| Objective:Estrogen responsive finger protein（Efp,also known as tripartite motif protein 25,TRIM25）is an important ubiquitin E3 ligase,the preliminary work of our laboratory had confirmed that Efp could interact with 14-3-3ηand mediate the ubiquitination degradation of 14-3-3η.Efp is also one of the target of Estrogen receptor（ER）,In this study,we would further investigate whether the expression of Efp protein could affect the anti-A/R injury cardio protective effect of 17-β-estradiol on H9c2 myocardical-like cells and whether this effect is related to 14-3-3η.Methods:（1）H9c2 cardiomyocyte-like cells were used to establish hypoxia-reoxygenation injury model;17-β-estradiol pretreatment was used to establish an estrogen-pretreatmental model;Efp protein was up-regulated or down-regulated by AD-Efp or AD-Efp-RNAi recombinant adenoviral vectors,respectively.The expression of Efp and 14-3-3ηprotein were detected by Western Blotting to determine the effect that the expression of 14-3-3ηprotein was affected by17-β-estradiol.（2）H9c2 myocardical-like cells were randomly divided into two large groups after culture,the first group was consist of Control group,A/R group,E2+A/R group,E2+AD-Efp+A/R group,AD-Efp+A/R group and AD-Efp-NC+A/R group,and the second group was made up of Control group,A/R group,E2+A/R group,E2+AD-Efp-RNAi+A/R group,AD-Efp-RNAi+A/R group and AD-Efp-RNAi-NC+A/R group.（3）The cell viability of H9c2 myocardical-like cells was tested by MTS assay,LDH,SOD activity and mitochondrial permeability transition pore（mptp）were detected by spectrophotometric method,while the reactive oxygen species（ROS）,mitochondrial membrane potential（Δψm）and mptp was analyzed by the flow cytometry.These index were used to evaluate the the anti-A/R injury cardio protective effect of each group.Results:（1）Compared with the A/R group,the cell viability was increased,the LDH activity was decreased,the SOD activity was increased,the ROS production was reduced,the mitochondrial membrane potential（Δψm）was increased and the opening of the mitochondrial permeability transition hole was reduced in the E2+A/R pretreatment group（P<0.01）.（2）Compared with E2+A/R group,the cell viability was decreased,LDH activity was increased,SOD activity was decreased,ROS production was increased,mitochondrial membrane potential（Δψm）was decreased,and the opening of mitochondrial permeability transition pores（mptp）was increased in the E2+AD-Efp+A/R group.While compared with E2+AD-Efp+A/R group,the cell viability was decreased,LDH activity was increased,SOD activity decreased,ROS production was increased,mitochondrial membrane potential（Δψm）was decreased,and the opening of mitochondrial permeability transition pores（mptp）was increased in the AD-Efp+A/R group（P<0.05）.（3）While compared with E2+A/R group,the cell viability was increased,LDH activity was decreased,SOD activity was increased,ROS production was decreased,mitochondrial membrane potential（Δψm）was increased,and the opening of the mitochondrial permeability transition pore（mptp）was reduced in the E2+AD-Efp-RNAi+A/R group（P<0.05）.Compared with the E2+AD-Efp-RNAi+A/R,the cell viability was decreased,LDH activity was increased,SOD activity was decreased,ROS production was increased,and mitochondrial membrane potential（Δψm）was decreased,and the opening of mitochondrial permeability transition pores（mptp）was increased in the AD-Efp-RNAi+A/R group（P<0.05）.（4）Compared with the E2+A/R group,14-3-3ηprotein was downregulated in the E2+AD-Efp+A/R group,while 14-3-3ηprotein was upregulated in the E2+AD-Efp-RNAi+A/R group.Compared with the E2+AD-Efp+A/R group,14-3-3ηprotein was upregulated in the AD-Efp+A/R group,and compared with E2+AD-Efp-RNAi+A/R group,14-3-3ηprotein was upregulated in the AD-Efp-RNAi+A/R group（P<0.05）.Conclusion:The expression of Efp protein could affect the anti-A/R injury cardio protective effect of 17-β-estradiol in H9c2 myocardical-like cells,and this effect may be related to 14-3-3η. |
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