Z. morio Hemolymph Relieves E. coli -Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood-Milk Barrier.

Escherichia coli ( E. coli ) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio ( Z. morio ) hemolymph against mastitis pathogens. In this study, we established E. coli -induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli -induced mastitis in vivo and in vitro. In mice with E. coli , Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4 + T/CD8 + T, and increased the production of IL-2 triggered by E. coli . Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli -induced mastitis. In E. coli -stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli -induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli -induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli -induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.