非酒精性脂肪肝大鼠肝细胞凋亡与线粒体膜通透性转换孔开放的关系

Hepatocyte apoptosis and mitochondrial permeability transition pore opening in rats with nonalcoholic fatty liver

目的通过研究肝细胞凋亡及线粒体膜通透性转换孔(MPTP)开放在大鼠非酒精性脂肪性肝病(NAFLD)形成中的作用,探讨NAFLD的发病机理。方法 30只雄性SD大鼠随机分为正常对照组和高脂饲料4、8、12周组;流式细胞仪检测肝细胞凋亡;紫外分光光度计检测MPTP开放程度;免疫组织化学法检查Bcl-2、Bax在肝组织中表达情况;Western Blot检测肝脏Bax表达及变化情况。结果与正常对照组比较,高脂4～12周组肝细胞凋亡指数增加;紫外分光光度计检测显示高脂组随着造模时间延长,MPTP开放增加;免疫组化显示Bcl-2在4、8、12周高脂组表达,但组间比较阳性细胞数增加不明显;Bax在高脂组随造模时间延长阳性细胞数增加;Western Blot进一步证实Bax在高脂组随造模时间延长蛋白表达量增加;并且随着脂肪肝的进展,Bcl-2/Bax比率进行性下降。结论 NAFLD大鼠模型中存在肝细胞凋亡,线粒体损伤与肝细胞凋亡密切相关,MPTP开放是NAFLD大鼠重要线粒体损伤机制,而Bax表达量增加、Bcl-2/Bax比率异常是MPTP开放的分子基础。

Objective To investigate the role of hepatocye apoptosis and mitochondrial permeability transition pore （MPTP） opening in the pathogenesis of nonalcoholic fatty liver disease （NAFLD）. Methods Thirty male SD rats were randomized into normal diet group and high-fat diet group. At 4, 8 and 12 week of feeding. The hepatocyte apoptosis index （AI） was measured using flow cytometry, and MPTP opening was evaluated with ultraviolet spectrophotometry. Immunohistochemistry was employed to detect hepatic expressions of Bcl- 2 and Bax, and Western blotting was used to detect Bax protein expression changes. Results High- fat feeding resulted in significantly increased hepatocyte AI at 4- 12 weeks and gradually increased MPTP opening. In the high-fat diet group, hepatic Bcl-2 expression was detected but the positive cell number remained stable, whereas Bax- positive cell number increased steadily with time with progressively increased intensity of Bax protein expression, resulting in gradually decreased Bcl- 2/Bax ratio. Conclusion Hepatocyte apoptosis occurs in the rat model of NAFLD in close correlation with mitochondrial damage. Increased MPTP opening as the result of increased Bax expression and aberrant Bcl-2/Bax ratio is an important mechanism of hepatocye mitochondrial damage in NAFLD.