Mechanism of miR-16-5p alleviates airway inflammation in asthma rats by targeting the TXNIP/NLRP3 signaling axis

Objective To investigate the mechanism of miR-16-5p alleviates ovalbumin (OVA)-induced airway inflammation in asthmatic rats by targeting thioredoxin-interacting protein (TXNIP)/NOD-like receptor thermoprotein structural domain-related protein 3 (NLRP3) signaling axis.Methods Male Sprague-Dawley rats were randomly divided into the control, model, miR-16-5p mimic, NC mimic, miR-16-5p inhibitor, and NC inhibitor groups, with 6 rats in each group. A mixture of 10% OVA and 10% aluminum hydroxide was used for sensitization, and 4% OVA was given by nebulized inhalation to stimulate asthma. Dual luciferase reporter assay was performed to verify the target binding relationship; microscopic was used to observe inflammatory cell numbers in bronchoalveolar lavage fluid (BALF); ELISA was used to detect IL-1β, IL-6, TNF-α, and MCP-1 levels; H&E staining was used to observe morphological changes of lung tissue; Masson staining was used to observe bronchial collagen deposition; Immunohistochemical staining was used to detect TXNIP and NLRP3 expression; Western blot was used to detect TXNIP, NLRP3, ASC, cleaved-caspase-1, and IL-1β proteins expression.Results Luciferase reporter assay showed that miR-16-5p could target and regulate TXNIP. Compared with the control group, the model group rats had damaged lung tissues, thickened airway walls, and massive collagen deposition; an increased number of inflammatory cells, significantly higher expression levels of IL-1β, IL-6, TNF-α and MCP-1 (p < 0.05); TXNIP, NLRP3, ASC, cleaved-caspase-1, and IL-1β protein expression levels were significantly elevated in lung tissues (p < 0.05). Compared with the model group, lung histopathological damage was significantly improved in asthmatic rats after overexpression of miR-16-5b, and collagen deposition was reduced; the number of inflammatory cells was decreased; the levels of IL-1β, IL-6, TNF-α and MCP-1 were significantly reduced (p < 0.05); the levels of TXNIP, NLRP3, cleaved-caspase-1, and IL-1β protein expression levels were significantly reduced (p < 0.05). MiR-16-5p inhibitor further aggravated the airway inflammatory response in asthmatic rats.Conclusion miR-16-5p can alleviate airway inflammation in asthmatic rats by targeting the TXNIP/NLRP3 signaling axis.

目的 探讨miR-16-5p（microRNA-16-5p）通过靶向硫氧还蛋白相互作用蛋白（TXNIP）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）信号轴，缓解卵清蛋白（OVA）诱导的哮喘大鼠气道炎症的作用机制。方法 将雄性Sprague-Dawley大鼠随机分为对照组、模型组、miR-16-5p模拟物组、阴性对照（NC）模拟物组、miR-16-5p抑制剂组及阴性对照（NC）抑制剂组，每组6只。采用10% OVA与10%氢氧化铝的混合液进行致敏，随后以4% OVA雾化吸入激发哮喘模型。采用双荧光素酶报告基因实验验证靶标结合关系；采用显微镜观察支气管肺泡灌洗液（BALF）中炎性细胞数量；采用酶联免疫吸附试验（ELISA）检测IL-1β、IL-6、TNF-α及MCP-1水平；采用苏木精-伊红（H&E）染色观察肺组织形态学变化；采用马松（Masson）染色观察支气管胶原沉积情况；采用免疫组织化学染色检测TXNIP与NLRP3的表达水平；采用蛋白质印迹法（Western blot）检测TXNIP、NLRP3、ASC、裂解型caspase-1及IL-1β蛋白的表达水平。结果 双荧光素酶报告基因实验证实，miR-16-5p可靶向调控TXNIP。与对照组相比，模型组大鼠肺组织损伤明显，气道壁增厚，胶原沉积显著增多；支气管肺泡灌洗液中炎性细胞数量增加，IL-1β、IL-6、TNF-α及MCP-1水平均显著升高（P<0.05）；肺组织中TXNIP、NLRP3、ASC、裂解型caspase-1及IL-1β蛋白表达水平均显著上调（P<0.05）。与模型组相比，过表达miR-16-5p后，哮喘大鼠肺组织病理损伤显著改善，胶原沉积减少；炎性细胞数量降低；IL-1β、IL-6、TNF-α及MCP-1水平均显著下降（P<0.05）；肺组织中TXNIP、NLRP3、裂解型caspase-1及IL-1β蛋白表达水平均显著降低（P<0.05）。miR-16-5p抑制剂可进一步加重哮喘大鼠的气道炎症反应。结论 miR-16-5p可通过靶向TXNIP/NLRP3信号轴，缓解卵清蛋白诱导的哮喘大鼠气道炎症。