高同型半胱氨酸血症经内质网途径影响ApoE-/-鼠肝细胞凋亡

Effect of hyperhomocysteinemia in hepatocyte apoptosis by endoplasmic reticulum pathway in ApoE^(-/-) mice

目的观察高同型半胱氨酸血症(HHcy)对ApoE-/-鼠肝细胞凋亡的影响并探讨其可能机制。方法 36只ApoE-/-小鼠随机分为3组,每组12只:①ApoE-/-对照组(ApoE-/-group):普通饮食饲养;②蛋氨酸饮食组(methionine diet group):普通饮食+1.7%蛋氨酸饲养;③干预组(treatment group):普通饮食+1.7%蛋氨酸+0.006%叶酸及0.0004%VitB12饲养。另取12只正常C57 BL/6J小鼠给予普通饮食,作为正常对照组(normal control group)。饲养20周后,ELISA测定血清同型半胱氨酸(Hcy)浓度和肝组织Caspase-12含量;全自动生化分析仪测定血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)活性;DAPI染色法观察肝组织细胞核形态;电镜观察肝脏组织细胞超微结构。结果与正常对照组和ApoE-/-对照组相比,蛋氨酸饮食组Hcy浓度分别增高2.4倍(P<0.01)和2.5倍(P<0.01);ALT活性分别增高2.0倍(P<0.05)和1.2倍(P<0.05);各组AST活性无明显差异;肝组织切片DAPI染色及电镜结果显示,蛋氨酸饮食组小鼠肝细胞出现凋亡的形态学改变,蛋氨酸饮食组小鼠肝细胞线粒体肿胀、浓缩,内质网大量增生,可见细胞凋亡前体及凋亡小体;Caspase-12活性检测显示蛋氨酸饮食组较正常对照组增高(P<0.05)。与蛋氨酸饮食组比较,干预组小鼠血清Hcy浓度降低,Caspase-12活性下降,肝细胞病理变化减轻,血清ALT活性明显降低。结论 HHcy可能通过影响ApoE-/-鼠内质网功能改变,引起肝细胞凋亡。

Abstract： Aim To observe the effect of hyperhomo-cysteinemia on hepatocyte apoptosis of ApoE-/- mice and to explore its possible mechanism. Methods Thirty-six ApoE-/- mice were randomly divided into 3 groups ： （ 1 ） ApoE -/- control group, （ 2 ） methionine diet group, and （3）folic acid and VitB12 treatment group. （4）another 12 normal C57 BL/6J mice were chosen as control group. After 20 weeks of feeding, ELISA determination of plasmic homocysteine concen- trations and the contents of Caspase-12 in liver tissue was performed; chemical method was used to measure serumal alanine aminotransferase （ALT） and aspartate aminotransferase （AST） activity ; DAPI staining was used to observe liver tissue nuclear morphology; elec-tron microscopic observation of hepatocyte uhrastruc-ture was also used. Results Compared with normal control group and ApoE-/- group, the serum homocys- teine concentrations of high methionine diet group in- creased by 2. 4 fold （ P 〈 0. 01 ） and 2.5 fold （ P 〈0.01） respectively; ALT increased by 2.0 fold （P 〈 0. 05） and 1.2 fold （ P 〈 0. 05 ） respectively. There was no significant change of AST activity in each group. DAPI staining showed that high methionine diet group showed apoptotic morphological changes in the nucleus. Electronic microscope results showed high methionine group liver mitochondrial swelling or con- centration, endoplasmic reticulum proliferated, show- ing precursors of apoptosis and apoptotic bodies. Com-pared with methionine diet group, the serum homocys-teine concentrations, Caspase-12 contents and ALT ac- tivity of folic acid and VitB12 in intervention group were reduced. Conclusion Hyperhomocysteinemia is closely related with liver cell apoptosis of ApoE-/-mice, and endoplasmic reticulum stress induced apop-tosis may be its critical path.