AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A recepto (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro . MET...AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A recepto (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 adul male Sprague-Dawley rats. IL-6 expression was evalu ated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated pro tein kinases (MAPK) and extracellular regulated pro tein 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 IL17RA 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 IL17RA 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.展开更多
BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory eff...BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells（ADSCs）on activation and proliferation of PSCs.METHODS：Pancreatic tissue was obtained from SpragueDawley rats for PSCs isolation.Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera- tion and apoptosis were determined using CCK-8 and flow cytometry, respectively, a-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor （NGF）, interleukin-10 （IL-10） and transforming growth factor-ill （TGF-[31）] in conditioned medium were detected by ELISA. Gene expression （MMP-2, MMP-9 and TIMP-1） was analyzed using qRT-PCR. RESULTS： This method produced 17.6_＋6.5 ~ 103 ceils per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhib- ited PSCs proliferation and induced PSCs apoptosis. Moreover, a-SMA expression was significantly reduced in PSCs＋ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins （e.g., MMP-2 and MMP-9） was upregulated and anti-fibrinolytic protein （TIMP-1） was downregulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the coculture conditioned medium compared with those in the PSC- only culture medium. CONCLUSIONS： This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.展开更多
基金Supported by The Zhejiang Extremely Key Subject of SurgeryThe Wenzhou Key Laboratory Project in Surgery
文摘AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A recepto (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 adul male Sprague-Dawley rats. IL-6 expression was evalu ated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated pro tein kinases (MAPK) and extracellular regulated pro tein 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 IL17RA 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 IL17RA 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.
基金supported in part by a grant from Zhejiang Province Key Surgery Projects(Zhejiang High-Tech 2008-255)
文摘BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells（ADSCs）on activation and proliferation of PSCs.METHODS：Pancreatic tissue was obtained from SpragueDawley rats for PSCs isolation.Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera- tion and apoptosis were determined using CCK-8 and flow cytometry, respectively, a-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor （NGF）, interleukin-10 （IL-10） and transforming growth factor-ill （TGF-[31）] in conditioned medium were detected by ELISA. Gene expression （MMP-2, MMP-9 and TIMP-1） was analyzed using qRT-PCR. RESULTS： This method produced 17.6_＋6.5 ~ 103 ceils per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhib- ited PSCs proliferation and induced PSCs apoptosis. Moreover, a-SMA expression was significantly reduced in PSCs＋ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins （e.g., MMP-2 and MMP-9） was upregulated and anti-fibrinolytic protein （TIMP-1） was downregulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the coculture conditioned medium compared with those in the PSC- only culture medium. CONCLUSIONS： This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.
