Bone Mesenchymal Stem Cells Deliver Exogenous MiR-19b Via Exosomes To Protect H9c2 Cardiomyocytes Against Hypoxia/reoxygenation-induced Autophagy And Mitophagy

Background: Ischemia-reperfusion injury(IR)can cause changes in cell structure and function in various tissues and organs,causing cell death.Among them,cardiomyocyte apoptosis is the main cause of cell death caused by ischemia-reperfusion injury.Bone Mesenchymal Stem cells(BMSCs)are stem cells that can significantly repair infarcted myocardium and improve myocardial remodeling and cardiac function.Bone marrow mesenchymal stem cells are now widely used in basic research,mainly because of its high proliferative activity,easy availability,anti-inflammatory effects,and minimal immune rejection.Transplantation of MSCs for myocardial infarction can improve ventricular remodeling and cardiac function.Its main role is the paracrine mechanism of cells,and exosomes(exo),which play a major role in the paracrine effect of stem cells.Exosomes are cell-derived microbubble structures that exert cellular information transmission by transferring proteins and genetic material.Endogenous stem cells secrete exosomes to the injured site to repair the myocardium during ischemia-reperfusion injury.Previous studies on the pathophysiology of hypoxia-hypoxia reoxygenation(HR)have focused on oxygen free radicals,calcium overload,inflammatory response,mitochondrial damage,apoptosis,and autophagy.Among them,autophagy and mitochondrial autophagy are thought to play a very important role in myocardial HR.Autophagy is a process in which damaged,denatured,aging long-lived proteins and organelles are transported to a lysosome for enzymatic digestion,and the substrate is reduced to its constituent components for reuse by the cells.The occurrence of mitochondrial autophagy is mainly caused by the production of reactive oxygen species(ROS)and the damage of mitochondria.Depolarization of the mitochondria in the cells is damaged,and the damaged mitochondria are specifically encapsulated into the autophagosome and fused with the lysosome to complete the degradation and maintain the stability of the intracellular environment.Oxidative stress can both activate mitochondrial autophagy and inhibit autophagy by disrupting the autophagy regulatory pathway.In vivoand in vitro experiments have confirmed that in the process of ischemia-reperfusion injury,moderately elevated levels of autophagy in hypoxia stage can alleviate the damage caused by hypoxia.When ischemic myocardial tissue restores nutrient and oxygen supply,A large accumulation of ROS shows that autophagy is significantly up-regulated,and with the excessive increase of autophagy levels,it is usually accompanied by an increase in autophagic death and cardiomyocyte apoptosis.At present,no studies have been reported on the regulation of cardiomyocyte apoptosis induced by hypoxia-reoxygenation(HR)by autophagy and mitochondrial autophagy in bone marrow mesenchymal stem cell exosomes.This study was to investigate the effects of bone marrow mesenchymal stem cell exosomes on H9c2 apoptosis,autophagy and mitochondrial autophagy based on HR-induced H9c2 cardiomyocyte apoptosis.To explore the mutual regulation mechanism of exosomes on the microenvironment of injured tissues,and provide new therapeutic targets and strategies for non-cell therapy of stem cells.Part Ⅰ Bone marrow mesenchymal stem cell exosome can improve cardiac function after myocardial infarction in ratsObjective: To investigate the effects of exosome from bone marrow mesenchymal stem cells(MSCs)on cardiac function in rats after acute myocardial infarction.Methods:(1)Rat MSCs required for the experiment were cultured by differential marrow centrifugation.Flow cytometry(FCM)was used to identify MSCs surface marker antigens(CD29 and CD90 and CD45)grown to P3-P6 generation;(2)Extracellular centrifugation(UC)was used to extract MSCs from cell culture media.Exo;Western Blot was used to detect the expression of transmembrane transmembrane proteins CD9,CD63 and Alix on exosomes;TEM was used to observe the morphological characteristics of exosomes;nanoparticle tracking analysis(NTA)The inner diameter and inner diameter distribution range of Exo were analyzed.(3)24 SD rats were randomly divided into 4 groups: SHAM group,MI group,PBS group and EXO group,6 rats in each group were established by ligation of left anterior descending coronary artery.Rat myocardial infarction model,exudate and PBS were injected into the peripheral area of myocardial infarction;(4)The improvement of cardiac function in rats 28 days after myocardialinfarction was observed;(5)HE staining and MASSON staining were performed for 14 days after myocardial infarction.Myocardial tissue and fibrinogen changes;(6)Myocardial protein was extracted on day 3 of myocardial infarction to observe changes in autophagy in myocardial tissue;(7)Heart tissue protein was extracted on day 14 of myocardial infarction to determine changes in apoptotic protein levels.Results:(1)Bone marrow mesenchymal stem cells isolated and cultured in vitro showed that some cells began to adhere to the wall after 48 hours of culture.The cells in the P3-P6 generation showed fish-like and swirling growth.The surface cells of FCM showed that the mesenchymal stem cells The positive surface markers CD29 and CD90 were up to 96.91% and 97.66%,respectively,and the negative marker CD45 was 1.45%(P<0.05).(2)The exosome of bone marrow mesenchymal stem cells was extracted by ultracentrifugation(UC).The results of protein electrophoresis showed that the surface markers of Exosome were positive for CD9,CD63 and Alix(P<0.05).TEM can observe different sizes,typical tea tray-like,double-layer membrane structure;NTA results show: Exosome average particle size is 87.5nm;(3)ECG results show ST-lead ST elevation after ligation of coronary artery,TTC The results showed that the heart became white after ligation of the coronary artery.(4)The cardiogram of the rats after 28 days of myocardial infarction showed that the ejection fraction of cardiac function in the EXO group was increased by 30.32%±7.21% compared with the MI group.Up to 55.31%±8.33%(P<0.05);(5)14 days after myocardial infarction,HE staining and MASSON staining were performed on the heart tissue.HE staining showed that the myocardial tissue of the MI group was disordered,and some of the myocardial cells had disappeared.The formation of a large number of fibrous tissues,the integrity of myocardial tissue in the EXO group was better than MI,and the fibrous scar tissue was less.The results of MASSON staining showed that the number of collagen fibers in the EXO group was significantly lower than that in the MI group.(6)The autophagy-associated protein in the myocardial tissue of each group was measured on the third day after myocardial infarction: the MI compared with the SHAM group.The ratio of LC3-II/ LC3-I in the group was significantly increased,and the P62 was decreased(P<0.05).Compared with MI group,the ratio of LC3-II/LC3-I protein in EXO group decreased,and the expression of P62 protein increased(P<0.05).(7)Apoptotic protein in myocardial tissue of each group was determined on the 7th day after myocardial infarction.The expression of the results showed that compared with the SHAM group,the ratio of active/pro caspase3 and Bax protein in the MI group were increased(P<0.05)and Bcl-2 was decreased(P<0.05),compared with the MI group.The ratio of active/pro caspase3 in EXO group,Bax protein expression level decreased,and Bcl-2 increased(P<0.05).Conclusion: Bone marrow mesenchymal stem cell exosomes can improve ventricular remodeling and cardiac function in rats after acute m yocardial infarction.Part Ⅱ Role of autophagy and mitochondrial autophagy in cardiomyocyte-derived mesenchymal stem cell exosome miR--19 b in improving H9c2 injury induced by hypoxia-reoxygenationObjective: Based on the improvement of cardiac function after myocardial infarction by MSCs source exosomes,a hypoxia-reoxygenation model was established in vitro to investigate whether exosomes inhibit H9c2 cardiomyocyte apoptosis and reduce autophagy and mitochondrial self in hypoxia-reoxygenation.bite.And to explore the regulatory role of the miR--19b/Bnip3 L signal axis.Methods:(1)The model of H9c2 cardiomyocyte apoptosis was established by hypoxia for 24 h.On this basis,400 ng/μl of Exosonme treated cells for 12 h was HR+EXO group,and 1nm rapamycin(Rapa)was added to treat cells for 12 h as HR+EXO+RAPA group.The experiment was divided into: Control group,HR group,HR+EXO group,HR+EXO+RAPA group.Apoptosis was detected by flow cytometry using Annexin V/PI staining.Mitochondrial membrane potential(ΔΨm)was measured by mitochondrial fluorescent probe JC-1,and cells were stained with mitochondrial ROS fluorescent probe Mitosox Red and ROS fluorescent probe DCFH-DA.Microscopic observation of changes in mitochondrial ROS and cellular ROS levels.The number of autophagosomes was observed by transmission electron microscopy.The protein changes of autophagy proteins LC3-I,LC3-II,P62 and mitochondrial marker proteins TOMM20 and COXIV were detected by Western blotting.(2)The microverexpression esicles were searched.And the NCBI literature library to screen out MSCs-derived exosomes with the most closely related micro RNAs of oxidative stress: miR--99 a,mi R-133,mi R-19 b,mi R-150,mi R-410,mi R-451;(3)The experiment was divided into Control group,HR group and HR+EXO group.RT-PCR was used to quantify the above candidate micro RNAs,and the micro RNAs with the most significant changes were screened out.That is,mi R-19b;(4)to verify the role of miR--19 b in anti-apoptosis,autophagy and mitochondrial autophagy in HR cardiomyocytes,mi R-19 b inhibitors and mimics and negatives using life2000 TM as a vector The control(50 n M)was transfected into cardiomyocytes.The experimental groups were: HR group,HR+mi R-19 b mimic group,HR+MNC group,HR+mi R-19 b inhibitor group,HR+INC group,using Annexin V-FITC/ PI detected apoptotic cells,DCFH-DA detected total ROS,Western Blot detected total cell protein LC3-I and LC3-II,detected mitochondrial marker protein TOMM20 and COX IV,JC-1 stained mitochondrial membrane potential changes,mitochondria ROS fluorescent probe Mitosox Red was used to detect changes in mitochondrial ROS;(5)To verify that exosomes exert anti-apoptosis,autophagy and mitochondrial autophagy by transmitting miR--19 b,the experiment was divided into HR group,HR+EXO group,HR.+EXO+mi R-19 b inhibitor group,HR+EXO +INC group,using the same method as(1)to detect changes in cell and mitochondrial ROS,mitochondrial membrane potential,autophagy levels and changes in mitochondrial membrane protein levels;In order to verify that Bnip3 L is a potential target gene of miR--19 b,the experiment is divided into Control group.mi R-19 b mimic group,mi R-19 b inhibitor group,Western Blot detection of Bnip3 L protein expression level changes;(7)to verify the regulatory relationship between miR--19 b and Bnip3 L,use life2000 TM to transfect Bnip3 L plasmid into cardiomyocytes Within the experiment,the experiment was divided into HR group,HR+mi R-19 b mimic group,HR+mi R-19bmimic+pc DNA-Bnip3 L,and the method of(1)was used to detect the total ROS,mitochondrial ROS,autophagy level and mitochondrial marker protein.(8)To verify the regulation of miR--19b/Bnip3 L signaling pathway in anti-apoptosis,autophagy and mitochondrial autophagy of MSCs-derived exosomes.The experiment was divided into HR group,HR+EXO group,HR+EXO+Bnip3L group,HR+EXO+pc DNA-Bnip3L+mi R-19 b mimc group,and the total ROS,mitochondrial ROS and autophagy levels were detected by the method of(1).Changes in mitochondrial marker proteins;(9)To verify whether miR--19 b is directlyinteracting with Bnip3 L,a dual luciferase reporter assay was designed.The previously designed r-Bnip3L-3UTR plasmid rno-mi R-19b-1-5p/Negative.Control was added to the helper transfection reagent.The experiment was divided into: NC mimic+r-Bnip3L-3UTR-WT,rno-mi R-19b-1-5p+r-Bnip3L-3UTR-WT,NC mimic+r-Bnip3L-3UTR-mu,rno-mi R-19b-1-5p+r-Bnip3L-3UTR-mu,four groups,using a fluorescence detection kit,Promega Dual-Luciferase system,to measure the fluorescence of each group.Results:(1)The results of in vitro tests showed that compared with the HR group,the apoptosis rate in the EXO group was significantly decreased(P<0.05),the number of autophagosomes was significantly decreased(P<0.05),total ROS in the cells and mitochondrial ROS.The level of significant decrease(P<0.05),the level of autophagy-related protein decreased(P<0.05),the levels of mitochondrial membrane proteins TOMM20 and COXIV protein increased significantly(P<0.05),and the autophagy agonist RAPA was added.Reversal of anti-apoptosis,anti-autophagy and mitochondrial autophagy of hypoxic-reoxygenated H9c2 cardiomyocytes from mesenchymal stem cell-derived exosomes;(2)Screening of miR--19 b,mi R-99,mi R-in combination with multiple databases 133,mi R-150,mi R-410,mi R-451 and other 6 micro RNAs;(3)in the Control group,HR group,HR+EXO group,compared with the Control group,the HR group miR--19 b decreased most significantly;Compared with the HR group,the expression of miR--19 b was significantly increased in the HR+EXO group(P<0.05).(4)To verify the anti-apoptosis,autophagy and mitochondrial autophagy of miR--19 b in HR cardiomyocytes.The results showed that compared with the HR group,the early apoptotic rate of the HR+mi R-19 b mimic group was significantly decreased(P<0.05),and the total cellular ROS.Mitochondrial ROS levels were decreased(P<0.05),the levels of autophagy proteins LC3-II/LC-I were decreased,and the levels of mitochondrial marker proteins TOMM20 and COXIV were increased,whereas the HR+mi R-19 b inhibitor group was opposite(P<0.05);(5)To verify that exosomes exert anti-apoptosis,autophagy and mitochondrial autophagy by transmitting miR--19 b,the results showed that: compared with HR+EXO group,HR+EXO+mi R-19 b inhibitor The early apoptotic rate of cells in the group increased significantly,the total ROS level,mitochondrial ROS level increased,the level of autophagy increased,and the mitochondrial outer membrane marker protein decreased significantly(P<0.05).(6)The potential for verifying Bnip3 L as miR--19 b The target geneshowed that compared with the Control group,the protein level of Bnip3 L in the miR--19 b mimic group was significantly reduced,and the protein level of Bnip3 L in the miR--19 b inhibitor was increased(P<0.05);(7)to verify the miR--19 b exerts anti-apoptotic effect by regulating Bnip3 L.The results showed that compared with HR+mi R-19 b mimic group,ROS+ mitochondrial ROS and autophagy increased in HR+mi R-19 b mimic+pc DNA-Bnip3 L group.Mitochondrial marker protein decreased(P<0.05)(8)To verify the regulation of miR--19b/Bnip3 L signaling pathway in anti-apoptosis,autophagy and mitochondrial autophagy of MSCs-derived exosomes,compared with HR+EXO group,HR+EXO+pc DNA-Bnip3 L group The early apoptosis rate of cells increased significantly,the total ROS level,mitochondrial ROS level increased,the level of autophagy increased,and the mitochondrial outer membrane marker protein decreased significantly(P<0.05).However,HR+EXO+pc DNA-Bnip3L+mi R-19 b mimc group This effect can be significantly reversed(P < 0.05).(9)The luciferase reporter assay of miR--19 b and Bnip3 L showed that rno-mi R-19b-1-5p failed to down-regulate the expression of luciferase in r-Bnip3L-3UTR,indicating that there was no direct Combination.Conclusion: Bone marrow mesenchymal stem cell-derived exosomes can down-regulate the expression of Bnip3 L in H9c2 cardiomyocytes under hypoxia-reoxygenation by transferring miR--19 b,thereby reducing autophagy and mitochondrial autophagy,and exerting anti-apoptotic effects.