Therapeutic effects and related mechanisms of erythropoietin sustained-release gelatin hydrogel microspheres on a murine model of hindlimb ischemia
10.3760/cma.j.issn.0253-3758.2016.06.013
- VernacularTitle:促红细胞生成素缓释明胶水凝胶微球治疗小鼠下肢缺血的效果及机制
- Author:
Jinwen XIAO
1
;
Longhu LI
;
Bingzhe HONG
;
Jianqi XIAO
;
Dongmei WEI
;
Zhe JIN
Author Information
1. 116001,大连大学附属中山医院心内科
- Keywords:
Ischemia;
Lower extremity;
Erythropoietin
- From:
Chinese Journal of Cardiology
2016;44(6):524-529
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the therapeutic effects of erythropoietin sustained-release gelatin hydrogel microspheres (EPO-GHM) on a murine model of hindlimb ischemia and related mechanisms.Methods Fifty two ten weeks old male C57BL/6J mice were assigned to 5 groups:shamoperated group (the right femoral artery suture was passed through the right femoral artery but not tied,n =8);saline group (right femoral artery ligation and intramuscular injection of saline at a dose of 4 ml/kg into the right hind limb,n =12);EPO group(right femoral artery ligation and intramuscular injection of EPO at a dose of 5 000 IU/kg into the right hind limb,n =12),empty GHM group (right femoral artery ligation and intramuscular injection of empty GHM at a dose of 4 ml/kg into the right hind limb,n =8);EPO-GHM group(right femoral artery ligation and intramuscular injection of EPO-GHM at a dose of 5 000 IU/kg into the right hind limb,n =12).The blood flow ratio of ischemic limb (right)/nonischemic limb (left) was measured using a laser Doppler perfusion imager.After 8 weeks,immunohistochemical analysis were used to evaluate the vessel density (vessel density of CD31 positive),arteriole density(vessel density of α-smooth muscle actin(α-SMA) positive) and muscle area(HHF35 positive area).The proliferating index of vessels was evaluated by double immunofluorescent labeling to evaluate effect of EPO-GHM on angiogenesis of ischemia limb.Western blot was used to evaluate the protein expression of EPO receptor,protein kinase B (AKT),p-AKT,endothelial nitric oxide synthase (eNOS),p-eNOS and matrix metalloproteinase 2(MMP-2).Results (1) Eight weeks later,the blood flow ratio of ischemic limb/nonischemic limb was significantly higher in the EPO-GHM group compared with other groups(0.810 ±0.080,0.563 ±0.051,0.570 ±0.056and 0.561 ± 0.052 respectively,all P ＜ 0.05).(2) CD31 antibody positive and α-SAM antibody positive densities were higher in the EPO-GHM group compared with other groups(P ＜ 0.01 or 0.05).(3)HHF35 positive area in saline group,EPO group,empty GHM group and EPO-GHM group were smaller than that of sham-operated group(all P ＜0.05).HHF35 positive area in saline group,EPO group,empty GHM group and EPO-GHM group were similar(all P ＞ 0.05).(4) The proliferating index of vessels was higher in the EPO-GHM group compared with other groups (P ＜ 0.01 or 0.05).(5) Compared with other groups,the protein levels of EPO receptor,AKT,p-AKT,p-eNOS and MMP-2 were significantly higher in EPO-GHM group(P ＜ 0.01 or 0.05) and level of eNOS was similar among five groups (P ＞ 0.05).Conclusion Results from present study suggest EPO-GHM could improve blood perfusion of ischemia limb in mice through increasing capillary and arteriolar densities and these beneficial effects are possibly mediated by EPOR up-regulation and AKT/p-eNOS/MMP-2 signaling pathway activation.
