南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (2): 181-189.doi: 10.12122/j.issn.1673-4254.2022.02.03

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抑制TAK1可加重甲苯二异氰酸酯诱导的哮喘小鼠气道炎症

杨淑銮，赵文驱，彭显如，蓝紫涵，黄俊文，韩慧珊，陈 颖，蔡绍曦，赵海金   

  1. 南方医科大学南方医院呼吸与危重症医学科，慢性气道疾病实验室，广东 广州 510515
  • 出版日期:2022-02-20 发布日期:2022-03-17

Inhibition of TAK1 aggravates airway inflammation by increasing RIPK1 activity and promoting macrophage death in a mouse model of toluene diisocyanate-induced asthma

YANG Shuluan, ZHAO Wenqu, PENG Xianru, LAN Zihan, HUANG Junwen, HAN Huishan, CHEN Ying, CAI Shaoxi, ZHAO Haijin   

  1. Laboratory of Chronic Airway Diseases, Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Online:2022-02-20 Published:2022-03-17

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摘要： 目的 探讨转化生长因子β激活激酶1（TAK1）对甲苯二异氰酸酯（TDI）哮喘小鼠气道炎症的影响。方法 建立TDI哮喘小鼠模型，为探讨TAK1在TDI哮喘模型中的作用，将32只小鼠随机分为4组（8只/组）：AOO组，AOO+5Z-7-Oxozeaenol组，TDI组，TDI+5Z-7-Oxozeaenol组；为进一步探讨RIPK1在体内模型的作用，将另外32只小鼠随机分为4组（8只/组）：AOO组，TDI组，TDI+5Z-7-Oxozeaenol组，TDI+5Z-7-Oxozeaenol+Necrostatin-1组。每次激发前使用TAK1抑制剂（5Z-7-Oxozeaenol，5 mg/kg）和/或RIPK1抑制剂（Necrostatin-1,5 mg/kg）。检测各组小鼠气道反应性、气道炎症及气道重塑等指标。体外实验：配制TDI-人血清白蛋白（TDI-HSA）复合物联合TAK1抑制剂处理小鼠单核巨噬细胞（RAW264.7），通过CCK8检测各组细胞活力，通过Western blot检测各组TAK1、丝裂原活化蛋白激酶（MAPK）及受体相互作用丝/苏氨酸蛋白酶1（RIPK1）相关信号通路分子表达情况；使用RIPK1抑制剂进一步评估对细胞活力及TDI哮喘模型的影响。结果 与单独TDI致敏激发哮喘小鼠相比，TAK1抑制剂加重了小鼠气道炎症、气道高反应性及气道重塑（P<0.05）。抑制TAK1降低了RAW264.7细胞活力，与TDI-HSA共同处理进一步降低（P<0.05）。抑制TAK1降低了TDI-HSA诱导的TAK1磷酸化水平及MAPK信号通路的活化（P<0.05）。TAK1抑制剂与TDI-HSA共同处理后，RIPK1磷酸化水平升高，同时Caspase 8持续活化（P<0.05）；使用RIPK1抑制剂可以恢复TAK1抑制剂与TDI-HSA共同处理引起的细胞活力的减低（P<0.05）。体内RIPK1抑制剂亦可以减轻TAK1抑制剂对TDI哮喘小鼠气道炎症的加重（P<0.05）。结论 抑制TAK1可能通过增加RIPK1的活性，引起Caspase 8的持续活化而增加巨噬细胞的死亡，参与促进TDI诱导的哮喘小鼠气道炎症。

关键词: 甲苯二异氰酸酯；哮喘；转化生长因子β激活激酶1；受体相互作用丝/苏氨酸蛋白酶1；丝裂原活化蛋白激酶

Abstract: Objective To explore the effect of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) on toluene diisocyanate (TDI)-induced allergic airway inflammation in mice. Methods Thirty-two mice were randomly divided into AOO group, AOO+5Z-7-Oxozeaenol group, TDI group, and TDI+5Z-7-Oxozeaenol group. Another 32 mice were randomly divided into AOO group, TDI group, TDI+5Z-7-Oxozeaenol group, and TDI+5Z-7-Oxozeaenol+Necrostatin-1 group. TAK1 inhibitor (5Z-7-Oxozeaenol, 5 mg/kg) and/or RIPK1 inhibitor (Necrostatin-1, 5 mg/kg) were used before each challenge. Airway responsiveness, airway inflammation and airway remodeling were assessed after the treatments. We also examined the effect of TDI-human serum albumin (TDI-HSA) conjugate combined with TAK1 inhibitor on the viability of mouse mononuclear macrophages (RAW264.7) using CCK8 assay. The expressions of TAK1, mitogen-activated protein kinase (MAPK) and receptor interacting serine/threonine protease 1 (RIPK1) signal pathway in the treated cells were detected with Western blotting. The effects of RIPK1 inhibitor on the viability of RAW264.7 cells and airway inflammation of the mouse models of TDI-induced asthma were evaluated. Results TAK1 inhibitor aggravated TDI-induced airway inflammation, airway hyper responsiveness and airway remodeling in the mouse models (P<0.05). Treatment with TAK1 inhibitor significantly decreased the viability of RAW264.7 cells, which was further decreased by co-treatment with TDI-HSA (P<0.05). TAK1 inhibitor significantly decreased the level of TAK1 phosphorylation and activation of MAPK signal pathway induced by TDI-HSA (P<0.05). Co-treatment with TAK1 inhibitor and TDI-HSA obviously increased the level of RIPK1 phosphorylation and caused persistent activation of caspase 8 (P<0.05). RIPK1 inhibitor significantly inhibited the reduction of cell viability caused by TAK1 inhibitor and TDI-HSA (P<0.05) and alleviated the aggravation of airway inflammation induced by TAK1 inhibitors in TDI-induced mouse models (P<0.05). Conclusion Inhibition of TAK1 aggravates TDI-induced airway inflammation and hyperresponsiveness and may increase the death of macrophages by enhancing the activity of RIPK1 and causing persistent activation of caspase 8.

Key words: toluene diisocyanate; asthma; transforming growth factor-β-activated kinase 1; receptor interaction serine/threonine protease 1; mitogen-activated protein kinase