SO_2衍生物通过NLRP3炎症小体促进气道上皮细胞炎症发生
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摘要
目的:观察二氧化硫(SO_2)衍生物亚硫酸钠和亚硫酸氢钠对支气管上皮细胞NLRP3炎症小体活化的影响。方法:使用不同浓度的SO_2衍生物作用于支气管上皮细胞16HBE,通过流式细胞术检测细胞内活性氧簇(ROS)的生成,Western blot检测细胞内NLRP3和caspase-1 p20蛋白水平,ELISA检测细胞上清中白细胞介素1β(IL-1β)的分泌水平,结合细胞毒性实验(MTT)确定2 mmol/L为SO_2衍生物的实验浓度。采用RNA干扰技术沉默16HEB细胞NLRP3基因及ROS清除剂N-乙酰半胱氨酸(NAC)预处理16HBE细胞,通过流式细胞术检测细胞内ROS, Western blot和ELISA分别检测NLRP3和caspase-1 p20蛋白表达及IL-1β分泌水平。结果:与对照组比较, 2 mmol/L和4 mmol/L SO_2衍生物组细胞内ROS水平、 NLRP3和caspase-1 p20蛋白表达及细胞上清液中IL-1β水平明显升高(P<0.05)。与2 mmol/L SO_2衍生物组比较,NLRP3 siRNA组细胞内的NLRP3和caspase-1 p20蛋白水平明显降低(P<0.05),且细胞上清液中IL-1β的浓度明显下降(P<0.05),ROS无明显变化;NAC组NLRP3和caspase-1 p20蛋白水平及IL-1β浓度均明显下降(P<0.05)。结论:SO_2衍生物激活支气管上皮细胞NLRP3炎症小体,促进IL-1β生成。
AIM: To investigate the effect of sulfur dioxide(SO_2) derivatives(sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS: SO_2 derivatives at different concentrations were applied to bronchial epithelial 16 HBE cells for 12 h. The production of reactive oxygen species(ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO_2 derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16 HBE cells was silenced by RNA interference technique or N-acetyl cysteine(NAC) was used to pretreat 16 HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS: Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO_2 derivative groups(P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group(P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased(P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found(P<0.05). CONCLUSION: SO_2 derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.
AIM: To investigate the effect of sulfur dioxide(SO_2) derivatives(sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS: SO_2 derivatives at different concentrations were applied to bronchial epithelial 16 HBE cells for 12 h. The production of reactive oxygen species(ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO_2 derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16 HBE cells was silenced by RNA interference technique or N-acetyl cysteine(NAC) was used to pretreat 16 HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS: Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO_2 derivative groups(P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group(P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased(P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found(P<0.05). CONCLUSION: SO_2 derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.
引文
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[8] Samukawa T, Arasidani K, Hori H, et al. c-jun mRNA expression and profilin mRNA amplification in rat alveolar macrophages exposed to volcanic ash and sulfur dioxide[J]. Ind Health, 2003, 41(4):313-319.
[9] Li R, Meng Z. Effects of SO2 derivatives on expressions of MUC5AC and IL-13 in human bronchial epithelial cells[J]. Arch Toxicol, 2007, 81(12):867-874.
[10] Wigenstam E, Elfsmark L, Bucht A, et al. Inhaled sulfur dioxide causes pulmonary and systemic inflammation leading to fibrotic respiratory disease in a rat model of chemical-induced lung injury[J]. Toxicology, 2016, 368-369:28-36.
[11] Huang Y, Hua M, Cui X. Fungal β-glucan activates the NLRP3 inflammasome in human bronchial epithelial cells through ROS production[J]. Inflammation, 2018, 41(1):164-173.
[12] Yun Y, Hou L, Sang N. SO2 inhalation modulates the expression of pro-inflammatory and pro-apoptotic genes in rat heart and lung[J]. J Hazard Mater, 2011, 185(1):482-488.
[13] Hirota JA, Hirota SA, Warner SM, et al. The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter[J]. J Allergy Clin Immunol, 2012, 129(4):1116-1125.e6.
[14] Hirota JA, Gold MJ, Hiebert PR, et al. The nucleotide-binding domain, leucine-rich repeat protein 3 inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under 10 μm[J]. Am J Respir Cell Mol Biol, 2015, 52(1):96-105.
[15] Chen L, Huang W, Li W, et al. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome regulates bronchial epithelial cell injury and proapoptosis after exposure to biomass fuel smoke[J]. Am J Respir Cell Mol Biol, 2016, 55(6):815-824.
[2] Schroder K, Tschopp J. The inflammasomes[J]. Cell, 2010, 140(6):821-832.
[3] Kim RY, Pinkerton JW, Essilfie AT, et al. Role for NLRP3 inflammasome-mediated, IL-1β-dependent responses in severe, steroid-resistant asthma[J]. Am J Respir Crit Care Med, 2017, 196(3):283-297.
[4] Pouwels SD, van Geffen WH, Jonker MR, et al. Increased neutrophil expression of pattern recognition receptors during COPD exacerbations[J]. Respirology, 2017, 22(2):401-404.
[5] Li R, Meng Z, Xie J. Effects of sulfur dioxide on the expressions of MUC5AC and ICAM-1 in airway of asthmatic rats[J]. Regul Toxicol Pharmacol, 2007, 48(3):284-291.
[6] Zielinski M, Gasior M, Jastrzebski D, et al. Influence of gaseous pollutants on COPD exacerbations in patients with cardiovascular comorbidities[J]. Adv Exp Med Biol, 2018, 1114:11-17.
[7] Tam E, Miike R, Labrenz S, et al. Volcanic air pollution over the Island of Hawai’i: emissions, dispersal, and composition. Association with respiratory symptoms and lung function in Hawai’i Island school children[J]. Environ Int, 2016, 92-93:543-552.
[8] Samukawa T, Arasidani K, Hori H, et al. c-jun mRNA expression and profilin mRNA amplification in rat alveolar macrophages exposed to volcanic ash and sulfur dioxide[J]. Ind Health, 2003, 41(4):313-319.
[9] Li R, Meng Z. Effects of SO2 derivatives on expressions of MUC5AC and IL-13 in human bronchial epithelial cells[J]. Arch Toxicol, 2007, 81(12):867-874.
[10] Wigenstam E, Elfsmark L, Bucht A, et al. Inhaled sulfur dioxide causes pulmonary and systemic inflammation leading to fibrotic respiratory disease in a rat model of chemical-induced lung injury[J]. Toxicology, 2016, 368-369:28-36.
[11] Huang Y, Hua M, Cui X. Fungal β-glucan activates the NLRP3 inflammasome in human bronchial epithelial cells through ROS production[J]. Inflammation, 2018, 41(1):164-173.
[12] Yun Y, Hou L, Sang N. SO2 inhalation modulates the expression of pro-inflammatory and pro-apoptotic genes in rat heart and lung[J]. J Hazard Mater, 2011, 185(1):482-488.
[13] Hirota JA, Hirota SA, Warner SM, et al. The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter[J]. J Allergy Clin Immunol, 2012, 129(4):1116-1125.e6.
[14] Hirota JA, Gold MJ, Hiebert PR, et al. The nucleotide-binding domain, leucine-rich repeat protein 3 inflammasome/IL-1 receptor I axis mediates innate, but not adaptive, immune responses after exposure to particulate matter under 10 μm[J]. Am J Respir Cell Mol Biol, 2015, 52(1):96-105.
[15] Chen L, Huang W, Li W, et al. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome regulates bronchial epithelial cell injury and proapoptosis after exposure to biomass fuel smoke[J]. Am J Respir Cell Mol Biol, 2016, 55(6):815-824.
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