Anti-inflammatory effects of recombinant TGF-arazyme protein on endotoxin-inflamed lung cell lines

Abstract

Background and Objectives: Pulmonary inflammation is central to the pathogenesis of various respiratory diseases, driving the need for novel therapeutics. Lipopolysaccharide (LPS) is a potent inflammatory trigger that induces proinflammatory cytokine synthesis via distinct signaling cascades. The objectives of this study were to evaluate the cytotoxic and anti-in- flammatory effects of recombinant TGF-Arazyme fusion protein on LPS-stimulated human lung A549 and MRC-5 cell lines.

Materials and Methods: A gene construct fusing Arazyme with a non-mitogenic TGFα fragment was expressed, purified via Ni-NTA chromatography, and validated enzymatically. Cytotoxicity was assessed by MTT assay. Inflammatory cytokine levels (TNF-α, IL-1β, IL-6, IL-10) were quantified by RT-PCR and ELISA. Anti-inflammatory efficacy was further validated in a murine (BALB/c) air pouch model infected with E. coli.

Results: At 20 μg/mL, the chimeric protein showed no significant toxicity in MRC-5 cells but dose-dependently reduced A549 viability (p<0.01). Co-treatment with LPS and the chimeric protein significantly downregulated proinflammatory cytokines (TNF-α, IL-1β, IL-6) at both mRNA and protein levels (p<0.01, p<0.001), while increasing IL-10 (p<0.05). In the air pouch model, TGF-Arazyme reduced leukocyte counts by 48.3% (p<0.01), comparable to dexamethasone (51.2%).

Conclusion: TGF-Arazyme exhibits both anti-inflammatory and selective cytotoxic properties, positioning it as a promising candidate for inflammatory lung diseases and lung cancer.