高糖应激促脂肪变性肝细胞凋亡的线粒体机制

目的:探讨高糖应激对脂肪变性肝细胞凋亡的作用及其可能机制。方法:C57BL/6J小鼠饲喂高脂饲料6周后,采用肝脏原位灌注技术分离得到脂肪变性原代肝细胞,在含有35 mmol/L葡萄糖的高糖DMEM培养基中孵育12 h,以正常DMEM培养基(添加30 mmol/L甘露醇)孵育的细胞作为对照,观察高糖处理对脂肪变性肝细胞活力、线粒体膜电位、凋亡蛋白酶caspase活性及凋亡相关信号通路的影响。结果:高糖应激使脂肪变性肝细胞活力下降,凋亡显著增加,而等渗甘露醇处理的对照细胞没有明显变化。高糖组细胞出现较为严重的线粒体去极化,导致线粒体膜电位降低,细胞色素C释放增多。线粒体介导凋亡关键酶caspase-9和caspase-3活性在高糖组有显著升高,抑制凋亡因子Bcl-2和Bcl-x L表达量明显降低,促凋亡蛋白Bax水平显著升高,而感受外源性凋亡信号的caspase-8的活性没有明显变化。结论:高糖应激会导致脂肪变性肝细胞线粒体膜电位下降,启动线粒体介导的内源性凋亡途径,引起肝细胞凋亡。这可能是高血糖加速非酒精性脂肪性肝病病程进展的一个重要原因。

Mitochondrial mechanism of hyperglycemia-induced apoptosis in primary mouse hepatocytes with steatosis

AIM: To investigate the role of high glucose in primary hepatocytes of mice fed with a high fat diet.METHODS: Male C57BL/6J mice were fed a high fat (45% of calories) diet ad libitum for 6 weeks to induce hepatic steatosis. Primary hepatocytes were isolated from the mouse liver by the 2 step collagenase perfusion method. The cells were incubated in low glucose (5 mmol/L), low glucose plus mannitol (30 mmol/L), or high glucose (35 mmol/L) DMEM medium for 12 h. The cell viability, apoptosis, mitochondrial membrane potential, and caspase enzymatic activities were measured. Furthermore, proteins related to the stress-sensitive signaling pathway of regulating high glucose-induced apoptosis in primary hepatocytes were determined by Western blotting.RESULTS: Incubation with 35 mmol/L glucose resulted in a significant decrease in cell viability and an increase in apoptosis, whereas mannitol had no significant effect on the cell viability or apoptosis. A progressive depolarization of the mitochondria, an increase in cytosol cytochrome C and a dramatic decrease in mitochondrial cytochrome C in high-glucose stressed hepatocytes were observed. The enzymatic activities of caspase-9 and caspase-3, but not caspase-8, were significantly increased in high glucose-stressed hepatocytes (P<0.05). High glucose treatment suppressed the expression of Bcl-2 and Bcl-xL, while it increased the expression of the pro-apoptotic factor Bax.CONCLUSION: High glucose stress reduces mitochondrial membrane potential, initiates mitochondria-mediated apoptotic pathways and promotes apoptosis of hepatocytes with steatosis. This may be an important pathological mechanism of hyperglycemia-induced progression of nonalcoholic fatty liver disease.