The role of PEP-1-SOD1 fusion protein on ischemia-reperfusion injury in isolated perfused rat hearts
10.3760/cma.j.issn.0253-3758.2009.03.016
- VernacularTitle:细胞穿透肽PEP-1介导人铜,锌-超氧化物歧化酶转导入大鼠离体心脏及其对缺血再灌注损伤的保护作用
- Author:
Zun-Ping KE
1
;
Jia-Ning WANG
;
Jun-Ming TANG
;
Jian-Ye YANG
;
Yong-Zhang HUANG
;
Ling-Yun GUO
;
Fei ZHENG
;
Xia KONG
;
Lei WANG
Author Information
1. 郧阳医学院附属人民医院
- Keywords:
Myocardial reperfusion injury;
Superoxide dismutase;
PEP-1 peptide
- From:
Chinese Journal of Cardiology
2009;37(3):268-274
- CountryChina
- Language:Chinese
-
Abstract:
Objective The transduction efficiency of the purified PEP-1-SOD1 fusion protein and the effects of PEP-1-SOD1 fusion protein on ischemia reperfusion injury in the isolated perfused rat hearts were investigated. Methods The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 were transformed into BL21 (DE3) for expression and purification of SOD1 and PEP-1-SOD1, respectively. Isolated perfusedrat hearts were subjected to 60 min of global ischemia and 30 min of reperfusion and treated with vehicle, 100 μmol/L SOD1 and 25, 50, 100 μmoL/L PEP-1-SOD1, respectively. The transduction efficiency was evaluated with immunofluorescent microscopy and Western blot The enzyme activity of the transduced PEP-1-SOD1 was measured with commercial SOD detection kit The MDA content in myocardial tissue and the CK activity in coronary exudate at 15 min after reperfusion were also measured. Cardiomyocyte apoptosis was detected with TUNEL The infarct size was determined in isolated hearts 60 min after reperfusion with TIC staining. Results Immunofluorescent microscopy and Western blot demonstrated PEP-1-SOD1 was transduced into myocardial tissue in a dose-dependent manner, whereas SOD1 could not be detected in SOD1 group. SOD activity in control, SOD1 group, 25,50,100 μmol/L PEP-1-SOD1 groups was (10.06±0.77) U/mg prot, (10.59±0.71) U/mg prot, (32.29±1.42) U/mg prot, (43.16±1.16) U/mg prot, (55.14±1.59) U/mg prot, respectively. MDA content in corresponding groups was (1.48±0.19) nmol/mg prot,(1.39±0.11) nmol/mg prot, (1.01±0.14) nmol/mg prot, (0.73±0.13) nmol/mg prot, (0.50±0.06) nmol/mg prot, respectively. CK activity in corresponding groups was (1.73±0.58)U/mg prot, (1.68±0.14) U/mg prot,(1.40±0.28) U/mg prot, (0.97±0.39) U/mg prot,(0.61±0.56) U/mg prot, respectively. Cardiomyocyte apoptotic index in corresponding groups was (17.25±0.75) %, (16.63±1.07) %, (11.50±0.57) U/mg prot, (6.50±0.63) U/mg prot, (4.13±0.52)%, repectively. The percentage of myocardial infarction area was (55.13±2.18)%, (52.13±2.59)%, (33.88±2.06)%, (25.50±2.16)%, (15.38±1.14)%, respectively. Compared with control group and SOD1 group, all P<0.01 These results demonstrated the enzyme activity of the transduced PEP-1-SOD1 was significantly increased in a dose-dependent manner and the MDA content, CK activity, the cardiomyocyte apoptotic index and the infarct size was decreased siginificantly in PEP-1-SOD1 pretreatment groups compared with SOD1 group. Conclusion The native, biologically active form of PEP-SOD1 fusion protein could be effectively transduced into the isolated rat hearts subjecting ischemia reperfusion injury in a dose-dependent manner. The transduced PEP-1-SOD1 has protective effects on ischemia reperfusion injury in the isolated rat hearts.
