己酮可可碱通过Hedgehog信号通路对日本血吸虫病肝纤维化形成的抑制作用
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摘要
目的:探讨己酮可可碱(PTX)通过Hedgehog信号通路抑制日本血吸虫病肝纤维化的作用机制。方法:PMA诱导单核细胞THP-1分化成巨噬细胞,用可溶性日本血吸虫虫卵抗原(SEA)刺激活化THP-1源性巨噬细胞,留取培养上清用于检测其活化状况。CCK-8检测培养上清及PTX对肝星状细胞LX-2的活性影响。设空白对照组、上清刺激组(加入活化的巨噬细胞培养上清)、PTX干预组(加入活化的巨噬细胞培养上清和PTX)。收集以上3组LX-2培养上清及细胞,上清用于ELISA检测TGF-β蛋白表达,细胞用于提取总RNA后RT-PCR检测TGF-β,shh,gli-1的基因表达。结果:SEA可活化THP-1源性巨噬细胞,其活化后培养上清可活化LX-2细胞。上清刺激组TGF-β,shh,gli-1的基因表达空白对照组明显上调,而PTX干预组相对于上刺激组则明显降低。结论:Hedgehog信号通路可能在血吸虫肝纤维化形成过程中起重要作用,己酮可可碱可通过该通路对血吸虫肝纤维化的形成起抑制作用。
OBJECTIVE To nvestigate the mechanism of pentoxifylline(PTX) in inhibiting liver fibrosis induced by schistosoma japonicum infection through the Hedgehog signaling pathway. METHODS Phorbol12-myristate13-acetate(PMA) was used to induce THP-1 monocytes to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen(SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8(CCK-8) was used to detect the effect of culture supernatant and PTX on the activity of LX-2 in hepatic stellate cells. Blank control group, supernatant stimulation group(added with activated macrophage culture supernatant), PTX intervention group(added with activated macrophage culture supernatant and PTX). LX-2 culture supernatants and cells were collected from the above three groups, and the supernatants were used for ELISA to detect TGF-β protein expression, and the cells were used for total RNA extraction and RT-PCR to detect the gene expression of TGF-β, shh, and gli-1. RESULTS The THP-1 derived macrophages were activated by SEA, and LX-2 cells were activated by SEA. The gene expression of TGF-β, shh, gli-1 in the supernatant stimulation group was significantly up-regulated in the blank control group, while it was significantly decreased in the PTX intervention group compared with the upper stimulation group. CONCLUSION Hedgehog signaling pathway may play an important role in the formation of schistosome liver fibrosis, and pentoxifylline can inhibit the formation of schistosome liver fibrosis through this pathway.
OBJECTIVE To nvestigate the mechanism of pentoxifylline(PTX) in inhibiting liver fibrosis induced by schistosoma japonicum infection through the Hedgehog signaling pathway. METHODS Phorbol12-myristate13-acetate(PMA) was used to induce THP-1 monocytes to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen(SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8(CCK-8) was used to detect the effect of culture supernatant and PTX on the activity of LX-2 in hepatic stellate cells. Blank control group, supernatant stimulation group(added with activated macrophage culture supernatant), PTX intervention group(added with activated macrophage culture supernatant and PTX). LX-2 culture supernatants and cells were collected from the above three groups, and the supernatants were used for ELISA to detect TGF-β protein expression, and the cells were used for total RNA extraction and RT-PCR to detect the gene expression of TGF-β, shh, and gli-1. RESULTS The THP-1 derived macrophages were activated by SEA, and LX-2 cells were activated by SEA. The gene expression of TGF-β, shh, gli-1 in the supernatant stimulation group was significantly up-regulated in the blank control group, while it was significantly decreased in the PTX intervention group compared with the upper stimulation group. CONCLUSION Hedgehog signaling pathway may play an important role in the formation of schistosome liver fibrosis, and pentoxifylline can inhibit the formation of schistosome liver fibrosis through this pathway.
引文
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[2] Qin Z.The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature[J].Atherosclerosis,2012,221(1):2-11.
[3] Luedde T,Schwabe RF.NF-kappaB in the liver--linking injury,fibrosis and hepatocellular carcinoma[J].Nature reviews Gastroenterology & hepatology,2011,8(2):108-118.
[4] Ueberham E,Low R,Ueberham U,et al.Conditional tetracycline- regulated expression of TGF-beta1 in liver of transgenic mice leads to reversible intermediary fibrosis[J].Hepatology,2003,37(5):1067-1078.
[5] Yang JJ,Tao H,Li J.Hedgehog signaling pathway as key player in liver fibrosis:newinsights and perspectives[J].Expert Opin Ther Targets,2014,18(9):1011-1021.
[6] Chen Y,Choi SS,Michelotti GA,et al.Hedgehog controls hepatic stellate cell fate by regulating metabolism[J].Gastroenterology,2012,143(5):1319-1329.e1-11.
[7] Syn W-K,Choi SS,Liaskou E,et al.Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis[J].Hepatology,2011,53(1):106-115.
[8]Mederacke I,Hsu CC,Troeger JS,et al.Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology[J].Nat Commun,2013,4(7):657-678.
[9] Seki E,Schwabe RF.Hepatic inflammation and fibrosis:functional links and key pathways[J].Hepatology,2015,61(3):1066-1079.
[10] Toda K,Kumagai N,Kaneko F,et al.Pentoxifylline prevents pig serum-induced rat liver fibrosis by inhibiting interleukin-6 production[J].J Gastroenterol Hepatol,2009,24(5):860-865.