甲硫氨酸负荷致同型半胱氨酸轻度升高对大鼠肝脏脂质代谢的影响及机制研究

Effects of moderate increase of homocysteine caused by methionine load on lipids metabolism of rat liver and possible mechanisms

目的了解高甲硫氨酸摄入对肝脏脂质代谢的影响及可能机制。方法选取6周龄健康SD雄性大鼠20只,随机分为对照组和甲硫氨酸负荷组。适应性喂养2周后,对照组饲以正常饮食并采用PBS灌胃,甲硫氨酸负荷组在正常饮食基础上采用甲硫氨酸灌胃,持续喂养8周,每周称取体质量,处死后收集血液和肝脏组织。肝脏称重并计算脏器系数;肝组织切片H-E染色进行组织学观察;血生化分析仪测定血清生化指标;采用ELISA法测定血清同型半胱氨酸水平;real-time PCR检测大鼠肝组织脂代谢相关酶基因的表达水平。在体外培养的Hep G2细胞,油红O染色光学显微镜观察甲硫氨酸负荷或同型半胱氨酸处理对细胞中脂质沉积的影响,Western blotting检测同型半胱氨酸处理后细胞中成熟型肝组织甾醇调节元件结合蛋白1c(n-SREBP1c)表达的变化。结果甲硫氨酸负荷组与对照组大鼠体质量的增长无明显差异,但甲硫氨酸负荷组的脏器系数显著高于对照组(P<0.05);甲硫氨酸负荷组大鼠肝脏出现轻微小泡性脂肪变性,但未出现炎症和明显肝功能损害;甲硫氨酸负荷组大鼠血清同型半胱氨酸、血清总胆固醇及高密度脂蛋白水平显著高于对照组(P<0.05);甲硫氨酸负荷组肝组织Fas、Acc、Scd1、Cpt1αmRNA表达与对照组比较差异无统计学意义(P>0.05)。在甲硫氨酸或同型半胱氨酸作用下,Hep G2细胞发生明显的脂质蓄积;同型半胱氨酸处理Hep G2细胞n-SREBP1c表达显著增高。结论甲硫氨酸摄入过量将导致肝脏轻微脂肪变性,且与血液中同型半胱氨酸水平升高有关。甲硫氨酸负荷导致的同型半胱氨酸水平轻度升高可通过调节肝细胞脂质代谢关键转录因子的活化,对肝脏脂质代谢造成影响。

Objective To explore the effects of the high methionine intake on lipids metabolism of liver and the possible mechanisms.Methods Twenty healthy 6-week old SD rats were selected and randomly divided into the control group and methionine group.After 2 weeks of acclimation,rats of the control group were fed with standard chow and received PBS gavages,while rats of the methionine group were fed with standard chow and received methionine gavages for 8 weeks.The body weight of rats was measured every week.The samples of blood and liver tissue were collected,the weight of livers was measured,and the organ coefficient was calculated after rats were sacrificed.The liver tissue was sliced and H-E stained and the morphologic observation was performed.Biochemical indexes of serum were detected by automatic biochemical analyzing system.The level of serum homocysteine was detected by ELISA.The gene expression levels of enzymes relevant to lipid metabolism were detected by quantitative real-time PCR.HepG2 cells were cultured in vitro and stained by oil red O.The effects of methionine load or homocysteine treatment on the lipids deposition in HepG2 cell were observed under optical microscope.The change of expression level of n-SREBPlc was detected by Western blotting after being treated by homocysteine.Results The difference of increase of body weight between the control group and methionine group was insignificant,while the organ coefficient of methionine group was significantly higher than that of the control group（P〈0.05）.Rats of the methionine group developed mild microvesicular steatosis,but no inflammation or obvious hepatic dysfunction was found.Levels of plasma homocysteine,TC,and HDL of the methionine group were significantly higher than those of the control group（P〈0.05）.The differences of mRNA expressions of Fas,Acc,Sodl,and Cptlα in the liver tissue between two groups were not statistically significant（P〉0.05）.Treatment with methionine or homocysteine led to obvious lipids deposition in HepG2 cells.The expression of n-SREBPlc in HepG2 cells significantly increased after being treated by homocysteine.Conclusion Excessive methionine intake may result in mild hepatic steatosis,which is associated with the increase of homocysteine in blood.Moderate increase of homocysteine level caused by methionine load can effect the lipids metabolism of liver by regulating the activity of a key transcription factor of lipids metabolism of hepatic cells.