神经酰胺对肝纤维化治疗作用的初步研究

Effects of ceramide on hepatic fibrosis

目的:探讨神经酰胺代谢酶抑制剂油酰乙醇胺(NOE)和丙咪嗪(Imi)对人源肝星状细胞(HSCs)LX-2中神经酰胺含量的影响,并探讨神经酰胺在肝纤维化治疗中的作用。方法:实验分为给药组(NOE组终浓度为50、75、100μmol/L;Imi组终浓度为10、20、30μmol/L),乙醇对照组(乙醇终浓度为1%),DMSO组(DMSO终浓度为1‰)。高效液相法测定HSCs内神经酰胺的含量,四甲基偶氮唑盐(MTT)法检测HSCs的增殖情况,比色法检测乳酸脱氢酶(LDH)的活性,酶消化法测定羟脯氨酸(Hyp)的含量。结果:与乙醇组比较,NOE各浓度组可显著提高HSCs内神经酰胺的含量(P<0.05);对HSCs的抑制作用呈剂量-效应关系;各浓度组LDH活性与乙醇组比较,无显著性差异(P>0.05);且各浓度组均可显著降低HSCs上清液中Hyp的含量(P<0.05)。30μmol/L Imi作用24 h后,与DMSO组比较,可降低HSCs内神经酰胺的含量,促进HSCs的增殖,并可升高HSCs上清液中Hyp的含量,且差异均具有统计学意义(P<0.05)。结论:神经酰胺代谢酶抑制剂对HSCs内神经酰胺的含量有显著影响,且神经酰胺可通过抑制HSCs细胞的增殖、减少胶原蛋白的合成来发挥抗肝纤维化作用。

Objective： To investigate the effects of ceramide metabolism inhibitors, N-oleoylethanolamine （NOE） and Imipramine（Imi） on intracellular contents in HSCs LX-2, and to explore the effect of ceramide on hepatic fibrosis. Methods：The experimental groups included three NOE-treated groups with 50, 75, 100 μmol/L NOE, three lmi-treated groups with 10, 20, 30 μmol/L Imi, one alcohol-treated group with 1% alcohol and one DMSO-treated group with 1‰ DMSO. The content of ceramide was determined by HPLC method, MTT assay was applied to detect the rate of cellular proliferation. The activity of LDH was determined by colorimetric method and the content of hydroxyproIine （Hyp） was obtained using enzyme digestion method. Results： Compared with alcohol-treated group, NOE significantly increased the content of ceramide in HSCs （P〈0.05）. MTT analysis revealed that HSCs treated with various concentrations of NOE （50-100 μmol/L） were inhibited on dose-effect relationships. LDH activity in NOE-treated groups had no significant difference compared with alcohol-treated group （P〉0.05） while content of Hyp in NOE-treated groups was significantly decreaced compared with alcohol-treated group （P〈0.05）. Compared with DMSO-treated group, 30 μmol/L Imi could decreace the concentration of ceramide in HSCs, promote the proliferation of HSCs and increase the content of Hyp in HSCs. All of these differences were significant （P〈0.05）. Conclusion： The ceramide metabolism inhibitors could change the concentration of ceramide effectively, and the anti-fibrogenic mechanisms of ceramide might inhibit the activation of HSCs, and decrease the synthesis of collagen.