To Explore The Anti-Apoptosis Activity And Its Mechanism Of Bone Marrow Mesenchymal Stem Cells By H₂O₂ Preconditioning And The Improvement Of Their Therapeutic Potential For Wound

ObjectiveTransplantation of BMSC is a promising strategy for improving wound healing. However, this treatment is limited due to the low survival rate of MSC after transplantation. Most of the grafted MSC died due to apoptosis in the early stages of transplantation. Therefore, it was necessary to protect MSC against apoptosis during transplantation. Various strategies have been studied to enhance the survival of the transplanted cells. So far, preconditioning-induced protection in stem cell transplantation therapy is the best strategy to enhance stem cell survival, apoptosis resistance, and improving stem cell therapeutic potential. Generation of ROS in mitochondria is one of the main triggers that induce ischemic tolerance in brain and heart. Many insults, including H₂O₂, H2 S and CO exert protective effects by inducing mitochondrial ROS production. Appropriate reactive oxygen species（ROS）production in mitochondria can effectively protect cells against consequent injurious oxidative stress induced by severe hypoxia and ischemia. It is reported that preconditioning human cardiac stem cells with 100μmol/L H₂O₂ in vitro significantly improved the left ventricular cardiac function and reduced cardiac fibrosis in ischemia–reperfusion injury of rat hearts after implantation, as compared to the nontreated groups. H₂O₂-induced preconditioning is reported to increase the migration of MSC through upregulation of CXCR4 and activation of extracellular signal-regulated kinase（ERK）. Hence, in the present study, we will observe the survival of BMSC, and activation of the pro-survival signaling pathway after preconditioned with low dose H₂O₂. After transplanting BMSC of preconditioned with H₂O₂, we will observe the homing efficiency of stem cells, the rate of apoptosis in wounds, finally, we will demonstrate the improvement in therapeutic potential of transplanted cells, promoting angiogenesis and accelerating wound healing.Methods1、BMSC was isolated from mice and identified using flow cytometry analysis. After the BMSC were treated with different low concentrations of H₂O₂（0、25、50、100、150and200μmol/L） for 48 h, the proliferation of BMSC was detected by MTT analysis, and the expression of SDF-1 and its CXCR4/CXCR7 receptors was assayed using western blotting.2、The apoptotic cells rate was measured by flow cytometry analysis, and the expression of phosphorylated PI3 K, Akt and mTOR was assayed using western blotting; For another part of the study, BMSC were primed with PI3 K inhibitor LY294002 for 30 min, then were preconditioned with or without 50μmol/L H₂O₂ for 12 h following treatment with 300μmol/LH₂O₂.. The apoptotic cells rate was measured by flow cytometry analysis and Hoechst33342 nucleus staining, the protein expression of Bcl-2, Bax, Caps-3, Cleaved-Caps-3,P-Akt,P-mTOR was analyzed by Western blot.3、Full-thickness skin wounds（diameter1 cm） were made on the backs of SD rats. Rats were randomly divided into three groups, sodium chloride group（NS）, BMSC group and H₂O₂ preconditiion group. BMSC were labeled with chloromethyl-1, 1’-dioctadecyl-3,3,3’,3’–tetramethylin docarbocyanine perchlorate（DiI）. Rats with full-thickness skin wounds received DiI-labeled BMSC via tail vein. Then, we observe e the DiI positive cell numbers in wounds, the rate of wound closure, microvessel density in each goups.Results1、The results by flow cytometry analysis showed that BMSC,the surface highly express CD29 and CD166,lowly express CD34 and CD45. Treatment with 25μmol/L and 50μmol/L H₂O₂ was markedly promoted BMSC proliferation（p<0.05）. However, the proliferation of BMSC was inhibitated markerdly after treatment with 200μmol/L H₂O₂ for 48h（p<0.05）. Furtherly, after treated BMSC with the concentrations of 25μmol/L and 100μmol/L H₂O₂, the expression of SDF-1 and its receptors CXCR4 and CXCR7 was markedly increased（p<0.05）.2、The results of flow cytometry analysis and Hoechst33342 nucleus staining showed that BMSC were induced apoptosis by H₂O₂ at the dose of 200⁵00μmol/L, the rate of apoptotic cells was markedly increased in H₂O₂ dose-dependence manner. However, the rate of apoptotic cells was markedly decreased after BMSC were preconditioned with low dose H₂O₂（50μmol/L）. It suggested that BMSC with low dose H₂O₂ preconditioning could enhance the activity of ant-apoptosis of stem cells when these cells exposed to following high dose H₂O₂ induced oxidative stress.3、The results of Wersten blot showed that the expression upregualtion of pro-apoptosis proteins Bax, Caps-3 and Cleaved-Caps-3, and downregulation of anti-apoptosis protein Bcl-2 induced by high dose H₂O₂ were markedly inhibited through BMSC with low dose H₂O₂ preconditioning. This suggested that the low dose H₂O₂ preconditioning could reinforce the activity of ant-apoptosis of stem cells4 、 The expression of phosphorylated PI3 K, Akt and mTOR and phosphorylated mTOR was significantly increased at the H₂O₂ dose of 25¹00μmol/L, but there was on changes of PI3 K, Akt protein, suggested that low dose H₂O₂ preconditioning activated the PI3K/Akt/mTOR pathway.5、The expression of key protein of the PI3K/Akt/mTOR pathway and anti-apoptosis protein Bcl-2 was significantly inhibted, and the expression of pro-apoptosis protein Cleaved-Caps-3 was increased when BMSC was induced with high dose H₂O₂ treated, however, which was reversed when BMSC were preconditioned with low dose H₂O₂. It was demonstrated that the low dose H₂O₂ preconditioning play a cytoprotect role in stem cell survival during oxidative stress. However, PI3 K inhibitor LY294002 could against the cytoprotect role induced by H₂O₂ preconditioning.6 、 The survival rate of transplanted BMSC with 50μmol/LH₂O₂ preconditioning in wounds was significant higher than that of BMSC without H₂O₂ preconditioning, concomipany with increase in microvessel density in wouds and the rate of wound closure, accelerating wound healing.ConclusionH₂O₂ preconditioning enhances the ability of anti oxidative stress injury of bone marrow mesenchymal stem cells and promotes wound healing.