Combating sepsis-induced acute lung injury: PARP1 inhibition mediates oxidative stress mitigation and miR-135a-5p/SMAD5/Nanog axis drives regeneration

Purpose: 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. Method: 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 (ADPribose) (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, RTqPCR, and immunohistochemistry. miR-135a-5p target validation using dual-luciferase assay. Results: 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-alpha) (*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. Conclusion: 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.