Sepsis-induced acute lung injury (ALI) accounts for about 40% of ALI, accompanied by alveolar epithelial injury. The study aimed to reveal the role of circular RNA_0114428 (circ_0114428) in sepsis-induced ALI. Human pulmonary alveolar epithelial cells (HPAEpiCs) were treated with lipopolysaccharide (LPS) to mimic a sepsis-induced ALI cell model. RNA expression of circ_0114428, miR-574-5p and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) was detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine (EdU) and flow cytometry analysis, respectively. The levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was analyzed by lipid peroxidation Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity detection assays. The interplay among circ_0114428, miR-574-5p and ROCK2 was identified by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. Circ_0114428 and ROCK2 expression were significantly increased, but miR-574-5p was decreased in blood samples from sepsis patients and LPS-stimulated HPAEpiCs. LPS treatment led to decreased cell viability and proliferation and increased cell apoptosis, inflammation and oxidative stress; however, these effects were relieved after circ_0114428 knockdown. Besides, circ_0114428 acted as a miR-574-5p sponge and regulated LPS-treated HPAEpiC disorders through miR-574-5p. Meanwhile, ROCK2 was identified as a miR-574-5p target, and its silencing protected against LPS-induced cell injury. Importantly, circ_0114428 knockdown inhibited ROCK2 production by interacting with miR-574-5p. Circ_0114428 knockdown protected against LPS-induced HPAEpiC injury through miR-574-5p/ROCK2 axis, providing a novel therapeutic target in sepsis-induced ALI.

中文翻译：

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Circ_0114428敲低抑制ROCK2表达，通过miR-574-5p减轻脂多糖诱导的人肺泡上皮细胞损伤

脓毒症引起的急性肺损伤(ALI)约占ALI的40%，并伴有肺泡上皮损伤。该研究旨在揭示环状 RNA_0114428 (circ_0114428) 在脓毒症诱发的 ALI 中的作用。用脂多糖 (LPS) 处理人肺泡上皮细胞 (HPAEpiCs)，以模拟脓毒症诱导的 ALI 细胞模型。通过 qRT-PCR 检测 circ_0114428、miR-574-5p 和含有蛋白激酶 2 (ROCK2) 的 Rho 相关卷曲螺旋的 RNA 表达。通过蛋白质印迹检查蛋白质表达。分别通过细胞计数试剂盒8、5-乙炔基-29-脱氧尿苷（EdU）和流式细胞术分析来研究细胞活力、增殖和凋亡。采用酶联免疫吸附试验（ELISA）检测促炎因子水平。通过脂质过氧化丙二醛 (MDA) 和超氧化物歧化酶 (SOD) 活性检测分析来分析氧化应激。通过双荧光素酶报告基因、RNA pull-down 和 RNA 免疫沉淀分析鉴定了 circ_0114428、miR-574-5p 和 ROCK2 之间的相互作用。在脓毒症患者和 LPS 刺激的 HPAEpiC 的血液样本中，Circ_0114428 和 ROCK2 表达显着增加，但 miR-574-5p 减少。LPS 处理导致细胞活力和增殖下降，细胞凋亡、炎症和氧化应激增加；然而，这些影响在 circ_0114428 击倒后得到缓解。此外，circ_0114428充当miR-574-5p海绵并通过miR-574-5p调节LPS治疗的HPAEpiC疾病。同时，ROCK2 被确定为 miR-574-5p 靶标，其沉默可以防止 LPS 诱导的细胞损伤。重要的是，circ_0114428 敲低通过与 miR-574-5p 相互作用抑制 ROCK2 的产生。Circ_0114428 敲低可通过 miR-574-5p/ROCK2 轴防止 LPS 诱导的 HPAEpiC 损伤，为脓毒症诱导的 ALI 提供新的治疗靶点。