A bioengineered CATH-2 (1–15)-Ta1 (1–9) hybrid peptide attenuates LPS-induced inflammation and oxidative stress through LPS neutralization and TLR4/MD-2 modulation

Excessive activation of the lipopolysaccharide (LPS)-mediated Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) signaling pathway plays a central role in the pathogenesis of sepsis and other inflammatory disorders by promoting oxidative stress and uncontrolled cytokine production. In the present study, a novel CATH-Ta1 hybrid peptide (CTHP), was rationally designed by combining the endotoxin-binding domain of chicken cathelicidin-2 (CATH-2, 1–15) with the immunomodulatory fragment of Thymosin alpha-1 (Ta1, 1–9). The objective was to develop a multifunctional peptide with enhanced anti-inflammatory, antioxidant, and endotoxin-neutralizing activities. Structural characterization showed that CTHP adopted an amphipathic α-helical conformation with favorable hydrophobicity and strong cationic character. Reverse-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry confirmed successful synthesis of the peptide with > 98.5% purity and a molecular mass consistent with the theoretical value (~3148.7 Da). Functionally, CTHP exhibited significantly greater LPS-neutralizing activity than its parental peptides, achieving approximately 90% endotoxin neutralization at 50 μg/mL (p< 0.05). Furthermore, CTHP displayed minimal hemolytic activity (<15%) and significantly reduced lactate dehydrogenase release in RAW 264.7 macrophages, indicating superior biocompatibility and cytoprotective properties. Ex vivo pharmacokinetic analysis revealed a prolonged plasma half-life (85–90 min) compared with CATH-2 and Ta1, demonstrating enhanced resistance to enzymatic degradation. In vivo , CTHP markedly attenuated LPS-induced liver injury by reducing serum alanine aminotransferase, aspartate aminotransferase, and histopathological damage. Additionally, CTHP significantly suppressed serum TNF-α, IL-6, and IL-1β production (p<0.01) and effectively reduced intracellular reactive oxygen species accumulation in LPS-stimulated macrophages. Molecular docking and surface plasmon resonance analyses demonstrated that CTHP interacts with the TLR4/MD-2 complex, while Western blot analysis showed attenuation of LPS-induced NF-κB signaling, while western blot analysis confirmed inhibition of p-IKKβ, p-IκBα, and NF-κB p65 activation. Collectively, our findings demonstrate that CTHP effectively neutralizes LPS, suppresses oxidative stress, and attenuates LPS-induced NF-κB signaling, potentially involving interaction with the TLR4/MD-2 complex, thereby attenuating inflammatory responses both in vitro and in vivo . These findings highlight the protective potential of CTHP against LPS-induced endotoxemia and support further investigation of its therapeutic efficacy in endotoxin-mediated inflammatory disorders.