end of the model.Compared with 35H/R group,Glycyrrhizin,an inhibitor of HMGB1,and 3-MA,an autophagy inhibitor,decreased the expression of BECN1 and BECN1,and increased the expression of SQSTM1 in the experimental group.7.Effects of HMGB1 and autophagy inhibitor on apoptosis: on the basis of the original grouping,HMGB1 inhibitor Glycyrrhizin intervention group(Gly group)and autophagy inhibitor 3-MA intervention group(3-MA group)were added.Flow cytometry was performed at the end of the model.The level of apoptosis was measured.Compared with 35H/R group,the level of early apoptosis in Gly group and 3-MA group decreased,but there was no difference between Gly group and 3-MA group and H/R group.Conclusion:The condition of high glucose activated the HMGB1/TLR4/ TRAF6/ NF-κB signaling pathway,and the excessive activation of autophagy resulted in the damage of cardiomyocytes. |
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