Khan Salman, Zaki Almaz, Masood Mohammad, Khan Aman, Mohsin Mohd, Verma Amit, Wilson Parker C, Ali Shakir, Syed Mansoor Ali
Translational Research Lab, Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi 110025, India.
Translational Research Lab, Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi 110025, India; Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India.
Int Immunopharmacol. 2025 Feb 20;148:114166. doi: 10.1016/j.intimp.2025.114166. Epub 2025 Jan 29.
The purpose of this study was to investigate the therapeutic potential of Poly (ADP-ribose) polymerase 1 (PARP1) inhibition combined with microRNA miR-135a-5p overexpression in sepsis-induced acute lung injury (ALI). Specifically, we aimed to elucidate combinatorial therapeutic potential of PARP1 inhibition in mitigating oxidative stress and inflammation across different models, simultaneously miR-135a-5p overexpression promoting regeneration through the SMAD5/Nanog axis.
We used C57BL/6 mice to create Cecal Ligation Puncture (CLP) model of Sepsis-induced Acute Lung Injury. RAW264.7 murine macrophages and MLE12 (Mouse Lung Epithelial) cells were stimulated through Lipopolysaccharide (LPS) to induce inflammation. miR-135a-5p mimic Transfection confirmed using one-step Real time quantitative PCR (RT-qPCR). PARP1 inhibition confirmed by western blotting using Poly (ADP-ribose) (PAR) expression. Reactive oxygen Species (ROS) generation measured through Dichlorofluorescein diacetate (DCF-DA) dye using fluorescent microscopy and Nitric Oxide (NO) via spectrophotometry. Bronchoalveolar Lavage Fluid (BALF) cytokine analysis was done using Enzyme-linked immunosorbent assay (ELISA). miRNA mediated signaling, inflammatory markers and cytokines were determined using immunoblotting, RT-qPCR, and immunohistochemistry. miR-135a-5p target validation using dual-luciferase assay.
Our results demonstrated that PARP1 inhibition significantly reduced oxidative stress (**P < 0.01) and inflammatory markers in sepsis-induced lung injury models. Specifically, we observed decreased protein levels of inducible nitric oxide synthase (iNOS) (***P < 0.001), cyclooxygenase-2 (COX2) (*P < 0.05), phospho-Akt (*P < 0.05), and Tumor necrosis factor-Alpha (TNF-α) (*P < 0.05) mRNA expression. We observed significant reduction in ROS and NO generation in macrophages. Moreover, histopathological evidence suggested improved lung health. Concurrently, miR-135a-5p overexpression decreased the expression of SMAD5 (*P < 0.05) which in turns increased the expression of Nanog and related pluripotency genes in epithelial cells and mice, thus promoting regeneration and repair.
The combination of PARP1 inhibition and miR-135a-5p overexpression showed significant potential as a therapeutic intervention by reducing inflammation alongside stimulating regenerative environment in Sepsis-induced ALI.
本研究旨在探讨聚（ADP - 核糖）聚合酶1（PARP1）抑制与微小RNA miR - 135a - 5p过表达联合应用于脓毒症诱导的急性肺损伤（ALI）的治疗潜力。具体而言，我们旨在阐明PARP1抑制在减轻不同模型中的氧化应激和炎症方面的联合治疗潜力，同时miR - 135a - 5p过表达通过SMAD5/ Nanog轴促进再生。
我们使用C57BL/6小鼠建立脓毒症诱导的急性肺损伤的盲肠结扎穿刺（CLP）模型。通过脂多糖（LPS）刺激RAW264.7小鼠巨噬细胞和MLE12（小鼠肺上皮）细胞以诱导炎症。使用一步实时定量PCR（RT - qPCR）确认miR - 135a - 5p模拟物转染。使用聚（ADP - 核糖）（PAR）表达通过蛋白质印迹法确认PARP1抑制。通过荧光显微镜使用二氯荧光素二乙酸酯（DCF - DA）染料测量活性氧（ROS）生成，并通过分光光度法测量一氧化氮（NO）。使用酶联免疫吸附测定（ELISA）进行支气管肺泡灌洗液（BALF）细胞因子分析。使用免疫印迹、RT - qPCR和免疫组织化学确定miRNA介导的信号传导、炎症标志物和细胞因子。使用双荧光素酶测定法进行miR - 135a - 5p靶标验证。
我们的结果表明，PARP1抑制在脓毒症诱导的肺损伤模型中显著降低了氧化应激（**P < 0.01）和炎症标志物。具体而言，我们观察到诱导型一氧化氮合酶（iNOS）的蛋白质水平降低（***P < 0.001）、环氧合酶 - 2（COX2）（*P < 0.05）、磷酸化Akt（*P < 0.05）以及肿瘤坏死因子 - α（TNF - α）（*P < 0.05）mRNA表达降低。我们观察到巨噬细胞中ROS和NO生成显著减少。此外，组织病理学证据表明肺健康状况得到改善。同时，miR - 135a - 5p过表达降低了SMAD5的表达（*P < 0.05），这反过来又增加了上皮细胞和小鼠中Nanog及相关多能性基因的表达，从而促进再生和修复。
PARP1抑制与miR - 135a - 5p过表达的联合应用显示出作为一种治疗干预的显著潜力，通过减少炎症并同时刺激脓毒症诱导的ALI中的再生环境。
