Mito-KATP对缺氧复氧大鼠心肌微血管内皮细胞凋亡的影响及作用机制

Effects and mechanism of Mito-KATP opening on apoptosis of rat myocardial microvascular endothelial cells undergoing hypoxia-reoxygenation

目的探讨线粒体ATP敏感性钾通道(mito-KATP)开放影响缺氧复氧大鼠心肌微血管内皮细胞(MMECs)凋亡的作用机制。方法培养大鼠心肌微血管内皮细胞,随机分为四组:对照组(N组)、模型组(H/R组)、开放剂组(DZ组)、阻断剂组(5-HD组)。DZ组加入100μmol/L二氮嗪预处理2 h,5-HD组在加入100μmol/L二氮嗪前,先用100μmol/L 5-羟葵酸预处理2 h,然后上述两组和H/R组同样进行缺氧2 h复氧2 h。Hoechst染色方法观察凋亡细胞形态,Annexin V-FITC/PI双标记法测定各组细胞凋亡率、RT-PCR法检测各组NF-κB、FKN和p53 mRNA转录水平。结果与N组比较,H/R组可见大量细胞坏死、脱落,细胞凋亡率升高(P<0.01),NF-κB、FKN和p53 mRNA表达上调(P<0.01);与H/R组比较,DZ组可见部分细胞坏死脱落,细胞凋亡率降低(P<0.01),NF-κB、FKN和p53 mRNA表达下调(P<0.01);5-HD组与H/R组比较无显著差异。结论 mito-KATP开放可抑制缺氧复氧所致MMECs凋亡,其机制可能与抑制NF-κB、FKN及p53 mRNA表达有关。

Objective To investigate the effects and mechanism of Mito-KATP opening on apoptosis of rat myocardial microvascular endothelial cells（MMECs） undergoing hypoxia-reoxygenation. Methods Rat MMECs were cultured and divided into four groups： control group（ N group）, modal group（ H/R group）, opener group（ DZ group）, blocker group（5- HD group）. MMECs in DZ group and 5-HD group were pretreated with 100 μmol/L DZ and 100μmol/LS-HD ＋ 100 μmol/L DZ respectively for 2 h, then MMECs in H/R group, DZ group and 5-HD group were exposed to 2 h hypoxia followed by 2 h reoxygenation. The morphology of apoptosis cells was observed by Hoechst staining method, the apoptosis rate was assayed by Annexin V-FITC/PI 2 h after reoxygenation. The expression of NF-KB, fractalkine （FKN） , and p53 mRNA were detected by RT-PCR. Results Compared with N group, a higher apoptosis rate （ P 〈 0. 01 ） and an increased expres- sion of NF-KB, FKN and p53 mRNA（ P 〈 0.01 ）were found in H/R group;compared with H/R group, a lower apoptosis rate （ P 〈 0. 0l ）, an decreased expression of NF-KB, FKN and p53 mRNA were found in DZ group （ P 〈 0.01 ） ; no signifi- cant difference was found between the indexes in 5-HD group and H/R group. Conclusion Opening of mito-KATP can in- hibit the apoptosis of MMECs undergoing hypoxia-reoxygenation, the mechanism may be related to the suppressing of NF- KB, FKN and p53 mRNA expression.