S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

AIM:To determine the role of c-Jun N-terminal kinase(JNK)activity in ethanol-induced apoptosis and themodulation of this signaling cascade by S-Adenosyl-methionine(AdoMet).METHODS:Primary hepatocyte cultures werepretreated with 100 μmol/L SP600125,a selective JNKinhibitor,1 mL/L DMSO or 4 mmol/L AdoMet and thenexposed to 100 mmo/L ethanol.Hepatocyte apoptosiswas determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMetwere determined by Western blot analysis of c-junphosphorylation and Bid fragmentation.SP600125 andAdoMet effects on the apoptotic signaling pathway weredetermined by Western blot analysis of cytochrome crelease and pro-caspase 3 fragmentation.The AdoMeteffect on glutathione levels was measured by Ellman'smethod and reactive oxygen species(ROS)generationby cell cytometry.RESULTS:The exposure of hepatocytes to ethanolinduced JNK activation,c-jun phosphorylation,Bidfragmentation,cytochrome c release and pro-caspase 3cleavage;these effects were diminished by SP600125,and caused a significant decrease in ethanol-inducedapoptosis(P<0.05).AdoMet exerted an antioxidanteffect maintaining glutathione levels and decreasing ROSgeneration,without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3cleavage. CONCLUSION:The JNK signaling cascade is a keycomponent of the proapoptotic signaling pathwayinduced by ethanol.JNK activation may be independentfrom ROS generation,since AdoMet which exertedantioxidant properties did not have a significant effect onJNK activity.JNK pathway modulator agents and AdoMetmay be components of promising therapies for alcoholicliver disease(ALD)treatment.

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 IJmol/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 EIIman＇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.