Acute respiratory distress syndrome (ARDS) is a disease with high mortality and morbidity. Regulator of G protein signaling protein 6 (RGS6), identified as a tumor suppressor gene, has received increasing attention owing to its close relationship with oxidative stress and inflammation. However, the association between ARDS and RGS6 has not been reported. Congruously regulated G protein-coupled receptor (GPCR)-related genes and differentially expressed genes (DEGs) in an acute lung injury (ALI) model were identified, and functional enrichment analysis was conducted. In an in vivo study, the effects of RGS6 knockout were studied in a mouse model of ALI induced by lipopolysaccharide (LPS). HE staining, ELISA, and immunohistochemistry were used to evaluate pathological changes and the degree of inflammation. In vitro, qRT‒PCR, immunofluorescence staining, and western blotting were used to determine the dynamic changes in RGS6 expression in cells. The RGS6 overexpression plasmid was constructed for transfection. qRT‒PCR was used to assess proinflammatory factors transcription. Western blotting and flow cytometry were used to evaluate apoptosis and reactive oxygen species (ROS) production. Organoid culture was used to assess the stemness and self-renewal capacity of alveolar epithelial type II cells (AEC2s). A total of 110 congruously regulated genes (61 congruously upregulated and 49 congruously downregulated genes) were identified among GPCR-related genes and DEGs in the ALI model. RGS6 was downregulated in vivo and in vitro in the ALI model. RGS6 was expressed in the cytoplasm and accumulated in the nucleus after LPS stimulation. Compared with the control group, we found higher mortality, more pronounced body weight changes, more serious pulmonary edema and pathological damage, and more neutrophil infiltration in the RGS6 knockout group upon LPS stimulation in vivo. Moreover, AEC2s loss was significantly increased upon RGS6 knockout. Organoid culture assays showed slower alveolar organoid formation, fewer alveolar organoids, and impaired development of new structures after passaging upon RGS6 knockout. In addition, RGS6 overexpression decreased ROS production as well as proinflammatory factor transcription in macrophages and decreased apoptosis in epithelial cells. RGS6 plays a protective role in ALI not only in early inflammatory responses but also in endogenous lung stem cell regeneration.

中文翻译：

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G蛋白信号蛋白6的调节剂通过抑制炎症和促进细胞自我更新减轻急性肺损伤

急性呼吸窘迫综合征（ARDS）是一种死亡率和发病率较高的疾病。G蛋白信号蛋白6（RGS6）的调节因子被认为是一种抑癌基因，由于其与氧化应激和炎症的密切关系而受到越来越多的关注。然而，ARDS 和 RGS6 之间的关联尚未报道。鉴定了急性肺损伤（ALI）模型中一致调节的G蛋白偶联受体（GPCR）相关基因和差异表达基因（DEG），并进行了功能富集分析。在一项体内研究中，在脂多糖 (LPS) 诱导的 ALI 小鼠模型中研究了 RGS6 敲除的影响。采用HE染色、ELISA、免疫组化等方法评价病理变化及炎症程度。在体外，采用qRT-PCR、免疫荧光染色和蛋白质印迹法测定细胞中RGS6表达的动态变化。构建RGS6过表达质粒用于转染。qRT-PCR 用于评估促炎因子转录。使用蛋白质印迹和流式细胞术评估细胞凋亡和活性氧（ROS）的产生。使用类器官培养来评估 II 型肺泡上皮细胞 (AEC2) 的干细胞性和自我更新能力。ALI模型中GPCR相关基因和DEG中总共鉴定出110个一致调节基因（61个一致上调基因和49个一致下调基因）。RGS6 在 ALI 模型中体内和体外均下调。LPS刺激后，RGS6在细胞质中表达并在细胞核中积累。与对照组相比，我们发现在体内LPS刺激后，RGS6敲除组的死亡率更高，体重变化更明显，肺水肿和病理损伤更严重，中性粒细胞浸润更多。此外，RGS6 敲除后，AEC2 损失显着增加。类器官培养测定显示，RGS6 敲除传代后，肺泡类器官形成较慢、肺泡类器官较少，且新结构的发育受损。此外，RGS6过表达会减少巨噬细胞中ROS的产生和促炎因子转录，并减少上皮细胞的凋亡。RGS6 在 ALI 中不仅在早期炎症反应中发挥保护作用，而且在内源性肺干细胞再生中也发挥保护作用。