Mitochondrial DNA deletion and apoptosis in alcoholic liver disease

背景：慢性饮酒可抑制肝脏ATP合成，诱导肝脏线粒体DNA(mt-DNA)缺失。细胞内的三磷酸腺苷含量可能在诱导细胞死亡、凋亡或坏死中起关键作用。然而，目前尚不清楚酒精性肝病发生哪种类型的细胞死亡。本研究通过检测乙醇处理后大鼠肝脏线粒体DNA(包括ATP合成酶编码区)的缺失和肝组织中的ATP含量，探讨了线粒体DNA缺失与肝组织ATP合成的关系。用免疫组织化学方法检测肝细胞单链DNA(ss-DNA)含量，以确定肝细胞ATP含量与细胞凋亡的关系。方法：雄性Wistar大鼠16只，分别饲喂含36%乙醇的流质饲料(AL组)和不含酒精的对照组(C组)，共5周。取新鲜肝组织提取液，测定肝组织三磷酸腺苷含量。肝切片中的ss-dna免疫…染色检测肝细胞的凋亡更确切地说是化学上的。计算每万个肝组织切片中SS-DNA阳性细胞数。用冰冻肝切片，用聚合酶链式反应(PCR)检测编码复合体I(ND4+ND5)、IV(COI、II、III)和V(ATPase 8/6)的mt-DNA缺失。结果：AL组1/3以上的小叶有脂肪改变，而C组未见。AL组肝组织三磷酸腺苷含量为0.44±0.10Mol/g肝，显著低于C组的0.84±0.31Mol/g肝(P&lt；0.005)。另一方面，在AL-和C-群中均未检测到编码复合体I、IV和V的mt-DNA缺失。两组大鼠肝细胞核内均可见明显的SS-DNA染色。SS-DNA阳性肝细胞数AL组为5.6±1.8/万肝细胞，明显少于C组的2 0.6±4.8/万肝细胞(P<0.0001)。肝组织三磷酸腺苷含量与单链DNA阳性细胞数呈正相关(r=0.674，P<0.005)。结论：慢性酒精摄入致线粒体DNA损伤前，线粒体功能至少部分受损。慢性酒精摄入可能不是肝细胞凋亡的原因。较少

Background : Chronic alcohol consumption depresses ATP synthesis and induces mitochondrial DNA (Mt-DNA) deletion in the liver. ATP content in cells may play a critical role in inducing cell death, apoptosis or necrosis. However, it is not known which type of cell death occurs in alcoholic liver disease. In this study, the deletions of hepatic Mt-DNA including ATPase (ATP synthase) encoding region and hepatic ATP content in rats treated with ethanol were determined to elucidate the relationship between Mt-DNA deletion and ATP synthesis. Single stranded DNA (ss-DNA) of hepatocytes was also stained immunohistochemically to determine the relationship between hepatic ATP content and apoptosis.Methods : Sixteen male Wistar rats were fed with a 36% ethanol-containing liquid diet (AL-group) or control diet without ethanol (C-group) for 5 weeks. Hepatic ATP content was measured in the extract of fresh liver tissue. To detect apoptosis of hepatocytes, ss-DNA in liver sections was stained immunoh … More istochemically. Ss-DNA positive cells per 10,000 hepatocytes of each liver tissue section were calculated. Using frozen liver sections, the deletions of Mt-DNA encoding complexes I (ND4 + ND5), IV (CO I, II and III) and V (ATPase 8/6) were determined by polymerase chain reaction (PCR).Results : Fatty change was observed in more than one third of lobules in AL-group, but not in C-group. Hepatic ATP content in AL-group was 0.44 ± 0.10 μmol/g liver which was significantly lower than that in C-group, 0.84 ± 0.31 μmol/g liver (P<0.005). On the other hand, no deletion of Mt-DNA encoding complexes I, IV and V was detected in either AL- or C-group. Ss-DNA staining was clearly observed in the nuclei of hepatocytes in both groups. The number of ss-DNA positive hepatocytes in AL-group was 5.6 ± 1.8/10,000 hepatocytes which was significantly less than that in C-group, 20.6 ± 4.8/10,000 hepatocytes (P<0.0001). There was a positive correlation between hepatic ATP content and the number of ss-DNA positive cells (r = 0.674, P<0.005).Conclusion : The results suggest that mitochondrial function, at least in part ATP level, was depressed before the damage of Mt-DNA by chronic ethanol consumption. Chronic ethanol consumption may not be responsible for the apoptosis of hepatocytes. Less