华支睾吸虫合并HBV感染对肝星状细胞和巨噬细胞作用的体外研究
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摘要
目的华支睾吸虫合并HBV感染对肝星状细胞和巨噬细胞的作用。方法以华支睾吸虫分泌排泄蛋白(CsESP)(10μg/mL)合并HepG2.2.15细胞(5×10~5个)共孵育人肝星状细胞(LX-2),同时设10μg/mL CsESP、5×10~5个HepG2.2.15单独处理组和PBS组。荧光定量PCR(qRT-PCR)法检测各组肝纤维化相关指标α-平滑肌蛋白(α-SMA)、胶原蛋白Collagen-Ⅰ、Collagen-Ⅲ的转录水平;利用CsESP(10μg/mL)合并乙型肝炎病毒e抗原(HBeAg)(5μg/mL)共同刺激巨噬细胞RAW264.7细胞,同时设10μg/mL CsESP、5μg/mL HBeAg单独处理组和PBS组,qRT-PCR法检测各组一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、白细胞介素6(IL-6)、NLRP3炎症小体、白细胞介素-1β(IL-1β)的表达情况,同时使用流式细胞术检测其凋亡水平变化。结果 qRT-PCR法检测结果显示,CsESP合并HepG2.2.15处理组中LX-2细胞α-SMA和Collagen-Ⅰ的m RNA水平与CsESP或HepG2.2.15单独处理组相比显著上升,差异有统计学意义(P<0.05),而Collagen-Ⅲ表达变化不明显。CsESP与HBeAg共处理诱导RAW264.7细胞中NLRP3、IL-1β、IL-6、TGF-β、TNF-α和iNOS的mRNA水平与CsESP或HBeAg单独刺激组相比显著上升,差异有统计学意义(P<0.05)。CsESP与HBeAg共处理组凋亡细胞比率为16.35%,明显高于CsESP、HBeAg单独处理组的6.03%和12.52%,差异有统计学意义(P<0.05)。结论华支睾吸虫合并HBV感染体外可促进肝星状细胞的激活,并可促进巨噬细胞炎症因子的释放及细胞凋亡,可能促进肝脏炎症、肝纤维化的进展。
Objective To explore the effects of Clonorchis sinensis(C.sinensis) and hepatitis B virus(HBV) co-infection on macrophage and hepatic stellate cell(HSC). Methods HSC LX-2 cell line was co-cultured with C. sinensis excretory/secrectory proteins( CsESP)+HepG2.2.15 or CsESP alone or HepG2.2.15 alone. The mRNA levels of alpha-smooth muscle actin(a-SMA),Collagen-Ⅰ, and Collagen-Ⅲ were detected by qRT-PCR. Mice macrophage cell line RAW264.7 cells were treated with CsESP and e antigen of HBV( HBeAg). Inducible nitric oxide synthase(iNOS), tumor necrosis factor-α( TNF-α), transforming growth factor-β(TGF-β), interleukin-6(IL-6), Nod-like receptor protein 3( NLRP3) and interleukin-1β(IL-1β) were detected by qRT-PCR. The cell apoptosis of RAW 264.7 were analyzed by flow cytometry(FACS).Results The transcriptional levels of α-SMA, Collagen-Ⅰ in LX-2 cell of CsESP+HepG2.2.15 group were increased when compared with CsESP alone group or HepG2.2.15 alone groups(P<0.05), while the Collagen-Ⅲ had no notable change.The inflammation related marker NLRP3,IL-1β, IL-6, TGF-β, TNF-α and iNOS in RAW264.7 cell were increased after treated with CsESP + HBeAg(P<0.05) compared to those in CsESP alone or HBeAg alone group. Moreover, the apoptosis rate of RAW264.7 cells was higher in the CsESP + HBeAg group than those of CsESP alone or HBeAg alone group.Conclusions Csinensis and HBV co-infection might directly activate HSC and promote transcriptions of inflammation related markers in macrophage. In addition, the co-infection might induce apoptosis of macrophage and cause more serious hepatic inflammation and acceleration of hepatic fibrosis.
Objective To explore the effects of Clonorchis sinensis(C.sinensis) and hepatitis B virus(HBV) co-infection on macrophage and hepatic stellate cell(HSC). Methods HSC LX-2 cell line was co-cultured with C. sinensis excretory/secrectory proteins( CsESP)+HepG2.2.15 or CsESP alone or HepG2.2.15 alone. The mRNA levels of alpha-smooth muscle actin(a-SMA),Collagen-Ⅰ, and Collagen-Ⅲ were detected by qRT-PCR. Mice macrophage cell line RAW264.7 cells were treated with CsESP and e antigen of HBV( HBeAg). Inducible nitric oxide synthase(iNOS), tumor necrosis factor-α( TNF-α), transforming growth factor-β(TGF-β), interleukin-6(IL-6), Nod-like receptor protein 3( NLRP3) and interleukin-1β(IL-1β) were detected by qRT-PCR. The cell apoptosis of RAW 264.7 were analyzed by flow cytometry(FACS).Results The transcriptional levels of α-SMA, Collagen-Ⅰ in LX-2 cell of CsESP+HepG2.2.15 group were increased when compared with CsESP alone group or HepG2.2.15 alone groups(P<0.05), while the Collagen-Ⅲ had no notable change.The inflammation related marker NLRP3,IL-1β, IL-6, TGF-β, TNF-α and iNOS in RAW264.7 cell were increased after treated with CsESP + HBeAg(P<0.05) compared to those in CsESP alone or HBeAg alone group. Moreover, the apoptosis rate of RAW264.7 cells was higher in the CsESP + HBeAg group than those of CsESP alone or HBeAg alone group.Conclusions Csinensis and HBV co-infection might directly activate HSC and promote transcriptions of inflammation related markers in macrophage. In addition, the co-infection might induce apoptosis of macrophage and cause more serious hepatic inflammation and acceleration of hepatic fibrosis.
引文
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[21]哈明昊,饶慧瑛,刘蜂,等.乙型肝炎病毒促进CTGF和TGF-β1在肝星状细胞中的表达[J].世界华人消化杂志,2008,16(9):924-928.
[22]Roingeard P,Lu SL,Sureau C,et al.Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells[J].Hepatology,2010,11(2):277-285.
[23]孔祥展.华支睾吸虫致病与巨噬细胞极化的相关性[J].热带医学杂志,2018,18(4):423-427.
[24]Hua H,Du Y,Ma R,et al.The regulatory roles of toll-like receptor 4 in secretions of type 1/type 2 relative cytokines by splenocytes and dendritic cells exposed to Clonorchis sinensis excretory/secretory products[J].Inflammation,2018,41(1):213-220.
[25]Ma H,Wei L,Guo F,et al.Clinical features and survival in Chinese patients with hepatitis B e antigen-negative hepatitis Bvirus-related cirrhosis[J].J Gastroenterol Hepatol,2010,23(8Pt 1):1250-1258.
[26]La Flamme AC,Macdonald AS,Pearce EJ.Role of IL-6 in directing the initial immune response to schistosome eggs[J].JImmunol,2000,164(5):2419-2426.
[27]Daniel C,Wiede J,Krutzsch HC,et al.Thrombospondin-1 is a major activator of TGF-beta in fibrotic renal disease in the rat in vivo[J].Kidney Int,2004,65(2):459-468.
[28]Lu YQ,Zhong S,Meng N,et al.NLRP3 inflammasome activation results in liver inflammation and fibrosis in mice infected with Schistosoma japonicum in a Syk-dependent manner[J].Sci Rep,2017,7(1):8120.
[29]Manuel R,Olaf G,Sarah K,et al.Schistosoma mansoni triggers Dectin-2,which activates the Nlrp3 inflammasome and alters adaptive immune responses[J].Proc Natl Acad Sci U S A,2010,107(47):20459-20464.
[30]Sebba A.Tocilizumab:the first interleukin-6-receptor inhibitor[J].Am J Health Syst Pharm,2008,65(15):1413-1418.
[2]Lun ZR,Gasser RBLai DH,Li AX,et al.Clonorchiasis:a key foodborne zoonosis in China[J].Lancet Infect Dis,2005,5(1):31-41.
[3]Qian MB,Chen YD,Song L,et al.The global epidemiology of clonorchiasis and its relation with cholangiocarcinoma[J].Infect Dis Poverty,2012,1(1):4.
[4]Zeng F,Guo P,Huang Y,et al.Epidemiology of hepatitis B virus infection:results from a community-based study of 0.15 million residents in South China[J].Sci Rep,2016,6:36186.
[5]郑培秋,何艳英,覃少洲,等.肝吸虫病肝胆管扩张与肝切除术后胆漏的关系[J].肝胆外科杂志,2011,19(3):222-224.
[6]陈钦艳,方钟燎,杨益超,等.广西乙型肝炎病毒感染及肝功能情况调查[J].广西医学,2013,35(11):1432-1434.
[7]牟洪香,王莉,和军,等.华支睾吸虫病感染者与乙型肝炎关系的探讨[J].黑龙江医药科学,2009,32(2):94.
[8]程占元,张显彬.人群感染华支睾吸虫病与乙型肝炎关系的探讨[J].中国病原生物学杂志,1997,10(2):99.
[9]何富强,朱胜利.乙型肝炎合并肝吸虫病60例治疗体会[J].实用肝脏病杂志,2000,5(2):114-115.
[10]Sandhu P,Haque M,Humphries-Bickley T,et al.Hepatitis Bvirus immunopathology,model systems,and current therapies[J].Front Immunol,2017,8:436.
[11]Lan S,Wu L,Wang X,et al.Impact of HBeAg on the maturation and function of dendritic cells[J].Int J Infect Dis,2016,46:42-48.
[12]Meng N,Xia M,Lu YQ,et al.Activation of NLRP3inflammasomes in mouse hepatic stellate cells during Schistosoma J.infection[J].Oncotarget,2016,7(26):39316-39331.
[13]Alegre F,Pelegrin P,Feldstein AE.Inflammasomes in Liver Fibrosis[J].Semin Liver Dis,2017,37(2):119-127.
[14]Wree A,Eguchi A,McGeough MD,et al.NLRP3 inflammasome activation results in hepatocyte pyroptosis,liver inflammation,and fibrosis in mice[J].Hepatology,2014,59(3):898-910.
[15]Pradere JP,Kluwe J,De MS,et al.Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice[J].Hepatology,2013,58(4):1461-1473.
[16]Li W,Dong H,Huang Y,et al.Clonorchis sinensis Co-infection could affect the disease state and treatment response of HBVpatients[J].PLoS Negl Trop Dis,2016,10(6):e0004806.
[17]Zhao L,Shi M,Zhou L,et al.Clonorchis sinensis adult-derived proteins elicit Th2 immune responses by regulating dendritic cells via mannose receptor[J].PLoS Negl Trop Dis,2018,12(3):e0006251.
[18]Wang X,Hu F,Hu X,et al.Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells[J].Parasitol Res,2014,113(8):3063-3071.
[19]胡凤玉,胡旭初,马长玲,等.华支睾吸虫分泌/排泄抗原致大鼠肝纤维化[J].南方医科大学学报,2009,29(3):393-396.
[20]Serriere-Lanneau V,Teixeira-Clerc F,Li L,et al.The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing[J].FASEB J,2007,21(9):2005-2013.
[21]哈明昊,饶慧瑛,刘蜂,等.乙型肝炎病毒促进CTGF和TGF-β1在肝星状细胞中的表达[J].世界华人消化杂志,2008,16(9):924-928.
[22]Roingeard P,Lu SL,Sureau C,et al.Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells[J].Hepatology,2010,11(2):277-285.
[23]孔祥展.华支睾吸虫致病与巨噬细胞极化的相关性[J].热带医学杂志,2018,18(4):423-427.
[24]Hua H,Du Y,Ma R,et al.The regulatory roles of toll-like receptor 4 in secretions of type 1/type 2 relative cytokines by splenocytes and dendritic cells exposed to Clonorchis sinensis excretory/secretory products[J].Inflammation,2018,41(1):213-220.
[25]Ma H,Wei L,Guo F,et al.Clinical features and survival in Chinese patients with hepatitis B e antigen-negative hepatitis Bvirus-related cirrhosis[J].J Gastroenterol Hepatol,2010,23(8Pt 1):1250-1258.
[26]La Flamme AC,Macdonald AS,Pearce EJ.Role of IL-6 in directing the initial immune response to schistosome eggs[J].JImmunol,2000,164(5):2419-2426.
[27]Daniel C,Wiede J,Krutzsch HC,et al.Thrombospondin-1 is a major activator of TGF-beta in fibrotic renal disease in the rat in vivo[J].Kidney Int,2004,65(2):459-468.
[28]Lu YQ,Zhong S,Meng N,et al.NLRP3 inflammasome activation results in liver inflammation and fibrosis in mice infected with Schistosoma japonicum in a Syk-dependent manner[J].Sci Rep,2017,7(1):8120.
[29]Manuel R,Olaf G,Sarah K,et al.Schistosoma mansoni triggers Dectin-2,which activates the Nlrp3 inflammasome and alters adaptive immune responses[J].Proc Natl Acad Sci U S A,2010,107(47):20459-20464.
[30]Sebba A.Tocilizumab:the first interleukin-6-receptor inhibitor[J].Am J Health Syst Pharm,2008,65(15):1413-1418.
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