Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis.
Rat hepatic stellate cells (HSC-T6) were incubated for 24 h with 10-180 microM of t10c12 (98%), c9t11 (96%) and a mixed form (c9,t11:t10,c12; 41%:44%) of conjugated linoleic acid (CLA). The MTS dye reduction was measured to verify cell viability in a dose-dependent manner. Among the three CLAs, c9,t11-CLA exhibited the most intense cytotoxic effect on HSCs, the survival rate of which was reduced to 60% under 80 microM of treatment, while cell survival was slightly affected by the mixed form. Three CLA-induced cell deaths were determined by measuring DNA fragmentation using 4',6-diamidino-2-phenylindole staining. The degrees of DNA fragmentation were the most severe in HSC treated with 80 microM of c9,t11-CLA. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase and mitogen-activated or extracellular signal-regulated protein kinase (MEK) 1 and 2 were not activated in the t10,c12-CLA treatment. This suggests that the MEK-dependent apoptosis signal is crucial in HSC, which is induced by c9,t11 and mixed CLA. In order to evaluate the protective effect of CLA on carbon tetrachloride (CCl4)-induced hepatic fibrosis in vivo, animals were treated with 10% CCl4 to induce hepatic fibrosis during all experimental periods. Rats were divided into two treatment groups: (1) control diet with tap water ad libitum (n=15) and (2) 1% CLA diet with tap water ad libitum (n=15). In the CLA-supplemented rat livers, alpha-smooth muscle actin-positive cells were significantly reduced around the portal vein. In addition, collagen fibers were not detected in the CLA-treated group. These results suggest that 9c,11t-CLA influences cytotoxic effect on HSC in an MEK-dependent manner and preserving liver from fibrosis.
Yun
Hae-Sun
HS
Department of Pathology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea.
Do
Sun-Hee
SH
Jeong
Won-Il
WI
Yang
Hai-Jie
HJ
Yuan
Dong-Wei
DW
Hong
Il-Hwa
IH
Lee
Hye-Rim
HR
Lee
In-Seon
IS
Kim
Yoo-Kyung
YK
Choi
Myung-Sook
MS
Kim
Hyeon-A
HA
Jeong
Kyu-Shik
KS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002251:Carbon Tetrachloride; D003094:Collagen / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D004032:Diet; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D044243:Linoleic Acids, Conjugated / Q000008:administration & dosage / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D044243:Linoleic Acids, Conjugated; D012333:RNA, Messenger; C503589:cis-9, trans-11-conjugated linoleic acid; C496197:trans-10,cis-12-conjugated linoleic acid; D003094:Collagen; D002251:Carbon Tetrachloride; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases