| Background and Objective:Ischemic heart disease(ischemic heart disease,IHD)is the leading cause of death worldwide.Thrombolysis and percutaneous coronary intervention have reduced the mortality of patients.However,reperfusion usually causes irreversible cardiomyocyte injury and arrhythmia,leading to myocardial ischemia-reperfusion injury((Myocardial ischemia reperfusion injury,MIR/I).MIR/I have become the main obstacle for patients to benefit from reperfusion therapy,which is an urgent clinical problem to be solved.Conditioned medium derived from bone marrow mesenchymal stem cells(BMSCs-CM,MSC-CM mentioned in this paper refers to BMSCs-CM)improves cardiac function after myocardial infarction.But the protective effect mediated by MSCs-CM was moderate and transient.Therefore,it is necessary to optimize MSCs-CM to improve its protective effect on cardiomyocytes,determine the effective components that play a cardioprotective effect,and clarify the mechanism of the therapeutic benefits of MSCs on cardiomyocytes.While it was reported that Lipopolysaccharides(LPS)pretreated MSCs enhanced MSCs-mediated cardioprotection,the molecular mechanism involved remains unknown.Whether MSCs-CM pretreated with LPS attenuates hypoxia/reoxygenation(H/R)-induced H9c2 cell injury and its regulatory mechanism need to be further elucidated.In addition,it is worth studying whether LPS pretreatment MSCs-CM can be optimized.High mobility group protein B1(HMGB1)is mainly found in the nucleus of eukaryotic cells.It is a highly conserved chromosome binding protein involved in gene replication and transcription.Studies have shown that HMGB1 can be actively secreted or passively released to extracellular,as a related molecular model to cause inflammation and participate in angiogenesis.Bach1(BTB and CNC homology 1,BACH1),a member of the basic leucine zipper protein family,is not only a key upstream transcription factor regulating angiogenesis,but also a factor for stem cells to maintain self-renewal and determine lineage differentiation.In this study,we hypothesized that MSCs,pretreated with LPS could promote the paracrine effect of MSCs through HMGB1/Bach1 signal pathway,and then enhance the protective effect of MSCs on H9c2 cells injured by H/R,and further optimize MSCs-CM,to identify the effective components that mediate myocardial protection,so as to provide a new direction for stem cells in the treatment of myocardial infarction.Methods:1.Construct H/R model of H9c2 cells.Put H9c2 cells in a hypoxic bag for 10 h,and then reoxygenate for 6,12 and 24 h,respectively.Detecting the apoptosis rate and the level of LDH and cTn.2.MSCs were treated with or without LPS,and the supernatant was collected.H9c2 cells were co-cultured with conditioned medium from MSCs and LPS-MSCs for24 h.The apoptosis rate,apoptosis protein and the expression of LDH and cTn were detected,and the cell morphology was observed.3.The supernatants of MSCs and LPS-MSCs were collected as conditioned medium to detect the levels of paracrine factors VEGF,HGF and IGF.The cells were collected to detect the expression of HMGB1 and Bach1 by Western Blot.4.To explore the relationship between Bach1 and HMGB1 in MSCs,the Bach1 and HMGB1 proteins in MSCs were silenced by siRNA interference technique,and then the knockout efficiency and the internal relationship between them were verified by Western Blot.5.MSCs and MSCs with Bach1 knockdown(MSCs/siBach1)were treated with or without LPS(400 ng/m L)for 48 h and collected supernatant as conditioned medium.H9c2 cells were co-cultured with CM derived from MSCs,MSCs/siBach1,LPS-MSCs and LPS-MSCs/siBach1 for 24 h and then subjected to hypoxia(10h)/reoxygenation(6h).The apoptosis rate,the expression of apoptosis protein and LDH and cTn were detected.6.Collect MSCs-CM,compare MSCs-CM optimization method and its effect on H/R-induced H9c2 cell damage(1)MSCs was treated with LPS for 48 h,collect the supernatant as LPS-CM.The LPS-CM was further treated with a LPS remover or concentration filter(3/10/50KDa).The expression of paracrine factor was detected.(2)H9c2 cells were co-cultured with conditioned medium from MSCs,LPS-MSCs(LPS-CM),LPS-CM treated with LPS remover(LPS Re-CM)and LPS-CM concentrated by 10 KDa ultrafiltration(10KDa-CM)for 24 h,respectively,and then subjected H/R.Observed cell morphology and detected cell apoptosis rate,apoptotic protein,LDH and cTn expression.7.Collect MSCs-CM,LPS-CM and 10KDa-CM.The composition of conditional medium was determined by quantitative proteomic analysis and its biological function was analyzed.Results:1.The H/R model of H9c2 cells was established: the cell injury was most obvious after hypoxia for 10 h and reoxygenation for 6 h,which was manifested by the increase of apoptosis rate and damage markers LDH and cTn.2.Both untreated and LPS pretreated MSCs-CM significantly alleviate H/R induced H9c2 cells injury,as evidenced by decreased apoptosis rate,improved cell morphology,and reduced LDH and cTn release.LPS pretreated MSCs-CM conferred better cardiomyocyte function recovery than untreated MSCs-CM.3.LPS decreased the expression of Bach1 and HMGB1 and promoted the expression of paracrine factors VEGF,HGF and IGF in MSCs.4.Si RNA silenced the expression of HMGB1 and decreased the expression of Bach1 in MSCs;after silencing HMGB1,LPS failed to further reduce the expression of Bach1;while silencing Bach1 almost did not affect the expression of HMGB1;silencing both HMGB1 and Bach1 by siRNA increased the levels of VEGF and IGF.5.Although MSCs/siBach1 exhibited a similar cardioprotection effect as untreated MSCs,LPS failed to further potentiate their cardioprotective effect.6.ELISA showed that among the filtrate and concentrate concentrated by3/10/50 KDa ultrafiltration,10KDa-CM detected a large amount of VEGF,HGF and IGF;compared with LPS-CM and LPS Re-CM,10KDa-CM significantly increased the levels of VEGF,HGF and IGF.7.CCK-8 displayed that viability of injured H9c2 cells enhanced with the increase of MSC-CM concentration,and the viability of H9c2 cells pretreated with10KDa-CM was significantly higher than that of MSC-CM,LPS-CM,LPS Re-CM.8.Compared with MSC-CM and LPS-CM,10KDa-CM significantly alleviated H/R induced H9c2 cells injury,as evidenced by improved cell morphology,increased Bcl-2/Bax ratio,decreased apoptosis rate and the release of LDH and cTn.9.Proteomic analysis identified 1059 proteins in 10KDa-CM,including 27up-regulated proteins and 14 down-regulated proteins,of which anti-inflammatory,angiogenesis and anti-apoptotic effects were associated with cardiac protection.Conclusions:1.LPS pretreated MSCs-CM protects H9c2 cells from H/R-induced injury partly through regulating HMGB1/Bach1 signaling.2.10KDa-CM(optimized with 10 KDa cutoff filters)protects H9c2 cardiomyocytes from H/R injury by retaining most of the cytokines in MSCs-CM. |
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