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Alfalfa polysaccharide (APS) has been proposed to exhibit growth-promoting and immune-enhancing bodily functions in vivo. However, little is known about its downstream immunomodulatory and intrinsic molecular mechanisms. Herein, mouse splenic lymphocytes were isolated to characterize the immunomodulatory effects and molecular mechanisms of APS in vitro. The results demonstrated that APS selectively improved the cell viability and IgM production of B cells, but no effects on T cell viability or secretion of IL-2, IL-4 and IFN-γ were observed in vitro. The receptor blocking assay showed that TLR4 was the primary receptor involved in APS-mediated B cell activation, which was confirmed by the results obtained using C57BL/10ScNJ (TLR4 gene-deficient) mice. Moreover, APS activated the TLR4-MyD88 signaling pathway at the translational level by significantly increasing the protein expression of TLR4 and MyD88. Downstream pathway blocking assay demonstrated that both the MAPK and NF-κB pathways were involved in APS-induced B cell activation. Additionally, APS significantly enhanced the phosphorylation of p38, ERK, and JNK and activated the nuclear translocation of the NF-κB p65 subunit. Therefore, we concluded that APS specifically activates the immune functions of splenic B cells by TLR4, acting through the MAPK and NF-κB signaling pathways, and potently activates the p38 pathway.

The effects of alfalfa polysaccharides (APS) on immunomodulatory and antioxidant functions, as well as intestinal morphology were investigated in vivo in this study. Sixty-four mice were randomly divided into four groups and administered 0, 200, 400 or 800 mg/kg/d body weight APS via gavage for 28 days. The blood parameters and metabolites, viscera indices, antioxidant enzyme activities and intestinal morphology were measured. The results showed that the oral administration of APS improved the immune functions of mice, significantly enhanced the white blood cells and lymphocyte counts, and led to improvements in spleen and thymus indices. APS exhibited significant antioxidant activity by enhancing total antioxidant capacity, superoxide dismutase and glutathione peroxidase activities in heart, kidney and liver, and decreasing the malondialdehyde levels of heart and liver. Moreover, administration of APS potently enhanced the small intestinal villous height and the villus-to-crypt ratio, and decreased the crypt depth of duodenum in mice. Therefore, we can conclude that APS possesses pronounced immunomodulatory activities, and plays an important role in the prevention of oxidative stresses and in the improvement of intestinal morphology in the immunological system in vivo. APS thus shows potential for the development as an effective natural immunomodulatory and antioxidant agent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alfalfa polysaccharide (APS), a bioactive compound extracted from alfalfa, has been proposed to exhibit potential growth-promoting and immune-enhancing functions. But, little is known about the cellular immunomodulatory and intrinsic molecular mechanisms. Here we extracted the APS, and performed in vitro experiments to characterize the immunomodulatory functions as well as the molecular mechanisms of APS on RAW 264.7 macrophages cells. Chemical analyses showed that APS was mainly composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid. The results of in vitro assays demonstrated that 50 and 100 μg/mL APS increased the cell viability of RAW 264.7 cells. The secretion and gene expression of NO/iNOS, IL-6 and TNF-α in APS-induced macrophage cell were significantly enhanced. However, APS-induced TNF-α production was decreased by blocking the MAPK or NF-κB signaling pathways, especially for the blockade of p38. Moreover, APS enhanced the phosphorylation of p38, ERK, and JNK, promoted the degradation of IκBα, and increased the nuclear translocation of NF-κB p65 subunit. Therefore, we demonstrated that APS could improve the immune functions of RAW 264.7 macrophages cells by promoting the cell viability and increasing secretion and gene expressions of NO/iNOS, IL-6 and TNF-α through the MAPK and NF-κB signaling pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alfalfa polysaccharide (APS) is a bioactive component extracted from alfalfa that exhibits potent antioxidant properties. However, the cellular and molecular mechanisms underlying these properties remain unclear. To explore the molecular mechanism by which APS exerts antioxidant effects, an H 2 O 2 -induced oxidative stress mouse embryonic fibroblast (MEF) model was established. Cell proliferation, antioxidant enzyme activity, immune cytokine expression, and related protein expression were examined in APS-supplemented or non-supplemented conditions. The results suggested that APS strengthened the antioxidative capacity of MEFs, increasing cell proliferation, superoxide dismutase activity (SOD), and the total antioxidant capacity (T-AOC). In addition, APS reduced the secretion of interleukin (IL)-6 and IL-8 as well as expression of the proinflammatory gene retinoic acid-inducible gene I (RIG-I). APS was also able to activate the mitogen-activated protein kinase (MAPK) pathway, which promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus. However, expression of nuclear factor-κB (NF-κB) was decreased after APS treatment. Overall, these results suggest that APS relieves H 2 O 2 -induced oxidative stress in MEFs by activating MAPK/Nrf2 signaling and suppressing NF-κB signaling. To the best of our knowledge, this is the first study to link APS with MAPK/Nrf2, NF-κB and RIG-I, thus providing new perspectives regarding the mechanisms of the antioxidant activity of APS.