色素上皮衍生因子和金属硫蛋白2A在心肌细胞缺氧损伤中的作用研究

Role of pigment epithelium⁃derived factor and metallothionein 2A in hypoxia induced myocardial cell injury

目的探究色素上皮衍生因子(PEDF)和金属硫蛋白2A(MT2A)在心肌细胞缺氧损伤中的作用。方法采用随机数字表法将20只健康C57 B/6小鼠分为4组,以酶消化法分离出心肌细胞并经MT2A处理后,A组常氧(95%空气+5%CO2)培养,B组、C组、D组均低氧(94%N2+1%O2+5%CO2)培养,其中C组过表达PEDF⁃MT2A,D组采用siRNA阻断PEDF⁃MT2A表达。应用DHR123标记后流式细胞仪检测细胞内活性氧自由基(ROS)表达,Western blotting检测心肌细胞氧化应激丝裂原活化蛋白激酶(MAPK)分子通路、纤维化基质金属蛋白酶(MMPs)/基质金属蛋白酶组织抑制物(TIMPs)及收缩功能Ca^(2+)⁃ATP酶mRNA表达水平。结果与A组相比,B、C、D组MT2A蛋白表达明显下降,其反应条带的校正灰度值分别为A组的35.33%、92.06%和49.16%;B组ROS,MAPK信号传导通路相关蛋白ERK、JNK、p38表达水平及Ca^(2+)⁃ATP酶mRNA表达水平较A组明显下降,Ca^(2+)荧光强度减弱,MMPs/TIMPs比值明显升高(P<0.05);C组仅ROS、MMPs/TIMPs比值表达水平较A组明显下降(P<0.05);D组ERK、JNK、p38表达水平及MMPs/TIMPs比值均显著高于A、B组(P<0.05)。与C组相比,D组ROS,MAPK信号传导通路相关蛋白ERK、JNK、p38表达水平及Ca^(2+)⁃ATP酶mRNA表达水平明显下降,Ca^(2+)荧光强度减弱,MMPs/TIMPs比值明显升高(P<0.05);结论PEDF可通过与MT2A的作用,调节相关分子通路,影响心肌细胞氧化应激、纤维化、收缩功能。

Objective To investigate the role of pigment epithelium⁃derived factor(PEDF)and metallothionein 2A(MT2A)in hypoxia⁃induced myocardial cell injury.Methods 20 healthy C57 B/6 mice were randomly divided into 4 groups.The myocardial cells were isolated by enzyme digestion.After treatment with MT2A,group A was cultured under normoxia(95%air+5%CO2),whereas groups B,C and D were cultured under hypoxia(94%N2+1%O2+5%CO2).PEDF⁃MT2A was overexpressed in group C,and the expression of PEDF⁃MT2A was blocked in group D by siRNA.The level of reactive oxygen species(ROS)in cells was determined by flow cytometry after labeling with DHR123.The myocardial cell oxidative stress mitogen⁃activated protein kinase(MAPK)molecular pathway,expression levels of fibrotic matrix metalloproteinases(MMPs)/tissue inhibitor of metalloproteinases(TIMPs)and systolic Ca^(2+)⁃ATPase mRNA were examined by Western blotting.Results Compared with group A,the expression of MT2A protein in groups B,C and D decreased significantly,and the corrected gray value of reaction bands was 35.33%,92.06%and 49.16%of that in group A,respectively.The expression levels of ERK,JNK and p38 proteins related to ROS and MAPK signal transduction pathways and the expression level of Ca^(2+)⁃ATPase mRNA in group B were significantly decreased compared with those in group A.The Ca^(2+)fluorescence intensity decreased,whereas the ratio of MMPs/TIMPs significantly increased in group B compared with those in group A(P<0.05).The expression level of ROS and MMPs/TIMPs ratio in group C significantly decreased compared with those in group A(P<0.05).The expression levels of ERK,JNK and p38 and MMPs/TIMPs ratio in group D were significantly higher than those in groups A and B(P<0.05).Compared with group C,the expression levels of ERK,JNK and p38 proteins related to ROS and MAPK signal transduction pathways and the expression level of Ca^(2+)⁃ATPase mRNA in group D were significantly decreased,whereas the Ca^(2+)fluorescence intensity was weakened,and the MMPs/TIMPs ratio significantly increased(P<0.05).Conclusion PEDF may regulate the related molecular pathways through the interaction with MT2A and affect the oxidative stress,fibrosis and systolic function.