Intestinal microbiota-derived d-(+)-malic acid promotes pBD1 expression via p-p38/ATF1 signaling pathway to maintain porcine intestinal health

This study aims to identify nutrients that enhance the expression of host defense peptides (HDPs) and to evaluate their application effects and regulatory mechanisms, thereby advancing the exploring of nutritional regulation of HDPs. To achieve this, we constructed 16 stable fluorescent reporter porcine epithelial cell lines, driven by promoters targeting eight porcine intestinal HDPs genes, using IPEC-1 and IPEC-J2 cells. Through untargeted metabolomics sequencing and high-throughput screening, 15 metabolites were identified as potential enhancers of pBD1 expression, with d-(+)-malic acid (DMA) emerged as the most effective candidate. Transcriptomic and western blot analysis suggested that DMA enhances pBD1 expression primarily via the p-p38/ATF1 signaling pathways. Functional studies demonstrated that DMA significantly improved intestinal barrier integrity and alleviated intestinal damage. Overall, this work successfully established promoter-driven fluorescent reporter cell lines and identified microbiota-derived metabolites enhancing pBD1 expression, such as DMA, as promising alternatives to antibiotics for maintaining porcine intestinal health.