Hypoxia Preconditioning Of Rat Bone Marrow Mesenchymal Stem Cells Regulation Of Calcium Homeostasis And Apoptosis In Cardiac Stem Cells Through The MicroRNA-133a/PLB Pathway Under Oxidative Stress

Objective: Stem cell therapy,especially C-kit pos cardiac stem cells（CSCs）transplantation therapy is an emerging approach to the treatment of acute myocardial infarction,which can significantly reduce the ventricular remodeling after myocardial infarction and improve heart function.Therefore,it is regarded as the most ideal seed cells for stem cell transplantation.However,one of the critical problems faced by CSCs is the poor engraftment after transplantation.So,there’s the question——How to enhance the engraftment and regenerative capabilities of these cells.Numerous studies had shown that exosomes derived from mesenchymal stem cells（MSCs）were proved to boost cell proliferation and angiogenic potency and resistance to apoptosis.Ischemic preconditioning or hypoxic preconditioning has been shown as a potent approach to enhance survival and regeneration of MSCs in an ischemic environment.It has been argued recently that stem cells exert their beneficial effects through release the beneficial factors.Related studies confirmed that exosome enrichment in microRNA133 a.In vitro studies have shown that microRNA133 a can inhibit the apoptosis of cardiac stem cells under oxidative stress,and may also be a target of PLB to inhibit cell calcium homeostasis.The aim of this study was to investigate the relationship between exosomal-microRNA133 a which derived from hypoxic preconditioning BMSC and the apoptosis and calcium homeostasis in C-kit poscardiac stem cells under oxidative stressMethods:Part one:1.Bone marrow MSCs were isolated from Sprague–Dawley（SD）rats（50g）as previously described.Use flow cytometry methods to identificate the purity for subsequent experiment.Hypoxic preconditioning MSCs with different concentration of H₂O₂（200,and 100,50,25 μM）for different time（1h,2h）.The control group was established with normal cells without H₂O₂.Detect MSCs activity with CCK-8.Then we choose the best reasonable concentration of H₂O₂ to enchance the MSCs’ activity.2.The extraction of bone marrow mesenchymal stem cell derived exosomes by SBI kit method,PEG method and q EV kit method,using Western blotting（Wstern blot,WB）detect the marker proteins of exosomes（CD63,CD9 and ALIX）,using electron microscopy and Nanoparticle Tracking Analysis（NTA）analysis the averge size and the concentration of exosomes,comparison of various fitting methods and select the optimal extracting method of hypoxic preconditioning of bone marrow mesenchymal stem cells to secrete exosomes（H-exo）and normoxic condition of mesenchymal stem cells secreted exosomes（N-exo）..3.C-kitpos CSCs was cultured and selected in SD rats by the methods of enzyme digestion and magnetic bead which we have mastered before.Use immunofluorescence and flow cytometry methods to identificate the purity for subsequent experiment.Processed C-kitpos CSCs with different concentration of H₂O₂（200,and 100,and 50 μM）for different time（1h,2h,4h）to simulate hypoxia micro-environment of different degrees.The control group was established with normal cells without H₂O₂.Detect C-kitpos CSCs’ survival rate,early apoptosis rate,late apoptosis rate,total apoptosis rate and necrosis rate with flow cytometry.Finally,make sure that the best reasonable concentration of H₂O₂ induced C-kitpos CSCs’ early apoptosis and total apoptosis rate to simulate the hypoxia micro-environment.4.Di I-labeled exosomes were internalized with C-kitpos CSCs.Exosome was co cultured with stem cells at different times（0,1,6,12,24h）to determine the optimal time for cell uptake of exosome.5.Co cultured with different concentrations（0、100、200、400、800ng/μL）of different exosomes（N-exo and H-exo）and cardiac stem cells.After co culture for a certain period of time,adding H₂O₂ to make sure C-kitpos CSCs’ under oxidative stress.CCK-8 method was used to detect its activity,and the best solution was obtained to ensure the maximum cell activity.6.The part of experiment was divided into four groups in order to explore the role of H-exo and apoptosis of C-kitpos CSCs under oxidative stress status.:（1）.Normal Group:C-kitpos CSCs without any treatment.（2）Control group:C-kitpos CSCs’ under oxidative stress.（3）N-exo group: C-kitpos CSCs co culture with a certain concentration of N-exo for a certain period of time,then adding H₂O₂ to make sure C-kitpos CSCs’ under oxidative stress.（4）H-exo group: C-kitpos CSCs co culture with a certain concentration of H-exo for a certain period of time,then adding H₂O₂ to make sure C-kitpos CSCs’ under oxidative stress.Using fluorescent-activated cell sorting（FACS）to detect the rate of apoptosis of all groups.Using Western blot to the detection of apoptosis-related proteins,such as the expression of cleaved-Caspase-3,caspase8 and caspase9 in each group.Using Confocal laser scanning microscope（CLSM）to detect calcium homeostasis stained by fluo8.Part two:7.In order to clarify the mechanism of H-exo regulating the calcium homeostasis in CSCs under oxidative stress,the experiment was divided into three groups: group normal,group control,group H-exo.WB detection of exosome regulation of sarcoplasmic reticulum calcium base regulation system of major regulatory protein,namely myocardial sarcoplasmic reticulum Ca²⁺-ATPase2 a 2A enzyme（SERCA2a）,Phosphoprotein（PLB）,type 2 ryanodine receptor（Ryanodine Receptor2,Ry R2）the influence of protein level.8.In order to determine whether microRNA-133 a exists in exosomes,and whether it is in exosomes are differentially expressed,the experiment was divided into two groups,namely N-exo group and H-exo group,we used RT-PCR to detect the exosome microRNA-133 a expression level of m RNA.9.The part of experiment was divided into four groups in order to clarify how H-exo can inhibit the apoptosis and calcium homeostasis of cardiac stem cells.（1）.Normal Group（2）Control group.（3）N-exo group（4）H-exo group.Using RT-PCR to detected m RNA expression of microRNA-133 a in each group.10.The results of this study above eloquently showed that microRNA-133 a would be overexpressed in H-exo.At the same time H-exo could inhibiting apoptosis of c-kitpos CSCs at a certain extent.To further investigate whether the overexpressed microRNA133 a in H-exo involved this process,we divided the experiment into six groups :（1）H-exo（2）control.（3）mimic（microRNA-133 a mimic）: C-kitpos CSCs transfected mimic microRNA-133 a for a certain amount of time and concentration.（4）MNC（microRNA-133 a mimic negative control）: C-kitpos CSCs transfected mimic negative control for a certain amount of time and concentration;（5）Inhibitor（si-microRNA-133a）: C-kitpos CSCs transfectedmicroRNA-133 a si RNA as it inhibitor for a certain amount of time and concentration;（6）INC（microRNA-133 a inhibitor negative control）: C-kitpos CSCs transfected inhibitor negative control for a certain amount of time and concentration.All the above groups were treated with H₂O₂ to induce apoptosis after the above treatment.Using fluorescent-activated cell sorting（FACS）to detect the rate of apoptosis of all groups.Using Western blot to detect the intracellular signal molecules or apoptosis-related proteins,such as the expression of PLB,cleaved-Caspase-3,caspase8 and caspase9 in each group.Plb m RNA were examined by Real Time-Polymerase Chain Reaction（RT-PCR）.Using Confocal laser scanning microscope（CLSM）to detect calcium homeostasis stained by fluo8.11.In order to further clarify the mechanism of H-exo regulating the calcium homeostasis and apoptosis of C-kit⁺CSCs under oxidative stress,whether PLB can play a role,the following experiments will be divided into 4 groups,we divided the experiment into four groups :（1）control（2）H-exo.（3）si RNA（PLBsi RNA）: C-kitpos CSCs transfected PLB si RNA as it inhibitor for a certain amount of time and concentration.（4）SNC（PLB si RNA negative control）: C-kitpos CSCs transfected PLB si RNA negative control for a certain amount of time and concentratio.All the above groups were treated with H₂O₂ to induce apoptosis after the above treatment.Using fluorescent-activated cell sorting（FACS）to detect the rate of apoptosis of all groups.Using Western blot to detect of intracellular signal molecules or apoptosis-related proteins,such as the expression of PLB,cleaved-Caspase-3,caspase8 and caspase9 in each group.Plb m RNA were examined by Real Time-Polymerase Chain Reaction（RT-PCR）.Using Confocal laser scanning microscope（CLSM）to detect calcium homeostasis stained by fluo8.Results:Part one :1.Bone marrow mesenchymal stem cells were successfully cultured.The surface markers were detected by FACS.The results showed that the surface markers CD29 and CD90 of MSCs were strongly positive,but the expression of CD45 was negative.CCK-8 results showed that compared with Control group,the activity of BMSCs reached the maximum value at 100 μmol/L（P<0.05）for a period of 2 hours.2.Exoquick,q EV and PEG were used to extract exosome from BMSC culture medium.The results of WB showed that the expression of CD63 protein was positive in the three methods.Electron microscopy showed that the extracted exosome is like the cup or saucer,diameter between 30-150 nm.NTA results showed that exoqucik,q EV,PEG,respectively,the peak value of the body were: 139 nm,161nm,163 nm.Combined with the results of the concentration and purity of the exocrine,the exoqucik reagent kit was selected for the next experiment.The results of WB showed that CD63,CD9 and ALIX were positive by exoquick.3.Primarycultured CSCs were adhered to the wall within 48 hours.The primary cultured cells showed that some of the cells were round and bright.The cells then grow into fusiform or triangular shape.Immunofluorescence detected that c-kit was positive after the separation by the method of magnetic bead.What’s more,The signs of C-kitpos cells were detected by the Fluorescence activated Cell Sorting（FACS）,and the results showed that the expression of c-kit were 91.26%,CD45 0.65% and CD34 0.32%.After H₂O₂ induced apoptosis,CCK-8 and FACS results showed that compared with normal group,the concentration of H₂O₂ was 100 mol/L after treatment for 2 hours,the apoptosis reached the maximum（P<0.05）.4.After exosome staining by Di I,Immunofluorescence results showed that after 24 hours of culture,exosomes which markered by Di I was enriched in the cells.5.After co cultured two groups exosome with cells,apoptosis was induced by H₂O₂,CCK-8 results showed that compared with control group,both N-exo and H-exo could increase the activity of C-kitpos CSCs under oxidative stress（P<0.05）,and the effect of H-exo was more significant（P<0.05）.In addition,when the concentration of H-exo was 400ng/μl,the activity of C-kitpos CSCs under oxidative stress could reach the maximum value（P<0.05）.So we select 400 ng/μl as the treatment concentration.6.Apoptosis was detected by AV-PI FACS,the results showed: compared with normal group,the apoptosis rate of control group increased significantly（P<0.05）;compared with control group,N-exo group and H-exo group could inhibit the apoptosis of the C-kit⁺CSCs under oxidative stress and the rate of early apoptosis rate（P<0.05）,and H-exo the anti apoptotic effect was more significant（P<0.05）;in addition,the H-exo group could significantly inhibit the C-kit⁺CSCs late apoptosis rate（P<0.05）.WB results showed: Compared with normal group,the expression of caspase8,caspase9 and cleaved-caspase3 increased significantly in control group（P<0.05）;Compared with the control group,H-exo can significantly inhibit the expression of cleaved-caspase3,caspase8,caspase9 protein（P<0.05）,while N-exo can only inhibit the expression of cleaved-caspase3 and caspase9 protein（P<0.05）,and H-exo is better than N-exo（P<0.05）.CLSM results showed that both H-exo and N-exo could significantly inhibit the calcium homeostasis（P<0.05）compared with the control group,and the effect of H-exo is better than N-exo（P<0.05）.Part two7.WB was used to detect the expression of SERCA2 a,PLB and Ry R2 protein levels.The results showed:compared with normal group,the expression of SERCA2 a protein levels were significantly decreased in control group and H-exo group（P<0.05）,the expression of Ry R2 protein levels were significantly inecreased in control group and H-exo group（P<0.05）,the expression of PLB protein was significantly increased in control group.Compared with control group,the expression level of SERCA2 a protein in H-exo group was significantly increased（P<0.05）,the expression of PLB and Ry R2 decreased significantly（P<0.05）.8.The expression of microRNA-133 a in exosome of each group was compared,The results of RT-PCR showed that the expression of microRNA-133 a in H-exo was significantly higher than that of in N-exo（P<0.05）.9.In order to detect in microRNA-133 a m RNA expression after co cultered with H-exo and N-exo,the RT-PCR results showed: compared with normal group, micorRNA-133 a m RNA expression increased significantly in control group（P<0.05）.Compared with the control group,the expression of micor RNA-133 a m RNA was increased significantly in the H-exo group（P<0.05）.10.To establish the over expression and inhibition model of microRNA-133 a in C-kitpos CSC,and then add hydrogen peroxide to induce cell apoptosis,and the results of FACS shows that : Compared with mimic group,the early apoptosis rate,the late apoptosis rate and the total apoptosis rate of control group were significantly increased（P<0.05）,the early apoptosis rate,the late apoptosis rate and the total apoptosis rate in H-exo group were significantly decreased（P<0.05）.Compared with the inhibitor group,the apoptosis rate of control group increased significantly（P<0.05）,early apoptosis and total apoptosis rate decreased significantly（P<0.05）,the necrosis rate decreased significantly（P<0.05）;group H-exo,the early apoptosis rate increased significantly（P<0.05）,late apoptosis and total apoptosis rate decreased significantly（P<0.05）,necrosis the rate of decrease（P<0.05）.WB results showed that: Compared with the mimic group,the expression of caspase8,cleaved-caspase3 protein level was significantly decreased in group H-exo（P<0.05）,the expression of caspase9,cleaved-caspase3 protein levels in inhibitor group were significantly increased（P<0.05）;compared with inhibito group,caspase8,caspase9,cleaved-caspase3 protein expression were significantly decreased in H-exo group（P<0.05）,caspase9 and cleaved-caspase3 protein levels were significantly decreased in mimic group（P<0.05）.The results showed that overexpression of microRNA-133 a had little effect on caspase8 protein level,and could significantly inhibit the expression of caspase9 and cleaved-caspase3 protein.The results of CLSM showed that the fluorescence difference ratio in mimic group increased significantly（P<0.05）,compared with control group,suggesting that calcium homeostasis in mimic group decreased significantly,but compared with H-exo group,there are still significant gaps（P <0.05）.11..WB results showed that: compared with mimic group,the expression of PLB protein in control group,MNC group,inhibitor group and INC group were significantly increased（P <0.05）,and the expression of PLB protein in H-exo group was significantly decreased（P<0.05）.Compared with inhibitor group,the expression level of PLB protein in control group,the expression of PLB in H-exo group and mimic group was significantly decreased（P<0.05）.RT-PCR results showed that compared with mimic group,the expression of PLB m RNA in H-exo group decreased significantly（P<0.05）.Compared with inhibitor group,the expression level of PLB m RNA in H-exo group was significantly decreased,the expression of PLB m RNA in mimic group,control group,MNC group and INC group had no significant difference（P>0.05）.In this regard,we consider that microRNA-133 a regulation of PLB in C-kitpos CSCs under oxidative stress may be limited to protein level.12.After transfection of si RNA into C-kitposCSCs,the results of RT-PCR showed that the expression level of PLB m RNA in control group,H-exo group and SNC group were significantly higher than those in group si RNA（P<0.05）.The results of WB showed that the expression of PLB protein in control group,H-exo group and SNC group were significantly higher than those in group si RNA（P<0.05）.Illustrates the success of the model that inhibiting the expression of PLB by si RNA.WB to detect the apoptosis protein,the results showed: compared with si RNA group,the expression of caspase8、caspase9、cleaved-caspase3 in H-exo was significantly decreased（P<0.05）.compared with control group,the expression of caspase9、cleaved-caspase3 in si RNA group was significantly decreased（P<0.05）.The results showed that inhibition of PLB may inhibit the expression of cleaved-caspase3 and caspase9 protein in the state of C-kitpos CSC under oxidative stress,but there was no significant effect on the expression of caspase8 protein.CLSM results showed that compared with the si RNA group,control group and SNC group had significant increase in calcium homeostasis（P<0.05）.The results suggest that PLB may play an important role in the regulation of cell calcium homeostasis.Conclusion:1.The exosome secreted by hypoxic preconditioning BMSC has an anti apoptotic effect on C-kitpos CSCs under oxidative stress,and its anti apoptotic effect may be related to the regulation of calcium homeostasis;.2.H-exo may inhibit the calcium homeostasis and apoptosis of C-kit⁺CSCs in oxidative stress via microRNA-133a/PLB signaling pathway.