目的 探究大蒜主要活性成分二烯丙基三硫醚(diallyltrisulfide,DATS)体外对氧化应激诱导肝星状细胞(HSC)活化的影响。方法 大鼠肝星状细胞体外培养,以噻唑蓝法筛选过氧化氢(H2O2)刺激肝星状细胞增殖的最佳作用浓度,建立氧化应激诱导肝星状细胞活化的体外细胞模型。在此基础上,以乳酸脱氢酶释放实验检测二烯丙基三硫醚对肝星状细胞的毒性作用,以噻唑蓝法检测二烯丙基三硫醚在非毒性浓度下对肝星状细胞增殖的影响;以流式细胞仪检测二烯丙基三硫醚对肝星状细胞细胞周期的影响;以Hoechst 33258染色与流式细胞仪检测二烯丙基三硫醚对肝星状细胞凋亡的影响;以Western blot法检测二烯丙基三硫醚对肝星状细胞中凋亡相关蛋白及纤维化标志物表达的影响。结果 H2O2在5 μmol·L-1剂量下即可显著促进肝星状细胞增殖,将其作为体外刺激肝星状细胞活化的有效浓度。二烯丙基三硫醚可剂量依赖性地抑制H2O2诱导的肝星状细胞增殖,在1 μmol·L-1剂量下即有显著抑制效应。二烯丙基三硫醚可阻滞H2O2诱导活化的肝星状细胞的细胞周期于G2/M期,该作用与下调细胞周期调控蛋白Cyclin B1和CDK1相关。二烯丙基三硫醚剂量依赖性地促进H2O2诱导活化的肝星状细胞发生凋亡,与调控凋亡相关蛋白Bax、Bcl-2、Caspase-9及Caspase-8相关。此外,二烯丙基三硫醚还抑制肝星状细胞中一系列纤维化标志物的蛋白表达。结论 二烯丙基三硫醚体外对于氧化应激诱导的肝星状细胞活化具有显著的抑制作用,表现为抑制肝星状细胞增殖、阻滞细胞周期、诱导凋亡以及减少纤维化标志物的表达。本实验为将二烯丙基三硫醚作为潜在的抗肝纤维化药物研究提供实验依据。
Abstract
OBJECTIVE To investigate the effects of the primary bioactive component diallyltrisulfide (DATS) contained in garlic on the activation of hepatic stellate cells (HSCs) induced by oxidative stress in vitro. METHODS Rat HSCs were cultured in vitro, and MTT assay was used to screen the effective dose of hydrogen peroxide (H2O2) for stimulating HSC proliferation. Then lactate dehydrogenase assay was used to examine the toxic effect of DATS on HSCs, and MTT assay to evaluate the anti-proliferative effects of DATS at non-toxic concentrations on HSCs. Flow cytometry was used to determine DATS effects on cell cycle. Hoechst33258 staining and flow cytometry were used to analyze apoptosis. Western blot assays were used to examine the expression of relevant proteins and fibrotic markers. RESULTS H2O2 at 5 μmol·L-1 significantly promoted HSC proliferation, and this concentration was selected to establish the oxidative stress-induced HSC activation model. MTT assay showed that DATS dose-dependently inhibited HSC proliferation in the presence of H2O2. DATS arrested the H2O2-treated HSC at G2/M check point associated with downregulation of Cyclin B1 and CDK1. DATS also dose-dependently stimulated H2O2-treated HSCs to undergo apoptosis, which was associated with modulation of apoptosis-related molecules including Bax, Bcl-2, Caspase-9 and Caspase-3. Furthermore, DATS was found to reduce the protein abundance of a series of fibrotic marker proteins. CONCLUSION DATS effectively inhibits oxidative stress-induced HSC activation in vitro demonstrated by suppressed proliferation, arrests cell cycle, increases apoptosis and reduces fibrotic marker expression. These findings provide novel insights into DATS as a potential antifibrotic candidate for further development.
关键词
肝纤维化 /
肝星状细胞 /
大蒜 /
二烯丙基三硫醚 /
细胞周期 /
凋亡
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Key words
hepatic fibrosis /
hepatic stellate cell /
garlic /
diallyltrisulfide /
cell cycle /
apoptosis
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81270514,31401210);江苏省自然科学基金青年基金资助项目(BK20140955);江苏省高校自然科学研究面上项目(14KJB310011);2013年江苏高校优秀科技创新团队计划;江苏高校优势学科建设工程资助项目(ysxk-2010)
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