The Metabolic Reprogramming Mechanism of Astragalus Polysaccharide-Induced Immune Memory in HD11 Cells: Integrated Analysis Based on Glycolysis and the TLR2/mTOR Pathway.

This study aimed to investigate the immunomodulatory effects of APS on HD11 cells and its potential mechanisms. This study aimed to investigate the immunomodulatory effects of APS on HD11 cells and its underlying mechanisms. CCK-8 assays revealed that APS significantly promoted HD11 cell proliferation in a dose-dependent manner. Griess and lactate dehydrogenase (LDH) assays indicated that APS dose-dependently increased nitric oxide (NO) release and LDH activity, although these levels remained lower than those in the lipopolysaccharide (LPS) group. RT-qPCR analysis demonstrated that APS significantly upregulated the mRNA levels of IL-8, CXCL13, and iNOS, while downregulating CCR5. Additionally, APS enhanced the expression and activity of glycolysis-related enzymes while suppressing those of the pentose phosphate pathway. Western blot analysis revealed that APS upregulated the protein expression of TLR1, MyD88, p-mTOR, and HIF-1α, but downregulated TLR2, ERK, and NF-κB p65. Metabolic assays showed a dose-dependent increase in Acetyl-CoA concentration and a decrease in glutathione (GSH) content following APS treatment. Mechanistically, small interfering RNA (siRNA)-mediated knockdown of TLR2 and mTOR demonstrated that APS regulates the mTOR signaling pathway via TLR2. Furthermore, mTOR silencing significantly attenuated the APS-induced expression of immune-related factors, accumulation of Acetyl-CoA, and reduction of GSH, confirming the central role of the mTOR signaling pathway in APS-mediated immunomodulation. In conclusion, APS enhances the immune response of HD11 cells by regulating TLRs signaling pathways, promoting glycolytic metabolism, and activating the mTOR pathway, providing a theoretical basis for the application of APS in poultry immunomodulation.