ABSTRACT
Objective To investigate the role of CXC chemokine receptor 4-focal adhesion kinase (CXCR4-FAK) signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) towards damaged tissues resulting from spinal cord ischemia-reperfusion (I/R) injury in rats.Methods Part I Rat BMSCs transfected with recombinant adenovirus-mediated green fluorescent protein gene 3 were seeded in 24-well plates and randomly divided into 5 groups (n =18 wells each):control group (group C),normoxia-incubated group (group N),HP group (group H),HP + CXCR4 antagonist AMD3100 group (group HA) and HP + FAK inhibitor FAK-related nonkinase group (group HF).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to 21％ O2-74％ N2-5％ CO2 for 36 h.In group H,BMSCs were exposed to 0.5％O2-94.5％ N2-5.0％CO2 for 24 h followed by 12 h exposure to normoxia.In groups HA and HF,5 μg/ml AMD3100 and 10 μg/ml FAK-related nonkinase were added to the culture medium before HP,respectively.The expression of stromal derived factor-1α (SDF-1α),CXCR4 and phosphorylated FAK (p-FAK) in BMSCs was determined by Western blot.The migratory capability and adhesive ability of BMSCs were measured by Transwell invasive assay and fibronectin adhesive assay,respectively.Part Ⅱ Two hundred and sixteen male Sprague-Dawley rats weighing 300-350 g were used and 210 out of the 216 rats underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Thirty-six rats were chosen and sacrificed before spinal cord I/R and at 12 h and 1,3,5 and 7 days of reperfusion (T0-5) and the lumbar segment of spinal cord was removed for detection of the content of SDF-1α.The left 180 rats with spinal cord I/R were randomly divided into 5 groups (n =36 each).IT DMEM medium 300 μl was injected in group C and BMSC suspension 300 μl (1 × 106/ml) was injected in groups N,H,HA and HF immediately after onset of reperfusion.Neurological function was scored at T0-5.The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the degree of BMSC aggregation.Results There was no significant difference in the expression of SDF-1α,CXCR4 and p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation between groups C and N (P ＞ 0.05).Compared with group N,the expression of SDF-1α,CXCR4 and p-FAK was significantly up-regulated,and migratory capability and adhesive ability of BMSCs,neurological function scores and the degree of BMSC aggregation were increased in group H,while no significant change was found in the expression of p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation in HA and HF groups (P ＞ 0.05).Compared with group H,the expression of p-FAK was down-regulated and the migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation were decreased in groups HA and HF (P ＜0.05).The content of SDF-1α was significantly higher at T2,3 than at T0.Conclusion HP can promote migration and adhesion of BMSCs towards damaged tissues resulting from spinal cord I/R injury through CXCR4-FAK signaling pathway in rats; thus CXCR4-FAK signal pathway provides the protective effect on spinal cord.
ABSTRACT
Objective To investigate the effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells (BMSCs) in rats through in vitro and in vivo experiments.Methods In vitro experiment The isolated rat BMSCs were cultured by whole bone marrow adherence method.The cells at passage 3 were seeded in 24-well plates at a density of 1 × 106 cells/ml and randomly divided into 5 groups (n =8 wells each) using a random number table:control group (group C),normoxia-incubated group (group N),hypoxic preconditioning group (group H),hypoxia preconditioning + STAT3 inhibitor Stattic group (group HS) and hypoxia preconditioning + anti-IL-10 monoclonal antibody group (group HA).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to21％ O2-74％ N2-5.0％ CO2 for48 h.In group H,BMSCs were exposed to 0.5％ O2-94.5％ N2-5.0％ CO2 for 24 h followed by 24 h exposure to normoxia.In HS and HA groups,500 μg/ml Stattic and 100 μg/rnl anti-IL-10 monoclonal antibody were added to the culture medium before hypoxia preconditioning,respectively.The expression of phosphorylated STAT3 (p-STAT3) and IL-10 was determined by Western blot.In vivo experiment Healthy male Sprague-Dawley rats,weighing 300-350 g,in which intrathecal catheters were successfully implanted without complications,underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Three hundred rats with spinal cord I/R injury were randomly divided into C,N,H,HS and HA groups (n =60 each) using a random number table.Immediately after onset of reperfusion,DMEM medium 300 μl was injected intrathecally in group C,and BMSC suspension 300 μl (1 × 106 cells/ml) was injected intrathecally in N,H,HS and HA groups.Neurological function was scored before ischemia and at 4,12,24 and 48 h of reperfusion (T0-,).The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the content of IL-10,TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1 (MCP-1),and macrophage inflammatory protein-1 α (MIP-1 α) (by ELISA) and the number of activated microglia (by immuno-histochemistry).Results Compared with C and N groups,the expression of pSTATβ and IL-10 was significantly up-regulated,the neurological function score and IL-10 content were increased,the content of TNF-α,IL-1β,IL-6,MCP-1 and MIP-1α and the number of activated microglia were decreased in group H (P ＜ 0.05).Compared with group H,the expression of p-STAT3 and IL-10 in group HS and expression of IL-10 in group HA was significantly down-regulated,and the neurological function score and IL-10 content were decreased,and the content of TNF-α,IL-13,IL-6,MCP-1 and MIP-1α and the number of activated microglia were increased in HS and HA groups (P ＜ 0.05).Conclusion Hypoxic preconditioning can enhance anti-inflammatory effects of BMSCs,thus increasing BMSCs-induced reduction of spinal cord ischemia-reperfusion injury in rats.