CC16 Ameliorates Inflammatory Responses and Acute Lung Injury in Mice by Modulating NF-κB/MAPK Signaling

Background: Acute lung injury (ALI) is a severe inflammatory lung condition characterized by alveolar damage, barrier disruption, and excessive inflammation. Current therapeutic strategies remain limited, highlighting the need for novel therapeutic targets. Dysregulated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling contributes to ALI progression. Club cell protein 16 (CC16) has emerged as a potential regulator of pulmonary inflammation and tissue protection; however, its role in ALI remains unclear. This study aimed to elucidate the protective effects and underlying mechanisms of CC16 in ALI.Methods: Lipopolysaccharide (LPS)-induced ALI mouse models were established and treated with varying doses of recombinant human CC16 (rhCC16). The evaluation of lung injury, inflammation, oxidative stress, and NF-κB/p38/extracellular regulated protein kinases (ERK) MAPK signaling activation was carried out using the lung wet-to-dry weight ratio (W/D), hematoxylin and eosin (H&E) staining, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), and Western blot assays. Meanwhile, an LPS-stimulated A549 cell inflammation model was also established to verify the protective effect of rhCC16 in vitro. Additionally, tumor necrosis factor-α (TNF-α) stimulation was performed to further investigate whether inflammatory signaling activation could attenuate the protective effects of rhCC16.Results: LPS-induced ALI significantly increased pulmonary edema (p < 0.05), inflammation, and oxidative stress in mice, accompanied by reduced CC16 expression (p < 0.05) and enhanced activation of NF-κB/p38/ERK MAPK signaling-related molecules. rhCC16 treatment significantly alleviated pulmonary edema and inflammatory damage, increased detectable CC16 levels, and reduced activation of NF-κB/p38/ERK MAPK signaling. In vitro, rhCC16 markedly enhanced cell viability, suppressed apoptosis, lactate dehydrogenase (LDH) release, oxidative stress, and inflammatory cytokine expression in LPS-stimulated A549 cells, while LPS and TNF-α pre-stimulation partially reversed these protective effects of rhCC16.Conclusion: Restoration of CC16 expression by rhCC16 attenuates inflammatory responses and oxidative damage accompanied by reduced activation of NF-κB/p38/ERK MAPK signaling, alleviating lung injury in ALI mice and LPS-exposed A549 cells. These results clarify both the protective effects of CC16 in ALI and the molecular pathways involved, highlighting its value as a potential intervention target.