title: Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6.
abstract: OBJECTIVE
  To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A receptor (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro.
  
  
  METHODS
  HSCs were derived from the livers of adult male Sprague-Dawley rats. IL-6 expression was evaluated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated protein kinases (MAPK) and extracellular regulated protein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting.
  
  
  RESULTS
  IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P < 0.01 in a dose-dependent manner). Suppression of IL-17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 ± 0.17 vs 4.07 ± 0.43, P < 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 ± 0.24, 2.01 ± 0.10 vs 4.08 ± 0.59, P < 0.01). Moreover, Lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 ± 0.17 vs 3.98 ± 0.68, P < 0.01). Lentiviral-mediated IL-17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure.
  
  
  CONCLUSIONS
  Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in the liver.
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, Zhejiang Province, China.
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Zhang
authors.forename: Yi-Hu
authors.initials: YH
authors.lastname: Zheng
authors.forename: Pan-Pan
authors.initials: PP
authors.lastname: Yu
authors.forename: Tan Hooi
authors.initials: TH
authors.lastname: Min
authors.forename: Fu-Xiang
authors.initials: FX
authors.lastname: Yu
authors.forename: Chao
authors.initials: C
authors.lastname: Ye
authors.forename: Yuan-Kang
authors.initials: YK
authors.lastname: Xie
authors.forename: Qi-Yu
authors.initials: QY
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000166:cytology*; D007249:Inflammation; D015850:Interleukin-6 / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D008103:Liver Cirrhosis / Q000473:pathology; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053722:Receptors, Interleukin-17 / Q000378:metabolism*; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D015850:Interleukin-6; D034741:RNA, Small Interfering; D053722:Receptors, Interleukin-17; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases