异丙酚通过激活ERK信号通路减少HO引起L肝细胞凋亡.docVIP

异丙酚通过激活ERK信号通路减少H2O2引起的L02肝细胞凋亡 王浩 薛张纲 复旦大学附属中山医院麻醉科，上海 200032 Propofol protected hepatic L02 cells from H2O2-induced apoptosis via activation of Extracellular Signal-Regulated Kinases (ERK) Pathway WANG Hao, XUE Zhang-gang Department of Anesthesiology, Zhongshan Hospital, Fudan University, Shanghai 200032, China [摘要]：目的 已有报道证明异丙酚在缺血再灌注损伤 (ischemia/reperfusion I/R) 中对一些重要脏器具有保护作用，但是对肝细胞的作用报道甚少。此外，最近的研究显示ERK信号通路在氧化应激中发挥重要作用。因此我们用H2O2诱导肝细胞氧化损伤，观察异丙酚预处理对人L02细胞的影响并且进一步检测ERK通路在其中的作用。方法 L02细胞经异丙酚预处理后被H2O2诱导凋亡，用TUNEL法及Caspase-3切割程度来检测细胞的凋亡。用蛋白质印迹技术检测ERK及MEK的磷酸化情况。通过实时定量PCR技术检测Bcl-2, Bcl-xL, Bad, 和Bax mRNA表达量变化。结果 在H2O2诱导的凋亡过程中，经异丙酚预处理的L02细胞凋亡数量及Caspase-3切割量均明显减少。并且这种减少与异丙酚的剂量成正相关：当异丙酚剂量用至300 μM时，细胞的凋亡程度被降至最低。与此一致的是ERK的磷酸化被显著的激活。单用不同浓度的异丙酚 (10 - 300 μM) 处理细胞，检测到浓度依赖性的ERK及MEK磷酸化升高。不同时间点（0 - 4 h）收获经异丙酚处理的细胞，发现ERK及MEK在0.5小时内就可以被激活，随后逐渐降低，至4 h时，ERK及MEK的活性下降到峰值时的一半。用特异性的MEK抑制剂PD98059处理细胞完全抑制异丙酚诱导的ERK活性，并且加重了细胞的凋亡程度。异丙酚预处理减少了促凋亡基因Bad和Bax的mRNA表达，并且这种抑制作用可以被PD98059逆转。结论 我们的研究提示异丙酚对H2O2诱导的人肝细胞凋亡具有保护作用，并且这种保护作用至少部分是通过激活ERK信号通路进而抑制Bad和Bax的表达起作用的。 [关键词]：异丙酚；肝细胞；ERK；凋亡 [Abstract]：Objective Here we investigated the effect of propofol preconditioning on human hepatic L02 cells under hydrogen peroxide (H2O2)-induced oxidative stress and attempted to find out whether ERK pathway is involved in this process. Methods Preconditioned or nonpreconditioned human hepatic L02 cells were exposed to H2O2 and the changes of apoptosis were evaluated by TUNEL assay and Caspase-3 cleavage. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP Kinase/ERK Kinase 1/2 (MEK1/2) was measured by western blot. The mRNA expression of Bcl-2, Bcl-xL, Bad, and Bax was quantified by real-time PCR. Results Propofol preconditioning reduced population of apoptotic cells and Caspase-3 cleavage induced by H2O2 in hepatic L02 cells. The minimal amount of cell death was relevant to 300 μM propofol pretreatment, accompanying with significant activation of ERK1/2. L02 cells treated with propofol (10 - 300 μ