摘要
目的研究乙醇对原代肝细胞中P70S6K和ERK1/2活性的影响。方法分离成年雄性SD大鼠的肝细胞,分别以0、50、100和200 mmol/L的乙醇处理4 h,或以200 mmol/L乙醇处理0、1、2和4 h,用Western blot法检测P70S6K和ERK1/2的活性。在细胞中加入100 nmol/L的mTOR特异抑制剂雷帕霉素或10μmol/L的MEK1/2特异抑制剂U0126,培养24 h后收集细胞,用Western blot法检测P70S6K、ERK1/2及PPARγ的表达;用EMSA检测PPARγ的活性。结果随着乙醇浓度的增加或处理时间的延长,P70S6K的表达逐渐降低;乙醇也可抑制ERK1/2的活性;U0126同时抑制ERK1/2和P70S6K的活性,而雷帕霉素虽抑制P70S6K的活性,但增强ERK1/2的活性;乙醇不影响PPARγ的表达和活性。结论在大鼠原代肝细胞,乙醇对P70S6K的活性有抑制作用,且存在量效关系和时效关系。P70S6K的活性受ERK1/2的控制,但P70S6K也可反向调节ERK1/2。
Objective To study the effect of ethanol on the activity of P70S6K and ERK1/2 in rat primary hepato- cytes. Methods Primary hepatocytes were isolated from adult male Sprague-Dawley rat and then treated with in- creased concentrations of etnanol (0, 50, 100 and 200 mmoL/L) for 4 h, respectively, or with increased duration of treatment with 200 mmol/L ethanol (0, 1, 2 or 4 h). P70S6K activity was then evaluated by Western blot. Primary hepatocytes were also treated with roTOR inhibitor rapamycin (100 nmol/L) or MEK1/2 inhibitor U0126 ( 10 μmol/L) for 24 h and then analyzed for the activity of P70S6K and ERK1/2 and the expression of PPARγ by Western blot, and PPAR activity by electrophoretic mobility shift assay. Results The activity of P70S6K decreased with the increased concentration of ethanol and duration of ethanol exposure. Ethanol also inhibited activity of ERK1/2. Rapamycin inhibited the activity of P70S6K and enhanced the activity of ERK1/2. But U0126 inhibited both the activation of P70S6K and ERK1/2. Ethanol did not affect the expression and activity of PPARγ. Conclusions Ethanol inhibits the activity of P70S6K in primary hepatocytes in a time-and concentration-dependentmanner. The activity of P70S6K is regulated by ERK1/2 but P70S6K also negative regulate ERK1/2 in rat primary hepatocytes.
出处
《基础医学与临床》
CSCD
北大核心
2013年第4期434-438,共5页
Basic and Clinical Medicine
基金
广西自然科学基金(2010GXNSFA013170)