线粒体通透性转换孔改变致mtDNA释放在肠缺血再灌注损伤中的作用

Role of mitochondrial DNA release induced by altered mitochondrial permeability transition pore in intestinal ischemia reperfusion injury

目的:探讨线粒体通透性转换孔(mPTP)对肠缺血再灌注(IIR)损伤的影响及相关机制。方法:将C57BL/6L小鼠随机分为假手术组(S组)、肠缺血再灌注组(IIR组)、环孢素A+肠缺血再灌注组(CsA+IIR组)。采用夹闭肠系膜上动脉根部45 min,恢复灌注2 h的方法制备IIR模型。观察肠组织病理学变化并行Chiu’s病理学评分;检测肠组织湿干重比;观察组织凋亡并计算细胞凋亡率;检测肠组织中干扰素(IFN)-β、乳酸(LD)及丙二醛(MDA)含量。在体外,将Caco-2细胞随机分为对照组(C组)、缺氧复氧组(HR组)、环孢素A+缺氧复氧组(CsA+HR组)。通过缺氧12 h后复氧4 h建立体外HR模型。检测细胞钙离子含量、mPTP开放程度及胞质线粒体DNA(mtDNA)水平。结果:与S组相比,IIR组Chiu’s病理学评分与湿干重比显著增高;凋亡细胞增多;IFN-β的mRNA水平明显增高;LD及MDA含量明显增加,而使用mPTP抑制剂CsA可显著抑制小鼠IIR引起的上述指标增高。同时在体外HR后,Caco-2细胞钙离子含量明显增加,mPTP开放显著增加,胞质mtDNA释放增加,而CsA的处理可显著抑制mtDNA的胞质释放。结论:mPTP开放可促进IIR损伤,其机制可能与mPTP介导mtDNA释放及诱导IFN-β生成有关。

Objective:To investigate the effect and related mechanism of mitochondrial permeability transition pore(mPTP)on intestinal ischemia reperfusion(IIR)injury.Methods:C57BL/6L mice were randomly divided into sham operation group(S group),intestinal ischemia reperfusion group(IIR group),and CsA+intestinal ischemia reperfusion group(CsA+IIR group).The IIR mouse model was established by clipping the root of the superior mesenteric artery for 45 minutes and restoring the perfusion for 2 hours.Intestinal histopathological changes and Chiu's pathological scores were recorded.The wet dry weight ratio of intestinal tissue was detected.Tissue apoptosis was observed and cell apoptosis rate was calculated.The contents of interferon(IFN)-β,lactic acid(LD)and malondialdehyde(MDA)in intestinal tissues were detected.In vitro,Caco-2 cells were randomly divided into control group(C group),hypoxia reoxygenation group(HR group),and CsA+hypoxia reoxygenation group(CsA+HR group).The cellular HR model was established by hypoxia for 12 hours and reoxygenation for 4 hours.Calcium content,mPTP openness and cytoplasmic mitochondrial DNA(mtDNA)were detected.Results:Compared with the S group,the Chiu's pathological score and the wet dry weight ratio in the IIR group were significantly increased;the apoptotic cells were increased;the mRNA level of IFN-βwas significantly increased;the contents of LD and MDA were significantly increased.The use of mPTP inhibitor CsA can significantly inhibit the increase of the above indicators caused by IIR in mice.Meanwhile,after HR in vitro,Caco-2 cells significantly increased calcium ion content,mPTP opening,and cytoplasmic mtDNA content,while CsA treatment could significantly inhibit the cytoplasmic release of mtDNA.Conclusion:mPTP opening can promote IIR injury,and the mechanism may be related to mPTP-mediated mtDNA release and induction of IFN-βproduction.