桑黄多糖对人TLR4信号通路的激活作用研究
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摘要
目的观察桑黄多糖(polysaccharide from Phellinus igniarius,PPI)对人Toll-样受体4(Toll like receptor 4,TLR4)通路的激活作用及作用特点。方法采用转染了人TLR4基因、分泌型胚胎碱性磷酸酶报告基因的HEK-Blue~(TM)hTLR4细胞模型,快速评价PPI对TLR4通路的激活作用,并用PMA诱导的THP-1细胞模型观察药物对TLR4通路作用的特点;MTT法检测细胞活性;ELISA法检测细胞因子分泌量;RealtimePCR检测细胞mRNA表达水平。结果 PPI作用24h不影响细胞活性,但能剂量依赖性地增加HEK-Blue~(TM) hTLR4细胞分泌型胚胎碱性磷酸酶的表达,并使THP-1细胞TLR4通路相关细胞因子TNF-α,IP-10分泌量增加。TLR4受体阻断剂TAK-242能显著降低PPI诱导的细胞因子分泌量(P<0.01)。Real-time PCR结果显示PPI既可以增加MyD88通路TNF-α、IL-6、IL-12、IL-1β、COX-2等细胞因子的mRNA表达,又可以增加TRIF通路IP-10、IFN-β等细胞因子的mRNA表达。结论 PPI对人TLR4受体具有直接激活作用,能同时激活TLR4下游MyD88和TRIF 2条分支,对TLR4分支通路的激活没有选择性。
OBJECTIVE To observe the effect of polysaccharide from Phellinus igniarius(PPI) on human Toll like receptor 4(TLR4) pathway. METHODS The activation of TLR4 pathway was evaluated rapidly by using HEK-Blue~(TM) hTLR4 cells transfected with human TLR4 gene and secreted embryonic alkaline phosphatase reporter gene, and the PMA stimulatedTHP-1 cells were used to observe the effects of drugs on the TLR4 pathway. The cell viability was detected by MTT assay. The cytokine secretion was evaluated by ELISA. mRNA expression level of the cells was analysed by Real-time PCR. RESULTS The treatment of PPI for 24 h did not affect cell viability but dose-dependently increased the expression of secreted embryonic alkaline phosphatase in HEK-Blue~(TM) hTLR4 cells. In addition, the treatment of PPI leaded to increased secretion of TLR4 pathway-associated cytokines such as TNF-α and IP-10 in THP-1 cells. The TLR4 receptor blocker TAK-242 significantly reduced PPI-induced cytokine secretion(P<0.01). The results of Real-time PCR showed that PPI not only increased the mRNA expression of cytokines such as TNF-α, IL-6, IL-12, IL-1β and COX-2 in MyD88 pathway, but also increased the mRNA expression of cytokines such as IP-10 and IFN-β in TRIF pathway. CONCLUSION PPI is an agonist of the human TLR4, it can activate both MyD88 and TRIF branches on the downstream of TLR4 and show no selective activation of the two TLR4 branching pathways.
OBJECTIVE To observe the effect of polysaccharide from Phellinus igniarius(PPI) on human Toll like receptor 4(TLR4) pathway. METHODS The activation of TLR4 pathway was evaluated rapidly by using HEK-Blue~(TM) hTLR4 cells transfected with human TLR4 gene and secreted embryonic alkaline phosphatase reporter gene, and the PMA stimulatedTHP-1 cells were used to observe the effects of drugs on the TLR4 pathway. The cell viability was detected by MTT assay. The cytokine secretion was evaluated by ELISA. mRNA expression level of the cells was analysed by Real-time PCR. RESULTS The treatment of PPI for 24 h did not affect cell viability but dose-dependently increased the expression of secreted embryonic alkaline phosphatase in HEK-Blue~(TM) hTLR4 cells. In addition, the treatment of PPI leaded to increased secretion of TLR4 pathway-associated cytokines such as TNF-α and IP-10 in THP-1 cells. The TLR4 receptor blocker TAK-242 significantly reduced PPI-induced cytokine secretion(P<0.01). The results of Real-time PCR showed that PPI not only increased the mRNA expression of cytokines such as TNF-α, IL-6, IL-12, IL-1β and COX-2 in MyD88 pathway, but also increased the mRNA expression of cytokines such as IP-10 and IFN-β in TRIF pathway. CONCLUSION PPI is an agonist of the human TLR4, it can activate both MyD88 and TRIF branches on the downstream of TLR4 and show no selective activation of the two TLR4 branching pathways.
引文
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[12]FENG Z,WANG Z,YANG M,et al.Polysaccharopeptide exerts immunoregulatory effects via MyD88-dependent signaling pathway[J].Int J Biol Macromol,2016(82):201-207.
[13]ZHOU L,LIU Z,WANG Z,et al.Astragalus polysaccharides exerts immunomodulatory effects via TLR4-mediated My D88-dependent signaling pathway in vitro and in vivo[J].Sci Rep,2017(7):44822.Doi:10.1038/srep44822.
[2]LI X,JIAO L L,ZHANG X,et al.Anti-tumor and immunomodulating activities of proteoglycans from mycelium of Phellinus nigricans and culture medium[J].Int Immunopharmacol,2008,8(6):909-915.
[3]GAO W,WANG W,SUN W,et al.Antitumor and immunomodulating activities of six Phellinus igniarius polysaccharides of different origins[J].Exp Therap Med,2017,14(5):4627-4632.
[4]PARK S K,KIM G Y,LIM J Y,et al.Acidic polysaccharides isolated from Phellinus linteus induce phenotypic and functional maturation of murine dendritic cells[J].Biochem Biophys Res Commun,2003,312(2):449-458.
[5]KIM G Y,CHOI G S,LEE S H,et al.Acidic polysaccharide isolated from Phellinus linteus enhances through the upregulation of nitric oxide and tumor necrosis factor-αfrom peritoneal macrophages[J].J Ethnopharmacol,2004,95(1):69-76.
[6]KIM G Y,HAN M G,SONG Y S,et al.Proteoglycan isolated from phellinus linteus induces toll-like receptors 2-and4-mediated maturation of murine dendritic cells via activation of ERK,p38,and NF-κB[J].Biol Pharm Bull,2004,27(10):1656-1662.
[7]IWASAKI A,MEDZHITOV R.Toll-like receptor control of the adaptive immune responses[J].Nat Immunol,2004,5(10):987-995.
[8]KAWAI T,ADACHI O,OGAWA T,et al.Unresponsiveness of My D88-deficient mice to endotoxin[J].Immunity,1999,11(1):115-122.
[9]YAMAMOTO M.Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway[J].Science,2003,301(5633):640-643.
[10]AKIRA S.Toll-like receptors in innate immunity[J].Advances Immunol,2001,78(1):1-56.
[11]AKIRA S,TAKEDA K.Toll-like receptor signalling[J].Nat Rev Immunol,2004,4(7):499.
[12]FENG Z,WANG Z,YANG M,et al.Polysaccharopeptide exerts immunoregulatory effects via MyD88-dependent signaling pathway[J].Int J Biol Macromol,2016(82):201-207.
[13]ZHOU L,LIU Z,WANG Z,et al.Astragalus polysaccharides exerts immunomodulatory effects via TLR4-mediated My D88-dependent signaling pathway in vitro and in vivo[J].Sci Rep,2017(7):44822.Doi:10.1038/srep44822.