| Background: The main pathophysiological basis of cardiac insufficiency after reperfusion in acute myocardial infarction is myocardial cell apoptosis and necrosis during ischemia/reperfusion injury(I/R).As an ideal stem cell,cardiac stem cells(CSCs)can significantly repaired infarcted myocardium,improved myocardial remodeling and cardiac function.Current researches suggest that the improvement of cardiac function in CSC transplantation for ischemic cardiomyopathy mainly benefits from the paracrine mechanism of stem cells and Exosomes played a pivotal role in the paracrine effects of stem cells transplantation.Compared with CSC,exosomes,which is a non-cellular component of CSC,has stable performance and is not easily affected by the ischemic microenvironment.Exosomes avoided the problems such as formation of teratoma,immune reaction,and limited cell proliferation and differentiation during stem cell transplantation.Meanwhile,exosomes provided cytoprotective factors such as growth factors that are able to resist apoptosis,antifibrosis,promote angiogenesis,enhance cardiac differentiation,and repair damaged tissue.Such Researches have shown that hypoxic preconditioning(HPC)can not only improve the ability of stem cell repair,but also enhance myocardial protection of stem cell-derived exosomes.Previous studies on the pathogenesis of myocardial I/R mostly focused on oxygen free radicals,calcium overload,inflammatory reactions,mitochondrial damage,apoptosis,endothelial cell activation injury and autophagy,among which autophagy played a very important role in I/R.Autophagy is the process of transporting damaged,degenerative,and senescent long-lived proteins and organelles to the lysosome for enzymatic digestion,reducing the substrate to its constituent components and allowing the cells to be reused.In heart tissue,autophagy and apoptosis are essential for the maintenance of normal function of cardiomyocytes.Both animal and cell experiments confirmed that moderately elevated levels of autophagy during hypoxia can reduce the damage caused by hypoxia during ischemia-reperfusion injury.When the ischemic myocardium recovers nutrient and oxygen supply,ROS accumulates in large numbers and autophagy is significantly upregulated.As autophagy increases,autophagic death and myocardial cell apoptosis are usually aggravated.At present,the study of myocardial cell apoptosis induced by hypoxia-reoxygenation(HR)of autophagy-regulated exosomes in HPC CSCs has not been reported.This study was designed to investigate the effect of exosomes from HPC CSC on H9c2 apoptosis and autophagy on the basis of HR-induced H9c2 apoptosis,and to explore the mechanism of mutual regulation between exosomes and damaged tissue microenvironment to provide new treatment for cellular treatment to provide new therapeutic targets and strategies for the cellular treatment of stem cells.Part I Culturing of Rat CSCs and Identification of ExosomesObjective: The c-kit+CSCs required for the experiments were cultured and exosomes were extracted and identified from normoxic(Nor-Exo)and HPC c-kit+CSCs supernatants(HPC-Exo).Methods:(1)c-kitposCSCs were cultured by collagenase II digestion combined with magnetic activated cell sorting(MACS)and identified in different ways,flow cytometry(FCM)detected CSCs’ surface marker protein,immunofluorescence detected the expression of c-kit,real-time cell analyzed(RTCA)the growth curve of c-kitposCSCs,CCK-8 detected the effect of different hypoxic preconditioning(HPC)time on the activity of c-kitposCSCs,q RT-PCR detected the characteristics of stem cell after 12 h of HPC.(2)Nor-Exo and HPC-Exo were respectively extracted by ultracentrifugation(UC)from the conditioned supernatants of c-kitposCSCs.The expression of transmembrane proteins CD9,CD63 and Alix,exosomal marker protein,was detected by Western Blot.The morphology of exosomes was observed by transmission electron microscope(TEM).The size,distribution range and concentration of exosomes in the two groups were both analyzed by nano-particle tracking analysis(NTA).Results:(1)The cell density of primary cells achieved about 80%-90% when cultured for about 10 days.MACS further sorted the c-kitposCSCs,we found that a large number of long triangle and fusiform CSCs grew adherently when cultured for about 5 days.The positive rate of c-kit after MACS was approximately 88.83%±3.45(P<0.05)with the method of FCM while c-kit were positively expressed observing by IF.Cells began to adhere at about 6h after inoculation.The latent growth phase was observed at 6h to 22 h.After 1 day,the cells proliferated rapidly and entered the logarithmic growth phase.About 3 days later,the CI did not increased,c-kitposCSCs entered the platform for growth.The viability of c-kitposCSCs was significantly up-regulated compared with HPC 0h group(P<0.05).Compared with the control group,there was no significant difference in the expression of myocardial markers(P>0.05).(2)CD9,CD63 and Alix were positively expressed detecting by Western Blot.We saw plenty of round or oval discoid or lamellar vesicular structure wrapped by lipid membrane by TEM.NTA showed that the size of Nor-Exo and HPC-Exo were both between 30-100 nm.Compared with Nor-Exo group,the size of exosomes in HPC-Exo group had no significant difference(P>0.05),but the concentration was significantly increased(P<0.05).Conclusion:(1)High purity of c-kitposCSCs can be effectively cultured by Collagenase digestion combined with MACS.(2)HPC c-kitposCSCs 12 h made ideal time point and had no effect on the characteristics of CSCs.(3)c-kitposCSCs derived exosomes could be successfully extracted by UC and it did match the characteristics of exosomes when identified the size and morphology.(4)HPC could promote exosomes secretion of c-kitposCSCs.Part II Function of Exosomes Reduced HR-Induced H9c2 ApoptosisObjective: To observe the role of Nor-Exo and HPC-Exo in HR induced apoptosis of H9c2 cardiomyocyte.Methods:(1)To establish hypoxia-reoxygenation(HR)-induced apoptosis model of H9c2.FCM was used to detect the effect of different HR time on apoptosis of H9c2.The optimal HR time which induced the best apoptosis rate of H9c2 was chosen as the processing condition of subsequent experiment.(2)Di I-labeled Exosomes were co-cultured with H9c2 at different times,and the process of H9c2 internalized exosomes was detected by immunofluorescence dynamic monitored.The suitable co-culture time was selected as the treatment condition for subsequent co-culture of exosomes and H9c2.(3)The effects of different concentrations of Nor-Exo(0μg/ml,100μg/ml,200μg/ml,400μg/ml and 800μg/ml)on the apoptosis of H9c2 induced by H24h6 h were detected by FCM.(4)The effect of 400μg/ml Nor-Exo and HPC-Exo on the eversion of phosphatidylserine(PS)in the early apoptotic cell membrane of H9c2 induced by HR was detected by Annexin V-FITC/PI dual staining.(5)DCFH-DA and Western Blot were used to observe the effects of 400μg/ml Nor-Exo and HPC-Exo on HR-induced activation of the active oxygen species(ROS)and the apoptosis related proteins(Actived Caspase-3,Bcl-2 and Bax).(6)Finally,the effects of Nor-Exo and HPC-Exo on the DNA fragmentation induced by HR-induced late apoptosis in H9c2 cells were observed by Tunel staining.Results:(1)Compared with other groups,the early apoptotic rate of H9c2 and total apoptotic ratio of H24 h R6h group were significantly increased(P<0.05),but the mortality rate were not significantly increased(P>0.05),H24 h R6h was selected as the ideal HR treatment time point to induce H9c2 apoptosis.(2)Compared with co-culture for 1h and 6h,H9c2 uptake exosomes reached the maximum(P<0.05,P<0.05)after co-cultured for 12 h.Therefore,exosomes co-cultured with H9c2 for 12 h were used for the follow-up experimental conditions.(3)Compared with 0μg/ml,100μg/ml and 200μg/ml groups,the apoptotic rate of H9c2 in 400μg/ml group was significantly decreased(P<0.05).Therefore,400μg/ml exosomes was chosen as the best concentration for the intervention of HR induced H9c2 apoptosis.(4)HPC-Exo significantly inhibited the eversion of PS(P<0.05)compared with Nor-Exo group.(5)HPC-Exo significantly inhibited ROS release(P<0.05)compared to Nor-Exo group.The expression of Actived-Caspase-3 and Bax in HPC-Exo group were significantly decreased(P<0.05)while Bcl-2 was significantly increased compared with Nor-Exo group.(6)HPC-Exo significantly inhibited DNA fragmentation compared with Nor-Exo group(P<0.05).Conclusion:(1)H24h R6 h is the optimal HR treatment time for H9c2 apoptosis.(2)H9c2 can uptake exosomes derived from c-kitposCSCs.(3)Exosomes inhibited the apoptosis of H9c2 induced by HR in a concentration-dependent manner.(4)HPC enhanced c-kitposCSCs derived exosomes to inhibit the apoptosis of H9c2 induced by HR at different stages of apoptosis.Part III Mechanism of Autophagy and AMPK/m TOR Signal Pathway Mediated HPC CSCs Derived Exosomes Inhibition of HR-Induced H9c2 ApoptosisObjective: Anti-apoptotic effects of autophagy and AMPK/m TOR signal pathway on HPC-Exo were investigated based on the induction of H9c2 apoptosis by HR.Methods:(1)The experiment were divided into four groups:(1)Nor,(2)HR,(3)Nor-Exo and(4)HPC-Exo.The levels of autophagy of H9c2 in each group were detected through various methods.LC3-II,LC3-I and P62 in H9c2 cells were detected by Western Blot.The change of intracellular autophagosome(AV1)and autolysosome(AV2)in H9c2 cells after transfection with double fluorescent labeled Ad-m RFP-GFP-LC3 was observed by laser confocal microscopy to evaluate the effects of autophagy and autophagic flux.The morphological and quantitative changes of autophagic vacuoles(AV)in H9c2 cardiomyocytes were observed by TEM.(2)The effect of HPC-Exo on the apoptosis of H9c2 cells was observed on the basis of HR-induced H9c2 apoptosis induced or reduced by rapamycin(Rapa)or 3-methyladenine(3-MA).Groups:(1)Nor,(2)HR,(3)HPC-Exo,(4)HPC-Exo+Rapa and(5)HPC-Exo+3-MA.Western Blot was used to detect the level of autophagy in each group.After that,the changes of H9c2 apoptosis induced by HRC-Exo after the regulation of autophagy.Annexin V-FITC/PI dual staining,DCFH-DA,Western Blot and Tunel methods were used to detect the ratio of PS eversion ROS release,apoptosis-related proteins and DNA fragmentation in each group.(3)Western Blot explored the effect of HPC-Exo on the activation of AMPK/m TOR signaling pathway-related proteins on the basis of HR-induced H9c2 apoptosis.(4)Groups,(1)Nor,(2)HR,(3)HPC-Exo,(4)HPC-Exo+Met and(5)HPC-Exo+Dor.The effects of HPC-Exo on the autophagy of H9c2 cells were observed by Western Blot with specific induction of Metformin(Met)and Dorsomorphin(Dor)inhibition of phosphorylation of AMPK.Then,Annexin V-FITC/PI dual staining,DCFH-DA,Western Blot and Tunel methods were used to detect the ratio of PS eversion ROS release,apoptosis-related proteins and DNA fragmentation in each group after regulating of AMPK phosphorylation.Results:(1)LC3-II/LC3-I was significantly increased(P<0.05)and P62 was significantly down-regulated in HR group.Compared with HR group,autophagy was significantly down-regulated in Nor-Exo group(P<0.05).Compared with Nor-Exo group,autophagy was further significantly down-regulated in HPC-Exo group(P<0.05).The number of autophagosomes(AV1)and autolysosomes(AV2)which showed yellow or red fluorescence in H9c2 cells in each group was examined by confocal laser scanning microscopy.HR significantly increased the number of AV1 and AV2(P<0.05).Compared with HR group,the numbers of AV1 and AV2 in Nor-Exo group were significantly decreased(P<0.05).The number of AV1 in HPC-Exo group was significantly down-regulated(P<0.05)but there was no significant difference in AV2 number(P>0.05).TEM results found that AV significantly increased in the visual field of the HR group(P<0.05)in the form of monolayer or bilayer membrane wrapped with cytoplasmic components.The number of intracellular AV of H9c2 in Nor-Exo group was significantly lower(P<0.05).Compared with Nor-Exo group,the number of AV in HPC-Exo group further decreased significantly(P<0.05).(2)Compared with HPC-Exo group,autophagy in HPC-Exo+Rapa group was significantly increased(P<0.05),but in HPC-Exo+3-MA group there’s no significant difference(P>0.05).Effect of HPC-Exo on H9c2 apoptosis induced by HR in different groups after regulating of autophagy.1)The early apoptosis rate in HPC-Exo+Rapa group was significantly increased(P<0.05),while significantly decreased(P<0.05)in HPC-Exo+3-MA group.2)ROS releasing rate in HPC-Exo+Rapa group was significantly increased(P<0.05)and significantly decreased(P<0.05)in HPC-Exo+3-MA group.Western Blot detected the activation of apoptotic-related protein.The expression level of Actived-Caspase-3 and Bax in Rapa group was significantly increased(P<0.05)and Bcl-2 was significantly decreased(P<0.05)in HPC-Exo+Rapa group while significantly reversed in 3-MA group(P<0.05).3)The proportion of Tunel+ cells in Rapa group increased significantly(P<0.05),but there was no significant difference in 3-MA group(P>0.05).(3)Compared with Nor group,the expression of P-AMPK was significantly up-regulated(P<0.05),while P-m TOR(P<0.05),P-p70 S6K(P<0.05)and P-4E-BP1(P<0.05)was significantly down-regulated in HR group.Compared with HR group,P-AMPK in HPC-Exo group was significantly down-regulated(P<0.05),and expression of P-m TOR,P-p70 S6 K,and P-4E-BP1 was significantly up-regulated(P<0.05).HPC-Exo significantly inhibited HR-induced activation of the AMPK/m TOR signal pathway.(4)On the basis of HR-induced H9c2 apoptosis,autophagy in HPC-Exo+Met group was significantly enhanced(P<0.05)while significantly reduced(P<0.05)in HPC-Exo+Dor group.1)PS eversion in HPC-Exo+Met group was significantly increased(P<0.05)compared with HPC-Exo group,while significantly decreased in HPC-Exo+Dor group(P<0.05).2)ROS released in HPC-Exo+Met group was significantly increased(P<0.05)but significantly decreased in HPC-Exo+Dor group(P<0.05).Apoptotic-related proteins were significantly activated in HPC-Exo+Met group but in HPC-Exo+Dor group showed no significant difference(P>0.05).3)DNA fragmentation increased significantly in HPC-Exo+Met group(P<0.05),while significantly decreased(P<0.05)in HPC-Exo+Dor group.Conclusion:(1)Exosomes derived from CSCs inhibited autophagy on the basis of HR induced H9c2 apoptosis and HPCs enhanced autophagy and apoptosis inhibitory function.(2)AMPK/m TOR signaling pathway may be the molecular mechanism by which HPC-Exo inhibits HR-induced H9c2 apoptosis and autophagy. |
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