L-Malic Acid Facilitates Stem Cell-Driven Intestinal Epithelial Renewal through the Amplification of β-Catenin Signaling by Targeting Frizzled7 in Chicks.

Abstract

l-Malic acid (l-MA) contributes to energy metabolism and nutrient digestion, which is an alternative to antibiotics for livestock; however, it is not clear whether l-MA can replace antibiotics to promote intestinal development in chicks. To investigate the effects of l-MA on intestinal stem cells (ISCs) driving epithelial renewal, we employed in vivo chick feeding experiments, chick intestinal organoid (IO) models, and in vitro chick intestinal epithelial cell models. The results showed that the feed conversion rate and diarrhea scores were decreased with improved jejunal morphology and barrier function in the 0.5% l-MA group. l-MA promoted the proliferation and differentiation of ISCs, inhibited the cell apoptosis, increased the IO formation efficiency, surface area, budding efficiency, and number of buds, suggesting that l-MA promoted the expansion of ISCs. Furthermore, l-MA treatment dramatically upregulated the Wnt/β-catenin signaling pathway in the jejunum. Importantly, Wnt transmembrane receptor Frizzled7 (FZD7) mRNA abundance was increased in response to dietary 0.5% l-MA. In addition, molecular docking analysis using Autodock software and isothermal titration calorimetry revealed that l-MA binds to Lys91 of FZD7 with high affinity, indicating a spontaneous interaction. The chick intestinal epithelial cells treated with 10 μM l-MA significantly increased cell viability, and the Wnt/β-catenin signaling pathway was activated, but l-MA failed to upregulate the Wnt/β-catenin signaling when treated with the FZD7-specific inhibitor Fz7-21 in chick intestinal epithelial cells, indicating that FZD7 is indispensable for l-MA activation of the Wnt/β-catenin signaling. Collectively, l-MA stimulated β-catenin signaling by targeting transmembrane receptor FZD7, which promoted ISC expansion and inhibited cell apoptosis to accelerate intestinal epithelial renewal in chicks.