核因子-κB信号通路在自来水有机提取物致L02细胞炎性损伤中的作用
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摘要
[目的]观察自来水有机提取物致正常人肝细胞株(L02细胞)炎性损伤中核因子-κB(NF-κB)信号通路的激活情况及其调控作用。[方法]采用固相萃取法提取自来水中的有机污染物。将L02细胞分别暴露于培养液(空白对照)、0.1%二甲基亚砜(溶剂对照)和0.312 5、0.625 0、1.250 0、2.500 0、5.000 0 L/m L自来水有机提取物中24、48、72 h。采用免疫印迹方法观察NF-κB信号通路的激活情况;并使用ELISA法测定细胞培养液上清中白介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的水平。[结果](1)L02细胞染毒24、48、72 h,细胞活力在各剂量组均明显降低(P<0.05)。(2)L02细胞染毒24、48、72 h,NF-κB抑制蛋白的表达水平在0.625 0 L/m L以上剂量组均明显降低(P<0.05);p65蛋白表达水平在染毒24、48 h时点,仅在1.250 0 L/m L以上剂量组明显升高(P<0.05),而在72 h时点,0.625 0 L/m L以上剂量组即明显升高(P<0.05)。(3)L02细胞染毒24、48 h后,0.625 0 L/m L以上处理组的IL-8水平均明显升高(P<0.05);而染毒72 h,低剂量组(0.312 5 L/m L)的IL-8水平即可升高明显(P<0.05);染毒24 h,1.250 0 L/m L以上处理组的TNF-α水平明显升高(P<0.05);而染毒48、72 h,TNF-α水平在0.625 0 L/m L以上处理组即升高明显(P<0.05)。与24 h染毒组比较,48、72 h各染毒组的IL-8与TNF-α水平均明显升高(P<0.05)。(4)IL-8、TNF-α水平与NF-κB抑制蛋白表达水平均呈负相关关系(r=-0.851 0、-0.818 0,P<0.05),与p65蛋白表达水平呈正相关关系(r=0.816 0、0.865 0,P<0.05)。[结论]自来水有机提取物可剂量-依赖性及时间-依赖性地激活NF-κB信号通路,诱导IL-8及TNF-α的分泌,此可能为其诱发L02细胞炎性损伤的重要原因之一。
[Objective] To observe the activation of nuclear factor-κB(NF-κB) signaling pathway and its inflammatoryregulating effects in human normal liver cells(L02 cells) exposed to organic extracts from tap water. [Methods] Organic pollutants in tap water samples were extracted by solid phase extraction method. L02 cells were divided into seven groups: blank control group(culture medium), solvent control group(0.1% dimethyl sulfoxide, DMSO), and organic pollutant groups(0.312 5, 0.625 0, 1.250 0, 2.500 0, and 5.000 0 L/m L). Each group was treated for 24 h, 48 h, and 72 h. Western blot was used to observe the activation of NF-κB signaling pathway. ELISA assay was used to detect interleukin 8(IL-8) and tumor necrosis factor α(TNF-α) levels in supernatant. [Results](1) The activity of L02 cells decreased obviously after treated with all designed dosages of organic extracts and at all designed exposure time blocks(P < 0.05).(2) The protein expression levels of inhibitor of NF-κB decreased obviously after exposure to the dosages at organic extracts of 0.625 0 L/m L and above for all designed time blocks(P < 0.05). The protein expression levels of p65 were remarkably higher after administered with the 1.250 0 L/m L organic extracts and above at 24 h and 48 h or with the 0.625 0 L/m L and above at 72h( P < 0.05).(3) After 24 h-and 48 h-exposure, the IL-8 level of L02 cells increased significantly in the 0.625 0 L/m L and above groups(P < 0.05); while after 72 h-exposure, the IL-8 level significantly increased in 0.312 5 L/m L group(P < 0.05). After 24 h-exposure, the TNF-α level of L02 cells increased significantly in the 1.250 0 L/m L and above groups(P < 0.05); while after 48 h-and 72 h-exposure, the TNF-α level increased significantly in the 0.625 0 L/m L and above groups(P < 0.05). Compared with 24 h-exposure, the IL-8 and TNF-α levels in all exposed groups with 48 h-and 72 h-exposure significantly increased(P < 0.05).(4) The IL-8 and TNF-α levels were negatively associated with the protein expression level of inhibitor of NF-κB(r=-0.851 0,-0.818 0, P < 0.05), and positively associated with the protein expressions level of p65(r=0.816 0, 0.865 0, P < 0.05). [Conclusion] The study findings suggest that organic pollutants from tap water could dose-dependently and time-dependently activate NF-κB signaling pathway and induce secretion of IL-8 and TNF-α, which might result in inflammatory injury in L02 cells.
[Objective] To observe the activation of nuclear factor-κB(NF-κB) signaling pathway and its inflammatoryregulating effects in human normal liver cells(L02 cells) exposed to organic extracts from tap water. [Methods] Organic pollutants in tap water samples were extracted by solid phase extraction method. L02 cells were divided into seven groups: blank control group(culture medium), solvent control group(0.1% dimethyl sulfoxide, DMSO), and organic pollutant groups(0.312 5, 0.625 0, 1.250 0, 2.500 0, and 5.000 0 L/m L). Each group was treated for 24 h, 48 h, and 72 h. Western blot was used to observe the activation of NF-κB signaling pathway. ELISA assay was used to detect interleukin 8(IL-8) and tumor necrosis factor α(TNF-α) levels in supernatant. [Results](1) The activity of L02 cells decreased obviously after treated with all designed dosages of organic extracts and at all designed exposure time blocks(P < 0.05).(2) The protein expression levels of inhibitor of NF-κB decreased obviously after exposure to the dosages at organic extracts of 0.625 0 L/m L and above for all designed time blocks(P < 0.05). The protein expression levels of p65 were remarkably higher after administered with the 1.250 0 L/m L organic extracts and above at 24 h and 48 h or with the 0.625 0 L/m L and above at 72h( P < 0.05).(3) After 24 h-and 48 h-exposure, the IL-8 level of L02 cells increased significantly in the 0.625 0 L/m L and above groups(P < 0.05); while after 72 h-exposure, the IL-8 level significantly increased in 0.312 5 L/m L group(P < 0.05). After 24 h-exposure, the TNF-α level of L02 cells increased significantly in the 1.250 0 L/m L and above groups(P < 0.05); while after 48 h-and 72 h-exposure, the TNF-α level increased significantly in the 0.625 0 L/m L and above groups(P < 0.05). Compared with 24 h-exposure, the IL-8 and TNF-α levels in all exposed groups with 48 h-and 72 h-exposure significantly increased(P < 0.05).(4) The IL-8 and TNF-α levels were negatively associated with the protein expression level of inhibitor of NF-κB(r=-0.851 0,-0.818 0, P < 0.05), and positively associated with the protein expressions level of p65(r=0.816 0, 0.865 0, P < 0.05). [Conclusion] The study findings suggest that organic pollutants from tap water could dose-dependently and time-dependently activate NF-κB signaling pathway and induce secretion of IL-8 and TNF-α, which might result in inflammatory injury in L02 cells.
引文
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[2]Ghosh S,Karin M.Missing pieces in the NF-kappa B puzzle[J].Cell,2002,109(Suppl):S81-96.
[3]O’Neil L A.The IL-1 receptor/Toll-like receptor superfamily:crucial receptors for inflammation and host defense[J].Immunol Today,2000,21(5):206-209.
[4]Medzhitov R.Toll-like receptors and innate immunity[J].Nat Rev Immunol,2001,1(2):135-145.
[5]杨光红,桂晓玲,岑延利,等.饮用水有机提取物致大鼠肝脏损伤中GSTs活性及其编码基因表达的变化[J].环境与职业医学,2015,32(3):206-210.
[6]陈曦.P53非依赖性信号通路在邻苯二甲酸二(2-乙基已基)酯致体外培养人肝细胞毒性中的调控作用[D].武汉:华中科技大学,2011.
[7]杨光红,王士然,张爱华,等.G市某区管网末梢水中有机提取物对雄性大鼠生殖激素分泌水平的影响[J].环境与职业医学,2013,30(5):342-345.
[8]杨光红,张爱华,王士然,等.常规处理工艺对地表水有机提取物遗传毒性的影响[J].环境与健康杂志,2013,30(9):799-801.
[9]赵淑华,隋春生,王春华,等.饮用水有机提取物对正常人肝HL-7702细胞的毒性[J].环境与健康杂志,2011,28(11):984-986.
[10]Kim S,Takahashi H,Lin W W,et al.Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis[J].Nature,2009,457(7225):102-106.