亚砷酸钠对人胚肺成纤维细胞核因子κB信号通路的影响及其作用机制
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摘要
目的探讨核因子κB(NF-κB)信号通路在亚砷酸钠染毒致人胚肺成纤维细胞(HELF)损伤中的作用机制。方法将HELF细胞分别暴露于0(对照)、0.625、1.25、2.5、5、10、20、40μmol/L亚砷酸钠溶液染毒18、24、48 h,采用MTT法检测细胞存活率。将HELF细胞分别暴露于0(对照)、0.625、1.25、2.5、5、10、20、40μmol/L亚砷酸钠溶液染毒24 h,采用ELISA法检测上清液中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-1β(IL-1β)炎性因子的浓度。将HELF细胞分为2组,一组分别暴露于0(对照)、2.5、5、10、20μmol/L的亚砷酸钠溶液,另一组同时加入10μmol/L的NF-κB抑制剂PDTC溶液,染毒24 h后,采用Western blot法检测细胞NF-κB相关蛋白p-IKKβ和p-P65的表达水平。结果与对照组比较,2.5、5、10、20、40μmol/L亚砷酸钠染毒HELF细胞18、24、48 h后的细胞存活率均下降,除2.5 mol/L亚砷酸钠染毒48 h外,差异均有统计学意义(P<0.05);且随着亚砷酸钠染毒浓度的升高和染毒时间的延长,各染毒时间HELF细胞的存活率均呈下降趋势(均P<0.01)。与对照组比较,1.25、2.5、5、10、20、40μmol/L亚砷酸钠染毒组HELF细胞上清液中TNF-α的浓度均升高,5、10、20、40μmol/L亚砷酸钠染毒组HELF细胞上清液中IL-6的浓度均升高,各浓度亚砷酸钠染毒组HELF细胞上清液中IL-1β的浓度均升高,差异均有统计学意义(P<0.05);且随着亚砷酸钠染毒浓度的升高,HELF细胞上清液中TNF-α、IL-6、IL-1β的浓度均呈上升趋势。与对照组和相同浓度PDTC干预组比较,各浓度亚砷酸钠染毒组HELF细胞p-IKKβ和p-P65蛋白的表达水平均升高,差异有统计学意义(P<0.05)。结论亚砷酸钠暴露可激活NF-κB通路,促使炎性因子增加,从而造成HELF细胞损伤。
Objective To investigate the mechanism of nuclear factor κB(NF-κB) signaling pathway in the damage of human embryonic lung fibroblasts(HELF) induced by sodium arsenite. Methods HELF cells were exposed to sodium arsenite at the doses of 0(control group),0.625,1.25,2.5,5,10,20 and 40 μmol/L respectively for 24 h,and ELISA was used to detect concentration of inflammatory factors TNF-α,IL-6 and IL-1β in the supernatant. The HELF cells were divided into two groups,one group was exposed to sodium arsenite at the doses of 0(control group),2.5,5,10 and 20 μmol/L,and the other group was added 10 μmol/L pyrrolidine dithiocarbamate(PDTC),the inhibitor of NF-κB,and exposed to the same doses of sodium arsenite. After 24 h of exposure,the expression levels of NF-κB-related proteins p-IKKβ and p-P65 were detected by Western blot. Results Compared with the control group,the concentration of TNF-α in the supernatant of HELF cells exposed to 1.25-40 μmol/L sodium arsenite increased,the concentration of IL-6 in 5-40 μmol/L sodium arsenite groups increased,the concentration of IL-1β in all sodium arsenite exposure groups increased(P<0.05) with a dose dependent trend. Compared with the control group and the PDTC intervention group exposed to the same concentration of sodium arsenite,the expression level of p-IKKβ and p-P65 protein in HELF cells in all sodium arsenite exposure groups increased significantly(P<0.05). Conclusion Sodium arsenite may activate the NF-κB signaling pathway and promote inflammatory factors production,resulting in HELF cell damage.
Objective To investigate the mechanism of nuclear factor κB(NF-κB) signaling pathway in the damage of human embryonic lung fibroblasts(HELF) induced by sodium arsenite. Methods HELF cells were exposed to sodium arsenite at the doses of 0(control group),0.625,1.25,2.5,5,10,20 and 40 μmol/L respectively for 24 h,and ELISA was used to detect concentration of inflammatory factors TNF-α,IL-6 and IL-1β in the supernatant. The HELF cells were divided into two groups,one group was exposed to sodium arsenite at the doses of 0(control group),2.5,5,10 and 20 μmol/L,and the other group was added 10 μmol/L pyrrolidine dithiocarbamate(PDTC),the inhibitor of NF-κB,and exposed to the same doses of sodium arsenite. After 24 h of exposure,the expression levels of NF-κB-related proteins p-IKKβ and p-P65 were detected by Western blot. Results Compared with the control group,the concentration of TNF-α in the supernatant of HELF cells exposed to 1.25-40 μmol/L sodium arsenite increased,the concentration of IL-6 in 5-40 μmol/L sodium arsenite groups increased,the concentration of IL-1β in all sodium arsenite exposure groups increased(P<0.05) with a dose dependent trend. Compared with the control group and the PDTC intervention group exposed to the same concentration of sodium arsenite,the expression level of p-IKKβ and p-P65 protein in HELF cells in all sodium arsenite exposure groups increased significantly(P<0.05). Conclusion Sodium arsenite may activate the NF-κB signaling pathway and promote inflammatory factors production,resulting in HELF cell damage.
引文
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[4]Abernathy CO,Thomas DJ,Calderon RL.Health effects and risk assessment of arsenic[J].J Nutr,2003,133:1536-1538.
[5]Yoshida T,Yamauchi H,Sun GF.Chronic health effects in people exposed to arsenic via the drinking water:dose-response relationships in review[J].Toxicol Appl Pharmacol,2004,198:243-252.
[6]Aggarwall BB.Nuclear factor-kappaB:the enemy within cancer[J].Cancer Cell,2004,6:203-208.
[7]高艳芳,郭有,欧阳璐,等.亚砷酸钠致人肝细胞凋亡及NF-κB信号通路影响的研究[J].毒理学杂志,2016,30(5):345-348.
[8]刘世宜,胡楠楠,蔡思斯,等.亚砷酸钠对正常人类皮肤角质形成细胞蛋白激酶B及其下游信号因子IκB和NF-κB蛋白表达的影响[J].环境与健康杂志,2012,29(8):700-703.
[9]黄浩杰.转录因子NF-kB信号通路在胰腺炎发生中的机制研究[D].上海:第二军医大学,2012.
[10]苏鑫,王素华,刘建国,等.亚砷酸钠致雄性大鼠肺损伤机制探讨[J].中国职业医学,2016,43(2):143-147.
[11]Tchounwou PB,Patlolla AK,Centeno JA.Invited reviews:carcinogenic and systemic health effects associated with arsenic exposure:a critical review[J].Toxicol Pathol,2003,31:575-588.
[12]Xu Y,Li Y,Pang Y,et al.Blockade of p53 by HIF-2α,but not HIF-1α,is involved in arsenite-induced malignant transformation of human bronchial epithelial cells[J].Arch Toxicol,2012,86:947-959.
[13]Chen ZH,Lam HC,Jin Y,et al.Autophagy protein microtubuleassociated protein1 light chain-3B(LC3B)activates extrinsic apoptosis during cigarette smoke-induced emphysema[J].Proc Natl Acad Sci USA,2010,107:1880-1885.
[14]杨航.IL-6与NF-KB在炎症相关性大肠癌发生中的作用[D].郑州:郑州大学,2014.
[15]谢蓉,董文.核因子-κB的活化及其在急性肺损伤中的作用[J].福建医药杂志,2004,26(1):112-114.
[16]Duprez L,Wirawan E,Berghe TV,et al.Major cell death pathways at a glance[J].Microbes Infect,2009,11:1050-1062.
[17]Van Gammeren D,Damrauer JS,Jackman RW,et al.The IκB kinases IKKαand IKKβare necessary and sufficient for skeletal muscle atrophy[J].FASEB J,2009,23:362-370.
[18]苏剑东,吴灵飞.NF-kB与细胞凋亡[J].世界华人消化杂志,2007,15(12):1411-1416.
[19]Xiao G.Autophagy and NF-κB:fight for fate[J].Cytokine Growth Factor Rev,2007,18:233-243.