神经酰胺诱导LX-2细胞凋亡抑制肝纤维发生的作用  

Effect of ceramide on promoting LX-2 cell apoptosis and inhibitting hepatic fibrosis

作　　者：李立楠[1] 李杉杉[1] 刘悦[1] 卢娜[1] 方步武[1] 

机构地区：[1]天津医科大学基础医学院药理学系,天津300070

出　　处：《天津医科大学学报》2016年第4期283-287,共5页Journal of Tianjin Medical University

基　　金：国家自然科学基金资助项目(30772856)

摘　　要：目的:探讨神经酰胺对人源肝星状细胞株(HSCs)LX-2的影响以及在肝纤维化治疗中的作用,为寻求肝纤维化治疗的新方法提供理论依据。方法:体外培养人源肝星状细胞LX-2,分为正常细胞对照组及神经酰胺C2给药组(C2终浓度为30、45、60μmol/L),应用神经酰胺从头合成途径的限速酶即丝氨酸棕榈酰转移酶的特异性抑制剂多球壳菌素(myriocin)进一步分析神经酰胺的作用。四甲基偶氮唑盐(MTT)法检测LX-2的细胞增殖情况,酶消化法测定上清中羟脯氨酸(Hyp)的含量,比色法检测乳酸脱氢酶(LDH)的活性,Western blot法检测bax、bcl-2及caspase-3的蛋白表达量。结果:C2各浓度组对LX-2的增殖均有明显抑制作用,且具有剂量依赖性(P<0.05);LX-2经C2处理后细胞内羟脯氨酸含量显著下降(P<0.05);LDH活性与对照组比较均无显著性差异;C2下调LX-2中抗凋亡蛋白bcl-2的表达,上调促凋亡蛋白bax与凋亡执行效应分子caspase-3的表达(P<0.05),bax/bcl-2比值增大。但是,以myriocin 15μmol/L作用LX-2细胞24 h后,细胞增殖较对照组增多,羟脯氨酸的含量明显升高(P<0.05);LDH活性与对照组比较无显著性差异;bcl-2蛋白表达上调,bax、caspase-3蛋白表达下调(P<0.05),bax/bcl-2比值减小。结论:神经酰胺可通过上调bax和caspase-3的表达,下调bcl-2的表达而诱导LX-2细胞凋亡,同时抑制其增殖,从而减少羟脯氨酸的生成,产生抑制肝纤维发生的作用。Objective： To investigate the effects of ceramide on hepatic stellate cells（HSCs） （LX-2）, and to study the treatment on hepatic fibrosis to provide a theoretical basis for new treatment of hepatic fibrosis. Methods： The cultured LX-2 were divided into control group and C2-ceramide-treated groups with 30, 45, 60 μmol/L. Myriocin as the inhibitor of serine palmitoyl tranferase enzyme, ceramide de novo synthesis rate-limiting enzyme, was applied to further analyze the role of ceramide. The rate of cellular proliferation was detected by MTI＂ assay, the content of hydroxyproline （Hyp） was determined by enzyme digestion method, the activity of LDH was determined by colorimetric method and the expression of bax, bcl-2 and caspase-3 were detected by Western blot. Results＂ The various concentrations of C2 could significantly inhibit LX-2 proliferation with a dose-dependent manner （P〈0.05）; the content of Hyp was significantly decreased （P〈0.05）; Activity of LDH had no statistically significance compared with control group. After treated with C2, the expression of bcl-2 decreased, the expression of bax and caspase-3 increased in LX-2 （P〈0.05）. But 15 μmol/L myriocin could promote LX-2 proliferation and increase the content of Hyp after treated 24 h （P〈0.05）. Activity of LDH in myriocin group had no statistically significance. The expression of bcl-2 significantly increased, while the expression of bax and caspase-3 in LX-2 decreased （P〈0.05）. Conclusion： Ceramide could increase the expression of bax, caspase-3 and decrease the expression of bcl-2 to induce apoptosis of LX-2, also inhibit the proliferation and decreased the synthesis of hydroxyproline to inhibit hepatic fibrogensis.

关 键 词：肝纤维化 神经酰胺 多球壳菌素 羟脯氨酸 BAX bcl-2 caspase-3 

分 类 号：R96[医药卫生—药理学]