背景:急性肺损伤(ALI)表现为肺内血管通透性增加以及随后的肺泡气体交换受损。甲基强的松龙(MP)通常用作ALI的治疗以减轻炎症,但其分子机制尚不清楚。本研究旨在探讨MP在脂多糖(LPS)诱导的ALI模型中的作用机制。
方法:增殖,生存能力,凋亡,和miR-151-5p在肺泡Ⅱ型上皮细胞(AECII)中的表达,膜联蛋白V/PI凋亡试剂盒,计数试剂盒-8(CCK-8)测定,和RT-qPCR。Westernblot分析用于检测Usp38蛋白水平。ELISA法测定IL-6和TNF-α。miR-151-5p和USP38的组合通过染色质免疫沉淀(ChIP)-PCR和双荧光素酶报告基因测定来确定。
结果:MP大大改善了体内肺功能,减少炎症,并在体外促进肺泡Ⅱ型上皮细胞(AECII)的增殖。通过比较MP治疗组和对照组肺组织中microRNA的变化,我们发现miR-151-5p在LPS处理的AECII后表现出显著的增加,但MP治疗后下降。通过荧光素酶报告基因测定证实,USP38,被确定为miR-151-5p的下游靶标,发现MP给药后增加。在AECII中抑制miR-151-5p或USP38过表达显著提高了抗炎症,抗凋亡,和MP的增殖促进作用。
结论:总之,我们的数据表明,MP减轻了LPS诱导的AECII的炎症和凋亡,并通过miR-151-5p抑制和随后的USP38激活部分促进AECII的增殖。
BACKGROUND: Acute lung injury (ALI) is manifested by increased blood vessel permeability within the lungs and subsequent impairment of alveolar gas exchange. Methylprednisolone (MP) is commonly used as a treatment for ALI to reduce inflammation, yet its molecular mechanism remains unclear. This study aims to explore the underlying mechanisms of MP on ALI in a model induced by lipopolysaccharide (LPS).
METHODS: The proliferation, viability, apoptosis, and miR-151-5p expression of alveolar type II epithelial cells (
AECII) were detected using the cell EdU assay, Annexin V/PI Apoptosis Kit, counting kit-8 (CCK-8) assay, and RT-qPCR. Western blot analysis was used to detect the Usp38 protein level. IL-6 and TNF-α were measured by ELISA. The combination of miR-151-5p and USP38 was determined by chromatin immunoprecipitation (ChIP)-PCR and dual-luciferase reporter assay.
RESULTS: MP greatly improved pulmonary function in vivo, reduced inflammation, and promoted the proliferation of the alveolar type II epithelial cells (
AECII) in vitro. By comparing the alterations of microRNAs in lung tissues between MP treatment and control groups, we found that miR-151-5p exhibited a significant increase after LPS-treated AECII, but decreased after MP treatment. Confirmed by a luciferase reporter assay, USP38, identified as a downstream target of miR-151-5p, was found to increase after MP administration. Inhibition of miR-151-5p or overexpression of USP38 in
AECII significantly improved the anti-inflammatory, anti-apoptotic, and proliferation-promotive effects of MP.
CONCLUSIONS: In summary, our data demonstrated that MP alleviates the inflammation and apoptosis of
AECII induced by LPS, and promotes the proliferation of
AECII partially via miR-151-5p suppression and subsequent USP38 activation.