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Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6 #MMPMID22851861
Zhang SC; Zheng YH; Yu PP; Min TH; Yu FX; Ye C; Xie YK; Zhang QY
World J Gastroenterol 2012[Jul]; 18 (28): 3696-704 PMID22851861show ga
AIM: 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. CONCLUSION: 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.
|*Gene Expression Regulation[MESH]
|Animals[MESH]
|Base Sequence[MESH]
|Dose-Response Relationship, Drug[MESH]
|Enzyme Inhibitors/pharmacology[MESH]
|Genetic Vectors[MESH]
|Hepatic Stellate Cells/*cytology[MESH]
|Inflammation[MESH]
|Interleukin-6/*metabolism[MESH]
|Lentivirus/*genetics[MESH]
|Liver Cirrhosis/pathology[MESH]
|Male[MESH]
|Mitogen-Activated Protein Kinase 1/metabolism[MESH]
|Mitogen-Activated Protein Kinase 3/metabolism[MESH]
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World+J+Gastroenterol 2012 ; 18 (28): 3696-704
