Dilinoleoylphosphatidylcholine reproduces the antiapoptotic actions of polyenylphosphatidylcholine against ethanol-induced hepatocyte apoptosis.
BACKGROUND
Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, attenuates hepatocyte apoptosis induced by ethanol feeding of rats. Our aims were to evaluate whether dilinoleoylphosphatidylcholine (DLPC), the main component of PPC, reproduces the antiapoptotic actions of PPC against alcohol-induced apoptosis and to identify the apoptotic proteins that are affected by PPC and DLPC.


METHODS
Rats were fed Lieber-DeCarli liquid diets containing ethanol (35% of energy) or an isocaloric amount of carbohydrate for 4 weeks. Another group of rats were given the ethanol diet supplemented with PPC (3 g/liter) or DLPC (1.5 and 3 g/liter). Hepatocyte apoptosis was assessed by terminal transferase-mediated dUTP nick end labeling staining and by caspase 3 enzyme activity. Activity of caspases 3 and 9 was assayed by using fluorogenic peptide substrates. Cytochrome c was quantified by enzyme-linked immunosorbent assay. The protein contents of cytochrome c, procaspase 3, caspase 3, Bcl-x(L), and Bax were analyzed by Western blot and quantified by densitometry. Lobular localization of active caspase 3 was examined by immunoperoxidase staining.


RESULTS
PPC and DLPC decreased ethanol-induced increases in hepatocyte apoptosis, cytosolic cytochrome c, and caspase 3 content and its activity. Caspase 3 activity correlated with the number of apoptotic hepatocytes. Active caspase 3 was present predominantly in perivenular hepatocytes, and ethanol feeding extended it to lobular hepatocytes; this ethanol effect was reduced by PPC and DLPC. Ethanol significantly decreased Bcl-x(L) in homogenate, mitochondria, and cytosol, and there was a trend for increased Bcl-x(L) in these fractions after PPC and DLPC supplementation. Microsomal Bcl-x(L) did not differ between treatment groups. Bax was detected in homogenate and cytosol, and its level was not affected by ethanol.


CONCLUSIONS
DLPC, at a dose contained in PPC, reproduces the antiapoptotic actions of PPC through a reduction in cytosolic cytochrome c concentration and caspase 3 activity, possibly in association with up-regulation of Bcl-x(L) expression. Because DLPC is a pure and well defined compound, it may be more suitable than PPC for intervention against alcohol-induced apoptosis.
Mak
Ki M
KM
Alcohol Research and Treatment Center, Bronx Veterans Affairs Medical Center and Mount Sinai School of Medicine, New York 10468, USA.
Wen
Kefeng
K
Ren
Chaoling
C
Lieber
Charles S
CS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D008297:Male; D010713:Phosphatidylcholines / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D003574:Cytochrome c Group; D010713:Phosphatidylcholines; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C005152:lipostabil; D000431:Ethanol; C002448:1,2-linoleoylphosphatidylcholine; D020169:Caspases