布地奈德和乙酰半胱氨酸联合治疗通过 miR-381/NLRP3 分子轴缓解 LPS 诱导的急性肺损伤。
Combination therapy with budesonide and acetylcysteine alleviates LPS-induced acute lung injury via the miR-381/NLRP3 molecular axis.
机构信息
Department of Rehabilitation Medicine, Qujing No.1 Hospital, Qujing, Yunnan, China.
出版信息
PLoS One. 2023 Aug 9;18(8):e0289818. doi: 10.1371/journal.pone.0289818. eCollection 2023.
BACKGROUND
Acute lung injury (ALI) usually has a high morbidity and mortality rate, but the current treatment is relatively scarce. Both budesonide (Bud) and N-acetylcysteine (NAC) exhibit protective effects in ALI, so we further investigated whether they have a synergistic effect on ALI when used together.
METHODS
Establishment of a rat model of ALI with Lipopolysaccharide (LPS). Bud and NAC were administered by nebulized inhalation alone or in combination. Subsequently, HE staining was performed to observe the pathological changes in lungs of rat. Evans blue staining was implemented to assess alveolar permeability, and the pulmonary edema was assessed by measuring the ratio of wet to dry weight of the lung. Moreover, a TUNEL kit was served to test apoptosis in lung tissues. Western blot and immunohistochemistry were analyzed for expression of scorch-related proteins and NLRP3 in lung tissue, respectively. ELISA was implemented to detect inflammatory factor levels in BALF. and RT-qPCR was utilized to assess the expression level of miR-381. After stable transfection of miR-381 inhibitor or OE-NLRP3 in BEAS-2B treated with LPS, Bud and NAC, miR-381 expression was assessed by RT-qPCR, scorch death-related protein expression was measured by western blot, cell proliferation/viability was assayed by CCK-8, apoptosis was measured by flow cytometry, and ELISA was implemented to assess inflammatory factor levels. Furthermore, the Dual-luciferase assay was used to verify the targeting relationship.
RESULTS
Bud and NAC treatment alone or in combination with nebulized inhalation attenuated the increased alveolar permeability, pulmonary edema, inflammatory response and scorching in LPS-induced ALI rats, and combined treatment with Bud and NAC was the most effective. In addition, combined treatment with Bud and NAC upregulated miR-381 expression and inhibited NLRP3 expression in cellular models and LPS-induced ALI rats. Transfection of the miR-381 inhibitor and OE-NLRP3 partially reversed the protective effects of Bud and NAC combination treatment on BEAS-2B cell proliferation inhibition, apoptosis, focal death and the inflammatory response.
CONCLUSION
Combined Bud and NAC nebulization therapy alleviates LPS-induced ALI by modulating the miR-381/NLRP3 molecular axis.
背景
急性肺损伤（ALI）通常具有较高的发病率和死亡率，但目前的治疗方法相对较少。布地奈德（Bud）和 N-乙酰半胱氨酸（NAC）在 ALI 中均表现出保护作用，因此我们进一步研究了当两者联合使用时是否对 ALI 有协同作用。
方法
使用脂多糖（LPS）建立大鼠 ALI 模型。单独或联合使用雾化吸入布地奈德和 N-乙酰半胱氨酸。随后，进行 HE 染色观察大鼠肺脏的病理变化。进行伊文思蓝染色评估肺泡通透性，通过测量肺湿重与干重的比值评估肺组织水肿。此外，使用 TUNEL 试剂盒检测肺组织中的细胞凋亡。通过 Western blot 和免疫组织化学分别分析肺组织中 scorch 相关蛋白和 NLRP3 的表达。通过 ELISA 检测 BALF 中的炎症因子水平，通过 RT-qPCR 评估 miR-381 的表达水平。在 LPS 处理的 BEAS-2B 中转染 miR-381 抑制剂或 OE-NLRP3 后，使用 Bud 和 NAC 进行稳定转染，通过 RT-qPCR 评估 miR-381 的表达，通过 Western blot 测量 scorch 死亡相关蛋白的表达，通过 CCK-8 测定细胞增殖/活力，通过流式细胞术测量细胞凋亡，通过 ELISA 测定炎症因子水平。此外，还使用双荧光素酶报告基因实验验证了靶向关系。
结果
布地奈德和 NAC 单独或联合雾化吸入治疗均可减轻 LPS 诱导的 ALI 大鼠肺泡通透性增加、肺水肿、炎症反应和 scorch，且联合治疗效果最显著。此外，联合布地奈德和 NAC 治疗可上调细胞模型和 LPS 诱导的 ALI 大鼠中 miR-381 的表达，并抑制 NLRP3 的表达。转染 miR-381 抑制剂和 OE-NLRP3 部分逆转了 Bud 和 NAC 联合治疗对 BEAS-2B 细胞增殖抑制、凋亡、焦亡和炎症反应的保护作用。
结论
联合雾化布地奈德和 NAC 治疗通过调节 miR-381/NLRP3 分子轴缓解 LPS 诱导的 ALI。
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