Ghrelin protects against palmitic acid or lipopolysaccharide-induced hepatocyte apoptosis through inhibition of MAPKs/iNOS and restoration of Akt/eNOS pathways.
BACKGROUND
Ghrelin has been shown to exert various biological functions. However, the effect and mechanism of ghrelin on PA- or LPS-induced liver injury remains unknown.


METHODS
Normal human hepatocyte lines (LO2 and 7701) were pretreated with ghrelin (10-8M) for 30min before stimulation with lipopolysaccharide (LPS) or palmitic acid (PA). The proliferation and apoptosis of cells were detected with CCK8, Hoechst staining and flow cytometric analysis. Levels of NO of cell supernatants were examined by enzyme-linked immunosorbent assay (ELISA). The protein levels and mRNA of target genes of endothelial NOS (eNOS) and inducible NOS (iNOS) were measured by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Bax, Bcl2, caspase 3, p-Akt, p-P38 and p-JNK were detected by western blotting.


RESULTS
Results of CCK8, Hoechst staining and flow cytometric analysis showed that ghrelin-pretreatment attenuated LPS- or PA- induced cellular proliferation inhibition and apoptosis induction. ELISA results revealed that ghrelin pretreatment reduced levels of NO of cell supernatants (P<0.05). Results of western blotting and immunofluorescence showed that protein levels of iNOS in ghrelin- pretreated group were significantly reduced compared with LPS- or PA- treated group, while protein levels of eNOS were restored by ghrelin pretreatment. Results of qRT-PCR showed that mRNA levels of Bax, iNOS were reduced by ghrelin pretreatment, while levels of mRNA of Bcl2 and eNOS were increased (P<0.05). The protein levels of pAkt were significantly increased by ghrelin pretreatment, while the protein levels of p-JNK, p-P38 and caspase 3 were reduced. The restoration of eNOS could be reversed by an Akt inhibitor.


CONCLUSIONS
Ghrelin pretreatment attenuated LPS- or PA-induced hepatocyte apoptosis, which may least partly via inhibition of mitogen-activated protein kinases (MAPKs)/iNOS and restoration of Akt/eNOS pathways.
Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, 201508 Shanghai, China.
Yuqing
Y
Mao
Department of Gastroenterology and Hepatology, The Central Hospital of Lishui City, Wenzhou Medical University, Lishui 323000, Zhejiang, China.
Jianbo
J
Wang
Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, 201508 Shanghai, China.
Fujun
F
Yu
Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, 201508 Shanghai, China.
Zhengyang
Z
Li
Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, 201508 Shanghai, China.
Huanqing
H
Li
Department of Gastroenterology and Hepatology, Shanghai Tenth People's Hospital of Tongji University, 200072 Shanghai, China. Electronic address: guochuanyong@hotmail.com.
Chuanyong
C
Guo
Department of Gastroenterology and Hepatology, Jinshan Hospital of Fudan University, 201508 Shanghai, China. Electronic address: xiaomingfan57@hotmail.com.
Xiaoming
X
Fan
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D054439:Ghrelin / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019308:Palmitic Acid / Q000633:toxicity*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
D051017:Apoptosis Regulatory Proteins; D054439:Ghrelin; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D019308:Palmitic Acid; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases
Akt/eNOS pathway; Apoptosis; Ghrelin; MAPKs/iNOS pathway; NO