S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism
S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism
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DOI：
10.3748/wjg.v12.i12.1895
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发表时间：
2006-03-28
影响因子：
4.3
通讯作者：
Villa-Trevino, Saul
中科院分区：
文献类型：
--
作者：
del Pilar Cabrales-Romero, Maria;Marquez-Rosado, Lucrecia;Villa-Trevino, Saul
目的：探讨c-jun氨基末端激酶活性在乙醇诱导的肝细胞凋亡中的作用及S-腺苷甲硫氨酸(ADOMet)对这一信号转导通路的调节作用。方法：用100mU/L的JNK抑制剂L SP600125、1mL/L二甲基亚磺酸或4 mmol/L ADOMet处理原代培养的肝细胞，然后用100MMO/L乙醇处理。用原位末端标记法和DNA梯状条带法检测肝细胞凋亡率。通过c-jun磷酸化和Bid断裂的Western印迹分析，检测JNK活性以及SP600125和ADOMet对JNK活性的抑制作用。通过细胞色素c释放和caspase3原片段的Western印迹分析，确定SP600125和ADOMet对细胞凋亡信号通路的影响。结果：乙醇可诱导肝细胞JNK活化、c-jun磷酸化、Bid断裂、细胞色素c释放和caspase3酶原裂解，这些作用可被SP600125减弱，并使乙醇诱导的细胞凋亡显著减少(P<；0.05)。ADOMet具有维持谷胱甘肽水平和减少ROS生成的抗氧化作用，而对JNK活性无明显影响，并阻止细胞色素c的释放和caspase 3原的裂解。结论：JNK信号转导通路是乙醇诱导的促凋亡信号通路的关键组成部分。JNK的激活可能独立于ROS的产生，因为具有抗氧化特性的ADOMet对JNK活性没有显著影响。JNK途径调节剂和ADOMet可能是治疗酒精性肝病(ALD)的有前景的疗法的组成部分。(C)2006年WJG出版社。版权所有。
AIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine (AdoMet).METHODS: Primary hepatocyte cultures were pretreated with 100 mu mol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry.RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis (P < 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment. (C) 2006 The WJG Press. All rights reserved.