Interaction of osteopontin with α-lactalbumin and their synergistic effects in protecting intestinal barrier injury

With increasing emphasis on designing infant formulas to better mimic human milk，the collaborative effects of milk proteins have become a subject of great interest. Our study was designed to explore the interaction between osteopontin (OPN) and α-lactalbumin (α-LA) and their impact on intestinal barrier function. We discovered that OPN and α-LA can spontaneously combine to form an endothermic protein complex via hydrophobic interactions (pH 4.5, with an OPN to α-LA mass ratio of 1:1). This finding was corroborated through isothermal titration calorimetry and fluorescence quenching techniques. Additionally, we also employed circular dichroism and infrared spectroscopy to analyze the structure of the OPN-α-LA complex. Compared with OPN or α-LA alone, we observed that OPN-α-LA complex significantly alleviates lipopolysaccharide-induced CCD 841 CoN cells injury (p < 0.05). Specifically, OPN-α-LA complex markedly increased cell viability by 42.24 % and reduced lactate dehydrogenase release by 80.71 % (p < 0.05). As confirmed by Western blot and RT-qPCR assays, the OPN-α-LA complex could promote the proliferation of intestinal epithelial cells by activating crucial genes (β-catenin, c-Myc, and cyclin D1) within the Wnt/β-catenin signaling pathway. Moreover, the complex suppresses inflammation by decreasing pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and increasing anti-inflammatory cytokine IL-10 (p < 0.05). Additionally, it bolsters the intestinal barrier by enhancing the expression of tight junction proteins. These findings suggest the beneficial effects of OPN-α-LA complex in alleviating intestinal barrier injury, and provide new insight into the synergistic function of milk-derived bioactive proteins.