| Beckground: Repairing myocardial infarction by transplanting stem cells has become a research hot spot.However,stem cells transplantation is challengeable,that is,the stem cells “seed” are weakness in survival ability and the hosts are in bad microenvironment.Both of which will lead to the deaths of transplanted cells,and affect curative effects.However there are no effective approaches to cope with the dual challenges at the same time.Objective: Adropin has been demonstrasted to be an endogenous bioactive substances which can promote cells survival.Our previous study also found that Adropin can reduce myocardial ischemia-reperfusion injury in mice.This study aimed to explicit that stem cells transplantation via Adropin preconditioning and postconditioning,namely the dual regulation of “seed” cells themselves and microenvironment can promote the survival of the transplanted cells,so as to enhance curative effect,and to explore its mechanism.Methods: 1.MSCs apoptosis model was induced by H2O2 in vitro,using 10 ng/ml,25ng/ml and 50ng/ml Adropin was used to pretreat MSCs for 24 hours respectively.Cell counting method was used to detect cell activity and the apoptosis rate was detected by flowcytometry,the best dose of Adropin was determined in vitro,used for subsequent experiments in vivo.Signal pathway proteins PI3K/Akt,ERK1/2 by western-blot and anti-apoptotic proteins BCL-2 and Bcl-XL by western-blot,apply signal pathway proteins PI3 K inhibitor LY294002 and ERK1/2 inhibitor PD98059 to determine possible molecular mechanisms on which Adropin play a protecting role.2.MSCs were labeled by BrdU in vitro.Left main coronary artery of SD rats was ligated continuously to establish myocardial infarction model,which were randomly divided into four groups:(1)Sham: only thread and no ligation;(2)MI group;(3)MSCs groups: 50μl MSCs was injected without Adropin preconditioning in the area around ischemia 10 minutes after ligationing(1.0×106cells);(4)MSCs pretreated by Adropin transplantation joint with Adropin post-treated group(the Adropin group): 0.2mg/kg Adropin was injected by caudal vein followed by injecting 50μl Adropinpreconditioning MSCs(24 hours before transplantation to Adropin preconditioning,the final Adropin concentration was 25ng/ml,1.0×106cells),8 animals were executed 72 hours after transplanting,examining echocardiography 28 days after transplanting,2ml blood sample was left in each group separately.72 hours and 28 days after transplantion: Immunohistochemistry detected the survival BrdU-positive MSCs in area of stem cells transplantation.Cardiac function detected by echocardiographic,72 hours after transplantion: inflammatory factors including tumor necrosis factor(TNF)-α and interleukin(IL)-1β and anti-inflammatory factor IL-10 were assayed by enzyme-linked immunosorbent assay(ELISA).28 days after transplantion: collagen deposition by masson staining,angiogenesis were evaluated by immunohistochemistry;the expression of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)protein and mRNA were assessed by real-time PCR and western-blot respectivelyResults: 1.MSCs apoptosis model was established by 100 μmol/L H2O2 treated 4hours in vitro.No matter 25 ng/ml or 50 ng/ml Adropin pretreated MSCs can improve the cells survival and reduce early apoptosis ratio apparently compared with the 10ng/ml group,the 50 ng/ml group failed to promote cell survival compared the 25 ng/ml group.The phosphorylated Akt(p-Akt)/total Akt(t-Akt)and phosphorylated ERKl/2(p-ERKl/2)/total ERKl/2(t-ERKl/2)ratio increased obviously in the Adropin group and the MSCs group compared with the MI group,the Bcl-2 and Bcl-XL expression was increased obviously in the Adropin group and the MSCs group.The phosphorylation caused by Adropin on Akt and ERK1/2 respectively can be inhibited by LY294002 and PD98059,and the Bcl-2 and Bcl-XL expression can be reduced by LY294002 and PD98059 either.2.No matter 72 hours or 28 days after transplanting in the Adropin group,BrdU positive cells were apparently increased than the MSCs group [72hours,(100±16)/mm2 vs.(63±14)/mm2,P<0.05;28days,(51±11)/mm2 vs.(17±4)/mm2,P< 0.05)],the survival cells quantity gradually reduced as the time goes by.BrdU positive cells of both group were reduced obviously after 28 days transplantation compared with 72 hours.The cardiac function in MSCs group and Adropin group improved significantly compared with the Control [ Left ventricular ejection fraction(LVEF),35%±5% vs.46%±4%,57%±4%,P<0.05;Left ventricular fraction shortening rate(LVFS),14%±4% vs.20%±1%,26%±2%,P<0.05],the Adropin group improved more obviously than the MSCs group(P<0.05);The MSCs group and Adropin group attenuated the levels ofinflammatory factors and enhanced the level of anti-inflammatory factors after 72 hours transplantation,the Adropin group was superior than the MSCs group in the beneficial effect(P<0.05).The expression of VEGF and bFGF mRNA and proteins expression in the infarction area was increased obviously in the Adropin and MSCs group campared with the Control(P<0.05),and the density of new blood vessels were equally significantly increased [(23±2)/0.06mm2 vs.(50±8)/0.06mm2,(88±10)/0.06mm2,P<0.05).VEGF and bFGF mRNA and proteins expression in Adropin group was significantly enhanced compared with the MSCs group,and the density of new blood vessels in Adropin group were also significantly increased compared with the MSCs group(P<0.05).The degree of myocardial fibrosis by Masson staining reveal that both the MSCs group and Adropin group were extenuated compared with the MI group,especially obviously in the Adropin group.Conclusions: MSCs apoptosis can be reduced by Adropin via activating PI3K/Akt and ERK1/2 pathways in vitro so as to promote MSCs survival;2.The survival rate of the transplanted cells can be increased and the cardiac function can be improved by Adropin preconditioning MSCs transplantation combined with Adropin drug postconditioning,whose mechanism may be related to the inhibition of early inflammatory reaction,the promotion of the expression of paracrine factors including VEGF and bFGF,the facilitation of angiogenesis and the reduction of myocardial fibrosis. |
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