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目的探讨乙醇抑制蛋白酶体在酒精性肝病大鼠模型肝细胞线粒体凋亡中的作用。方法健康雄性Wistar大鼠40只,随机分为对照组、模型1组、模型2组、模型3组、模型4组各8只。模型组均采用乙醇灌胃构建酒精性肝病大鼠模型,对照组给予等量生理盐水。分别于造模6周(模型1组)、8周(模型2组)、10周(模型3组)、12周(对照组、模型4组)处死大鼠,取肝组织行组织病理学检查并应用电镜观察,采用流式细胞术检测肝细胞线粒体膜电位,采用RT-PCR法检测肝组织低分子多肽7(low molecular weight polypetide 7,LMP7)mRNA表达水平,采用Western blot检测肝组织LMP7蛋白表达水平。结果 (1)组织病理结果显示对照组肝小叶轮廓清晰,肝细胞围绕中央静脉呈正常索状排列,无变性、炎症及坏死;模型1组肝小叶中央区域少量脂肪变性;模型2组细胞内出现较多脂滴,肝细胞细胞核被推挤至细胞一侧;模型3组细胞内含大量脂滴,肝细胞肿大变圆;模型4组脂肪变进一步加重,可见不同程度肝细胞水样变性和炎细胞浸润及Mallory小体;(2)电镜结果显示模型组均成功构建酒精性肝病模型,线粒体在结构和形态上出现损伤,有凋亡表现;(3)模型组罗丹明123(rhodamine 123,Rho123)荧光强度[(239.46±12.72)%]明显低于对照组[(377.59±16.81)%](P<0.05);(4)模型1、2、3、4组肝组织LMP7 mRNA相对表达量[0.51(0.31,0.87),0.49(0.15,0.79),0.08(0.03,0.14),0.02(0.01,0.03)]和LMP7蛋白表达水平(0.784±0.025、0.601±0.018、0.376±0.013、0.121±0.021)均明显低于对照组(1、0.997±0.031)(P<0.05),模型1、2、3、4组LMP7mRNA相对表达量及LMP7蛋白表达水平依次降低,两两比较差异均有统计学意义(P<0.05)。结论乙醇抑制蛋白酶体可能在酒精性肝病肝细胞线粒体凋亡中发挥重要作用。
Objective To investigate the effect of ethanol on mitochondrial apoptosis induced by proteasome in alcoholic liver disease rat model. Methods Forty male Wistar rats were randomly divided into control group, model group 1, model group 2, model group 3 and model group 4. The model group were fed with ethanol to build alcoholic liver disease rat model, the control group was given the same amount of saline. The rats were sacrificed at 6 weeks (model 1), 8 weeks (model 2), 10 weeks (model 3), 12 weeks (control, model 4), and histopathological examination The mitochondrial membrane potential of hepatocytes was detected by electron microscopy. The expression of LMP7 mRNA in liver tissue was detected by RT-PCR. The expression of LMP7 protein The expression level. Results (1) Histopathological results showed that the contours of hepatic lobules in the control group were clear, and the hepatocytes arranged in normal cord around the central vein with no degeneration, inflammation and necrosis. A small amount of steatosis was found in the central lobe of model group 1; More lipid droplets, the liver cell nucleus pushed to the cell side; model group 3 cells contain a large number of lipid droplets, hepatocyte enlargement and rounding; model 4 group of fat further aggravate, showing varying degrees of liver cell degeneration and Inflammatory cells infiltration and Mallory bodies; (2) Electron microscopy results showed that model group were successfully constructed alcoholic liver disease model, mitochondria damage and apoptosis in structure and morphology, (3) model group rhodamine 123 (rhodamine 123, (P <0.05); (4) The relative expression of LMP7 mRNA in model groups 1, 2, 3, 4 were significantly lower than that in control group [(377.59 ± 16.81)%] (0.51 (0.31,0.87), 0.49 (0.15,0.79), 0.08 (0.03,0.14), 0.02 (0.01,0.03)] and LMP7 protein expression levels (0.784 ± 0.025,0.601 ± 0.018,0.376 ± 0.013,0.121 ± 0.021 ) Were significantly lower than those in the control group (1, 0.997 ± 0.031) (P <0.05). The relative expression of LMP7 mRNA in model groups 1, 2, White successively lower level of expression of pairwise differences were statistically significant (P <0.05). Conclusion Ethanol inhibits the proteasome may play an important role in mitochondrial apoptosis in liver cells of alcoholic liver disease.