Department of Emergency Medicine, The Second Xiangya Hospital of Central South University, Emergency and Difficult Diseases Institute of Central South University, Changsha, Hunan, PR China.
Department of Respiratory Medicine, Hunan Center for Evidence-Based Medicine, Research Unit of Respiratory Diseases, The Second Xiangya Hospital of Central South University, Changsha, PR China.
Life Sci. 2023 Nov 15;333:122148. doi: 10.1016/j.lfs.2023.122148. Epub 2023 Oct 5.
To investigate the role and mechanisms of methyltransferase-like 3 (METTL3) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI).
LPS intratracheally instillation was applied in alveolar epithelial cell METTL3 conditional knockout (METTL3-CKO) mice and their wild-type littermates. In addition, METTL3 inhibitor STM2457 was used. LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP.
METTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. Knockdown of METTL3 alleviated, while overexpression of METTL3 exacerbated LPS-induced apoptosis. LPS treatment reduced neprilysin expression, the intervention of neprilysin expression negatively regulated apoptosis without affecting METTL3 expression, and mitigated the promoting effect of METTL3 on LPS-induced apoptosis. Additionally, METTL3 could bind to the mRNA of neprilysin, and reduce its expression.
Our findings revealed that inhibition of METTL3 could exert anti-apoptosis and ALI-protective effects via restoring neprilysin expression.
研究甲基转移酶样 3（METTL3）在脂多糖（LPS）诱导的急性肺损伤（ALI）发病机制中的作用和机制。
采用气管内滴注 LPS 法对肺泡上皮细胞 METTL3 条件性敲除（METTL3-CKO）小鼠及其野生型同窝仔鼠进行处理。此外，还使用了 METTL3 抑制剂 STM2457。应用 LPS 处理小鼠肺上皮细胞 12（MLE-12）细胞，建立 LPS 诱导的 ALI 体外模型。采用 H&E 染色、肺湿干重比和总支气管肺泡灌洗液（BALF）浓度评估肺损伤。采用 m6A RNA 甲基化定量试剂盒和斑点印迹法测定整体 m6A 水平。采用免疫组化、免疫荧光、免疫荧光-荧光原位杂交和 Western blot 测定 METTL3 和 Neprilysin 的表达。采用 TUNEL、Western blot 和流式细胞术检测细胞凋亡。采用 RIP-qPCR 和 MeRIP 测定 METTL3 和 Neprilysin 的相互作用。
LPS 处理的小鼠肺泡上皮细胞中 METTL3 表达和细胞凋亡增加，METTL3-CKO 或 METTL3 抑制剂 STM2457 可减轻细胞凋亡和 LPS 诱导的 ALI。在 MLE-12 细胞中，LPS 诱导 METTL3 表达和细胞凋亡。METTL3 敲低可减轻，而过表达则加剧 LPS 诱导的细胞凋亡。LPS 处理降低 Neprilysin 表达，Neprilysin 表达的干预可负调控凋亡而不影响 METTL3 表达，并减轻 METTL3 对 LPS 诱导的细胞凋亡的促进作用。此外，METTL3 可与 Neprilysin 的 mRNA 结合，降低其表达。
本研究结果表明，抑制 METTL3 通过恢复 Neprilysin 表达可发挥抗凋亡和 ALI 保护作用。
