Regulation Of Calcitonin Gene-related Peptide On The Survival And Cardiomyogenic Differentiation Of Mesenchymal Stem Cells By GSK3-β/β-catenin

Objective: After acute myocardial infarction(MI), most myocardial cell deaded andreplaced by scar tissue, which severly impaired the cardiac systolic function. Fortunately,in recent years several researchers found that the bone marrow mesenchymal stemcells(MSCs) can effectively promote the regeneration of cardiomyocytes due to the MSCs’ablity of self-renewal, proliferation and multidifferentiation. But there were also negativeresult showed that, the cardiac function was not improved by MSCs’ transplantation in theacute phase of myocardial infarction, the reason mainly was that the heart was in anoxidative stress environment after MI, which weakened the MSCs’ capacity ofproliferation, differentiation and survival, so the MSCs could not play a good role in MI.The key to solve this problem was to improve the survival rate of transplantedMSCs.Calcitonin Gene-related Peptide(CGRP) could promote the regeneration ofmyocardium and decrease the infarction area according to paracrine factors that benefit forangiogenesis, restrain Vascular Smooth Muscle Cells’(VSMCs) proliferation andpromote it’s a poptosis. Wnt played a role in the development and growth of heart, whichwidely distributed in the heart. It can be activated when the heart was in a state of stress,following to participate in a series of pathophysiological process of MI. The differentiationmechanism of MSCs mainly depends on Wnt signal,Glycogen synthasekinase3-β(GSK3-β), the downstream signaling the of Wnt,promoted the accumulation ofβ-catenin in nuclear when it was phosphorylated, and then lead an expression of a seriestarget genes of proliferation, survival, and differentiation. The purpose of this ex perimentis to study whether the CGRP can control the fate of the MSCs and strengthen the effect of the treatment on MI, and the mechanism was related with GSK3-β/β-catenin signalingpathways?Methods:1.Mice was put to death with cervical vertebra dislocation, stripped the femur and tibia,rinsed marrow cavity with incom plete DMEM/F12medium, then collected bone marrowfluid, cultured and amplificated MSCs by using gradient centrifuge and adhesive culturemethods in vitro; Flow cytometry(FCM) analysis appraise the surface markers of MSCs;Drawing MSCs growth curve by cell counting.2.MSCs were pretreatmented with different concentration of H2O2(400,200100,50,25μmol/L) for2h, the control group was without H2O2, Flow cytometry(FCM) analysisdetected the survival and death of MSCs, to find a concentration of H2O2that made theMSCs’ early apoptosis more than that the late apoptosis; MSCs were pretreatmented withdifferent concentration of CGRP (10-6,10-7,10-8,10-9,10-10mol/L) for24h, addingappropriate concentration of H2O2to each group and the control group only with H2O2,after2h detected cell survival by MTT; Flow cytometry(FCM) tested survival and apoptosis; According to the result of MTT and Flow cytometry(FCM), found whichconcentration of CGRP can significantly suppress the apoptosis induced by H2O2, andMSCs were pretreatmented with the above concentration of CGRP added H2O2, westernblot detected the expression of apoptosis related proteins (Bcl2, survivin, caspase3andBax).3.MSCs were pretreatmented with different concentration of CGRP (10-6,10-7,10-8mol/L)+5-aza, control group only with5-aza, blank with nothing, after4weeks detected theexpression Nkx2.5and cTnI mRNA by RT-PCR; Flow cytometry(FCM) detected thesurvival and mortality of MSCs.4.The experimental group pretreatmented with10-6mol/L CGRP MSCs, control groupwith10-6mol/L CGRP+10-6mol/LCGRP8-37, blank group with nothing, western blotdetected the expression of phosphorylated GSK3-β and β-catenin.Results:1. MSCs cultured in vitro, distributed sus pendly and scatteredly at24h, it was small andround. Adhered to wall at48h, some individual cells becomed into fusiform, triangular orstar at4-5days. There are cell colonies formation, overlapping and fusion at9-10days, in which there were the "whirl pool-like" structure. MSCs that passaged can stick a wallcompletely after24h, growed like a fibroblasts sample form; The P3generation of MSCsgrowth curve showed MSCs went into rapidly proliferation in24-72h, the logarithmicphase was in48h,in6days, the cell growed slowly, and went into the plateau in8days;Flow cytometry identification results for MSCs surface antigens: CD2997.4%;CD9098.0%;CD452.3%.2.1. MSCs were pretreatmented with different concentration of H2O2, Flow cytometryanalysis found that the survival rates of experimental group were lower than the controlgroup (P<0.01), the survival difference between100μM H2O2and200μM H2O2groups hadno statistical significance (P>0.05), it found among other groups:The early apoptosis rateof control group（2.867±0.145） was less than25μM H2O2group（7.000±0.551）,50μMH2O2grou（p7.500±0.289）and100μM H2O2group（9.367±0.233）(P<0.01), but there wereno statistical difference between200μM H2O2group（3.533±0.233）and400μM H2O2group（3.733±0.252）(P>0.05), the early apoptosis rate in100μM H2O2group was the highest inall experimental groups (P<0.01); The late apoptosis rate+mortality of control group wereless than the experimental group(P<0.01), there were no statistically significant differenceof late apoptosis rate+mortality between the50μM H2O2group and100μM H2O2group(P>0.05), among other groups showed:25μM H2O2<50μM H2O2、100μMH2O2<200μM H2O2<400μM H2O2(P<0.01).According to the results it found that when theconcentration of H2O2was100μmol/L, it induced MSCs to peaked early apoptosis, and thedeath, late apoptosis and survival cells counted in the intermediate values.2.2. MSCs were pretreatmented with different concentration CGRP+100μM H2O2, testedby OD490and flow cytometry respectively. The result showed that the OD value of theexperimental group were greater than the control group(P<0.01); With the increaseing ofCGRP concentration, OD value increased gradually(10-6M CGRP group>10-7M CGRPgroup>10-8M CGRP>10-9M CGRP group)(P<0.01), there were no statisticalsignificance difference between10-9M CGRP group and10-10M CGRP group(P>0.05).Flow cytometry results showed that with the increaseing of CGRP concentration, thesurvival had been improved gradually(10-6M CGRP group>10-7M CGRP group>10-8MCGRP>10-9M CGRP group)(P<0.01), the group of10-9M CGRP and10-10M CGRP had no statistical difference(P>0.05). Consistented with the results of MTT.2.3. MSCs were pretreatmented with different concentration CGRP+100μM H2O2, westernblot tested the expression of apoptosis related proteins (Bcl2, survivin, Caspase3and Bax).The results indicated that the expression of antiapoptotic protein (survivin, Bcl2) inexperimental group were more than the control group (P<0.01), along with the increaseingof CGRP concentration, the expression of antiapoptotic proteins increased (10-6M CGRP>10-7M CGRP>10-8M CGRP group)(P<0.01). The apoptosis protein (Caspase3, Bax) incontrol group were higher than in experimental group (P<0.01),with the increaseing ofCGRP concentration,the expression of apoptosis proteins reduced (10-6M CGRP<10-7MCGRP<10-8M CGRP group)(P<0.01). And Bcl2/Bax was>1in experimental group, whileBcl2/Bax <1in control group.3.1. MSCs were pretreatmented with different concentration of CGRP+5-aza, controlgroup only with5-aza, and blank group with nothing. After4weeks, tested the expressionof Nkx2.5and cTnI mRNA by RT-PCR, the result showed that the10-6M CGRP+5-azagroup and10-7M CGRP+5-aza group were more than the control group and Blank(P<0.01),while there was no statistically significant difference among the10-8M CGRP+5-aza group,control group and Blank(P>0.05), it also found that the expression of Nkx2.5in controlgroup was more than that in Blank (P<0.01), while the expression cTnI between controlgroup and Blank had no difference(P>0.05). In CGRP+5-aza groups, it found that with therising of the concentrations of CGRP, the expression of Nkx2.5cTnI wereincreased(P<0.01).3.2Tested the survival and apoptosis rate of each groups by flow cytometry, which showedthat the Blank was higher than other groups(P<0.01), while the late apoptosisrate+mortality in Blank was lower than other groups (P<0.01); The cell survival rate inCGRP+5-aza groups were higher than5-aza group (P<0.01), and the late apoptosisrate+mortality in CGRP+5-aza groups were lower than5-aza group (P <0.01); AmongCGRP+5-aza groups, it found that with the rising of the concentrations of CGRP, thesurvival rate increased (P<0.01), while the late apoptosis rate+mortality reduceed(P<0.01).4. MSCs were pretreatmented with CGRP and CGRP+CGRP8-37, the control group with nothing, and then collected protein, tested the expression of P-GSK3β and β-cantenin withwestern blot. The results indicated that the expression of P-GSK3-β and β-catenin proteinin CGRP group were higher than control group(P<0.01), added CGRP8-37, the expressionof P-GSK3-β and β-catenin protein were reduced, and lower than the control group(P<0.01).Conclusion:1.It can successfully obtain high purity, good condition of MSCs by gradient centrifuge andadhesive culture methods.2.100μM H2O2induced MSCs to take early apoptosis, and made the late apoptosis anddeath of cells less;CGRP was concentration dependent to suppressed H2O2of inducing MSCsapoptosis; The mechanism of CGRP preventing H2O2from apoptosis was the increaseingexpression of Bcl2and survivin, and the decreasing expression of Caspase3and Bax.3. High concentrations of CGRP can promote MSCs to differentiat to myocardial cell, andinhibit the cytotoxic effect of5-aza.4. The mechanism of CGRP controling the fate of MSCs may that the CGRP binded withthe CGRP receptor and activated GSK3-β/β-catenin signal pathway.