安徽医科大学学报

目的 探讨DExD/H框RNA解旋酶58（DHX58）在急性呼吸窘迫综合征（ARDS）中的表达变化及其对巨噬细胞活化的调控机制，明确DHX58是否通过NOD样受体家族蛋白3（NLRP3）炎症小体介导ARDS炎症反应。方法 将C57BL/6J小鼠随机分为正常组和ARDS模型组，每组5只。通过气管滴注脂多糖（LPS）构建ARDS模型，采用HE染色、肺损伤病理评分、肺湿干比（W/D）及肺泡灌洗液（BALF）总蛋白浓度评估肺损伤；Western blot与qRT-PCR检测肺组织中DHX58的表达水平，免疫荧光双染检测DHX58与巨噬细胞标志物F4/80的共定位情况。利用GSE数据库分析LPS刺激后人肺泡巨噬细胞中DHX58的表达变化。通过气管内滴注Dhx58-shRNA腺病毒进行体内基因沉默，观察其对ARDS肺损伤的影响。体外培养小鼠肺泡巨噬细胞MH-S，采用Dhx58-shRNA沉默DHX58表达，qRT-PCR、Western blot及ELISA检测沉默效率及促炎细胞因子白细胞介素(IL)-6、IL-1β的表达变化。进一步检测沉默DHX58对NLRP3表达的影响，并在沉默DHX58的基础上加入NLRP3特异性激动剂BMS-986299，观察其对巨噬细胞炎症反应的逆转作用。结果 成功构建ARDS小鼠模型，模型组肺组织呈典型病理改变， 肺损伤病理评分、W/D比值及BALF总蛋白浓度均升高。与对照组相比，ARDS小鼠肺组织中DHX58的表达水平升高（P<0.05），且DHX58阳性信号与F4/80阳性信号存在部分共定位。GSE数据分析显示，LPS刺激的人肺泡巨噬细胞中DHX58 mRNA表达水平高于对照组（P<0.05）。体内沉默Dhx58可减轻LPS诱导的肺组织病理损伤，降低肺损伤病理评分、W/D比值及BALF中IL-1β、IL-6水平（P<0.01）。体外实验说明，Dhx58-shRNA可有效沉默MH-S细胞中DHX58的表达；沉默DHX58后，IL-6和IL-1β的mRNA、蛋白及细胞上清分泌水平均下调（P<0.05）。机制研究显示，沉默DHX58可降低NLRP3的蛋白及mRNA表达水平（P<0.05）；而加入NLRP3激动剂BMS-986299后，可部分逆转沉默DHX58所致的IL-6和IL-1β下调（P<0.05）。结论 DHX58在ARDS小鼠肺组织及LPS刺激的巨噬细胞中表达上调，且主要定位于肺泡巨噬细胞。体内及体外沉默DHX58均可抑制巨噬细胞活化及炎症因子释放，其机制与下调NLRP3炎症小体表达有关。

Objective To investigate the expression changes of DExD/H-box RNA helicase 58 (DHX58) in acute respiratory distress syndrome (ARDS) and its regulatory mechanism in macrophage activation， and to determine whether DHX58 mediates ARDS inflammatory responses through the NOD-like receptor family protein 3 (NLRP3) inflammasome. Methods: C57BL/6J mice were randomly divided into a control group and an ARDS model group. ARDS was induced by intratracheal instillation of lipopolysaccharide (LPS). Lung injury was assessed by hematoxylin and eosin (HE) staining， lung injury pathological score， lung wet/dry weight ratio (W/D)， and total protein concentration in bronchoalveolar lavage fluid. The expression of DHX58 in lung tissue was detected by western blot and qRT-PCR， and co-localization of DHX58 with the macrophage marker F4/80 was examined by immunofluorescence double staining. The GSE database was used to analyze changes in DHX58 expression in human alveolar macrophages after LPS stimulation. In vivo gene silencing was performed by intratracheal administration of Dhx58-shRNA adenovirus to evaluate its effect on ARDS-induced lung injury. Mouse alveolar macrophage MH-S cells were cultured in vitro， and Dhx58-shRNA was used to silence DHX58 expression. The knockdown efficiency and the expression changes of pro-inflammatory cytokines IL-6 and IL-1β were measured by qRT-PCR， western blot and ELISA. The effect of DHX58 silencing on NLRP3 expression was further examined， and the NLRP3-specific agonist BMS-986299 was added on the basis of DHX58 silencing to observe its reversal effect on the macrophage inflammatory response. Results: The ARDS mouse model was successfully established. Lung tissues in the model group showed typical pathological changes， and the lung injury score， W/D ratio and BALF total protein concentration were all significantly increased. Compared with the control group， the expression of DHX58 in lung tissue was significantly elevated in ARDS mice (P < 0.05)， and partial co-localization of DHX58-positive signals with F4/80-positive signals was observed. GSE data analysis showed that DHX58 mRNA expression was significantly higher in LPS-stimulated human alveolar macrophages than in the control group (P < 0.05). In vivo silencing of Dhx58 significantly alleviated LPS-induced lung histopathological damage， and reduced the lung injury score， W/D ratio， and BALF levels of IL-1β and IL-6 (P < 0.01). In vitro experiments confirmed that Dhx58-shRNA effectively silenced DHX58 expression in MH-S cells. After DHX58 silencing， the mRNA， protein and supernatant secretion levels of IL-6 and IL-1β were all significantly downregulated (P< 0.05). Mechanistic studies showed that silencing DHX58 markedly decreased the protein and mRNA expression of NLRP3 (P < 0.05); furthermore， addition of the NLRP3 agonist BMS-986299 partially reversed the downregulation of IL-6 and IL-1β caused by DHX58 silencing (P< 0.05). Conclusion: DHX58 is upregulated in the lung tissue of ARDS mice and in LPS-stimulated macrophages， and is mainly localized in alveolar macrophages. Both in vivo and in vitro silencing of DHX58 inhibits macrophage activation and the release of inflammatory cytokines， and the mechanism is associated with downregulation of NLRP3 inflammasome expression.