Abstract：
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.
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.
摘要：
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。
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。
