Immune organs are the core components of the immune system, comprising central immune organs (thymus, bone marrow) and peripheral immune organs (spleen, lymph nodes, and intestine). Central immune organs are the primary sites for the production, proliferation, differentiation, and maturation of immunoreactive cells, and they systematically shape the organization and phenotypic functionality of peripheral immune organs. In contrast, peripheral immune organs are the main sites of immune responses and are responsible for immune surveillance and defense (21).

Immune cells originate from immune organs and undergo differentiation, maturation, and immune response processes to identify and eliminate pathogens (e.g. bacteria and viruses) and abnormal cells (e.g. cancer cells). The major immune cell types include T and B lymphocytes, macrophages, natural killer cells, and dendritic cells (21). Tonifying polysaccharides enhance immune function by modulating immune cell activity, thereby benefiting immune-related diseases.

The NF-κB family includes core proteins p50, p65, RelB, c-Rel, and p52. NF-κB remains in the cytoplasm during the resting phase. Upon external stimulation, the signaling pathway is activated, leading to the activation of the IκB kinase (IKK) complex. Activation of IKK leads to the phosphorylation of IκB, which in turn results in IκB degradation and the subsequent release of NF-κB dimers. These dimers translocate to the nucleus, where they modulate inflammation, cell growth, and immune responses (68). SONG et al. (69) observed that treatment with alkali-extracted Bupleurum chinense polysaccharide (BCAP-1) in homozygous mice significantly inhibited the growth of sarcoma 180, promoted TNF-α, NO, and iNOS secretion, and enhanced macrophage phagocytic activity. Meanwhile, BCAP-1 activated NF-κB signaling via p65 phosphorylation and reduced IκB expression in macrophages, thereby exerting its anti-tumor immune regulatory mechanism. QIN et al. (70) identified that sHEP2 in Selenizing Hericium erinaceus polysaccharides (sHEPs) induced dendritic cell (DC) maturation and exerted immunomodulatory effects by inducing IκBα degradation, promoting the nuclear translocation of p65 and p50, and enhancing NF-κB signaling pathway activity. YANG et al. (61) demonstrated that Cnidium officinale polysaccharide significantly promoted NO secretion and up-regulated the expression of TNF-α, IL-1β, and IL-6 in RAW264.7 macrophages, while also activating p65 phosphorylation and exerting immunomodulatory effects via the NF-κB signaling cascade.

The NF-κB signaling pathway is regulated by various receptors, including Toll-like receptors (TLRs), the tumor necrosis factor receptor (TNFR) family, and the interleukin-1 receptor (IL-1R). TLRs are vital for innate immunity and activate downstream NF-κB and mitogen-activated protein kinases (MAPKs) via myeloid differentiation factor 88 (MyD88), prompting cells to synthesize and release large amounts of inflammatory cytokines (e. g., TNF-α, IL-1β, IL-6), chemokines, and interferons, thereby regulating immune signaling (71). WU et al. (72) uncovered that Tetrastigma hemsleyanum polysaccharide (THP) triggers specific signaling cascade reactions through its action on TLR4, resulting in strong p65 phosphorylation and weak IκBα phosphorylation, and significantly increases the relative values of p-p65/p65 and p-IκBα/IκBα. Meanwhile, THP promotes p65 nuclear translocation, thereby activating the NF-κB pathway. Lycium barbarum polysaccharides liposomes (LBPL) (66) exert immunomodulatory effects by engaging the TLR4 receptor, initiating the MyD88-dependent pathway, inducing TRAF6 phosphorylation, and thereby triggering the NF-κB pathway, thereby which facilitates dendritic cell (DC) maturation and Th1-type immune polarization. ZHANG et al. (73) revealed that Catharanthus roseus polysaccharides (CRPS-2) activate the NF-κB pathway by dose-dependently elevating TLR2 and TLR4 expression, which is one of the important mechanisms underlying its immunomodulatory effects.

The mitogen-activated protein kinase (MAPK) signaling pathway is a three-tiered kinase cascade mechanism, primarily composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. This pathway impacts inflammation, cell differentiation, proliferation, apoptosis, immune regulation, and stress response (74). The MAPK signaling pathway consists of three core pathways: the c-Jun N-terminal kinase (JNK) pathway, the p38 MAPK pathway, and the extracellular signal-regulated kinase (ERK) pathway (75). Different pathways regulate distinct cellular processes: the extracellular signal-regulated kinase (ERK) pathway predominantly responds to growth-promoting cytokines, thereby stimulating cell proliferation and differentiation; whereas the JNK and p38 pathways are associated with cellular stress responses and immuno-inflammatory processes. The three pathways in the MAPK pathway—ERK, JNK, and p38—are both independent and interact with each other, forming a complex regulatory network. GUO et al. (15) verified that Ganoderma lucidum polysaccharide (GLP) inhibits the phosphorylation of JNK and ERK triggered by lipopolysaccharides in both normal intestinal epithelial cells (NCM460) and colorectal cancer cells (HT-29), attenuates the expression levels of inflammatory mediators (e. g., iNOS, COX-2, IL-1β, TNF-α), and thereby inhibits the MAPK pathway, exerting inflammation-suppressing and anti-tumor effects. QIN et al. (70) found that selenized Hericium erinaceus polysaccharides (sHEPs) significantly enhanced the phosphorylation of ERK, JNK, and p38, and promoted the nuclear translocation of p-c-Jun, p-CREB, and c-Fos, thereby promoting dendritic cell (DC) proliferation and maturation and exerting immunomodulatory effects. Tetrastigma hemsleyanum polysaccharide (THP) treatment (72) significantly elevated the phosphorylation of ERK, JNK, and p38 MAPK in macrophages, indicating its potential to modulate immune responses via these MAPK pathways. ZHANG et al. (73) revealed that Catharanthus roseus polysaccharides (CRPS-2) promote the phosphorylation of ERK, JNK, and p38 proteins in a dose-dependent manner, thereby activating the MAPK pathway, which is one of the key mechanisms underlying its immunomodulatory effects.

The PI3K/Akt signaling pathway serves as a crucial modulator of cell growth, proliferation, differentiation, and metabolic processes (76). The canonical PI3K/Akt pathway is activated by the activation of phosphatidylinositol-3 kinase (PI3K) upon exposure to extracellular stimuli. Upon activation, PI3K catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 functions as an intracellular signal transducer, recruiting and activating protein kinase B (Akt) to the plasma membrane, thereby initiating a cascade of downstream reactions and regulating immune function. HU et al. (77) investigated the immunomodulatory activities of longan polysaccharide (CP1) and its fermentation products, including longan wine (CP2), vinegar (CP3), and CP4. The findings indicated that the CP3 vinegar fermentation product augmented the phosphorylation of PI3K p85, IκBα, and Akt in macrophages, while also stimulating cytokine release, including NO, TNF-α, and IL-6, thereby activating the immune response. Additionally, the immunomodulatory effects of CP3 are strongly associated with its low molecular weight and increased mannose and arabinose content. ZHANG et al. (73) revealed that Catharanthus roseus polysaccharides (CRPS-2) activate the Akt/mTOR (mammalian target of rapamycin) pathway through enhanced expression of phosphorylated Akt and mTOR, which is one of the key mechanisms underlying its immunomodulatory activity. QI et al. (78) observed that low-molecular-weight ginseng polysaccharides (LGP) alleviated spontaneous liver injury through suppression of the PI3K/Akt pathway. LGP inhibited the phosphorylation of PI3K, Akt, and mTOR proteins and reduced the mRNA expression of inflammatory mediators (e. g., IL-1β, IL-6, IL-18, TNF-α), thereby attenuating inflammation. Additionally, LGP diminished Bax expression, augmented Bcl-2 levels, and subsequently reduced the Bax/Bcl-2 ratio, thereby reducing hepatocyte apoptosis. WU et al. (79) showed that azuki bean (Vigna angularis) polysaccharide (ABP) significantly up-regulated the gene and protein expression of insulin receptor (INSR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glucose transporter 2 (GLUT-2) in the liver of diabetic rats. This mechanism involves activation of the insulin/PI3K/Akt pathway, thereby promoting glucose metabolism and maintaining immune homeostasis.

In conclusion, existing studies have demonstrated that both the NF-κB signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway are involved in the pathophysiological processes of inflammatory diseases and tumors (80). However, aberrant activation of the PI3K/Akt signaling pathway is closely associated with tumor development and the pathogenesis of metabolic diseases. The NF-κB signaling cascade predominantly governs the synthesis of pro-inflammatory cytokines at the transcriptional level. Among the MAPK pathways, the p38 MAPK and JNK signaling pathways are strongly correlated with neuroinflammation, apoptosis, and glial scarring. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway is implicated in neuroprotection, cell survival, and axonal regeneration (74). The immunomodulatory mechanisms of Tonifying polysaccharides are depicted in Figure 2 .

The tumor microenvironment (TME) significantly influences tumor development and progression. It enables tumor cells to evade immune surveillance and clearance through multiple mechanisms, thereby constructing an immunosuppressive microenvironment and promoting tumor cell proliferation, invasion, and metastasis. Investigations have revealed that tonifying polysaccharides possess the capacity to modulate the tumor microenvironment through multiple pathways, including stimulating the polarization of tumor-associated macrophages to the M1 phenotype; enhancing the maturation of dendritic cells and their antigen-presenting ability; enhancing the cytotoxic and proliferative activities of natural killer cells; and modulating T-lymphocyte activation, proliferation, and cytokine secretion. Through these mechanisms, Tonifying polysaccharides can remodel the tumor microenvironment, reverse its immunosuppressive state, and stimulate the body’s anti-tumor immune response, thereby exerting anti-tumor immunomodulatory effects (62, 80).

Macrophages polarized to the M1 phenotype suppress tumor growth, while those polarized to the M2 phenotype facilitate tumor progression (81). Therefore, shifting macrophage polarization to the M1 phenotype has emerged as a potential approach in cancer immunotherapy. Research has demonstrated that Astragalus polysaccharide (PG2) (82) markedly elevated the M1/M2 polarization ratio and suppressed the levels of pro-tumor cytokines IL-6 and IL-10 in non-small cell lung cancer cell lines H1299 and H441. Additionally, PG2 promotes dendritic cell (DC) maturation, thereby enhancing T-cell activity and mediating antitumor immune regulation. Clinical studies have shown that Astragalus polysaccharide injection (PG2) significantly reduces the neutrophil-lymphocyte ratio (NLR) in advanced lung cancer patients receiving immune checkpoint inhibitor therapy, especially in those with a high baseline NLR. Moreover, PG2 modulates macrophage polarization (M1/M2) and dendritic cell maturation, thus enhancing antitumor immunity (83). LIU et al. (84) confirmed that the combination of Ganoderma lucidum crude polysaccharides (GLCP) and Codonopsis pilosula crude polysaccharides (CPCP) followed by dacarbazine significantly reduced CD68⁺ macrophage numbers in murine melanoma tissue and inhibited tumor growth. Additionally, particular concentrations of digested Codonopsis pilosula polysaccharide (dCPP) enhanced the expansion of M1-type macrophages, inhibited the proliferation of M2-type macrophages, and induced the polarization of M2-type macrophages to the M1 phenotype by up-regulating the transcriptional levels of IL-1, IL-6, iNOS, and TNF-α, thereby inhibiting melanoma progression. Tonifying polysaccharides can regulate neutrophil polarization, inhibit the expression of pro-tumor phenotypes (N2 type), and promote the expression of anti-tumor phenotypes (N1 type), thereby exerting a substantial influence on anti-tumor immune regulation. XU et al. (85) demonstrated that Lentinan (LNT), either alone or in combination with Delta-like 1 (DLL1), inhibited the growth of both breast and lung cancer, with the combined treatment yielding superior results. Additionally, LNT enhances the antitumor effects of DLL1 through two immune mechanisms: it promotes the infiltration and activation of CD8⁺ T cells, thereby activating adaptive immunity, and induces neutrophils to polarize toward the N1 type, secreting cytokines such as IFN-γ to synergize with tumor suppression and remodel innate immunity. ZHAO et al. (86) demonstrated that the co-administration of Lentinan and cisplatin thoracic injection significantly enhances the clinical efficacy in patients with non-small cell lung cancer (NSCLC) and improves their quality of life. Lentinan has been demonstrated to stimulate immune cells such as T cells and NK cells, promote the secretion of cytokines including IL-6 and TNF-α, and inhibit the expression of VEGF. The combined mechanisms of immune regulation and anti-angiogenesis have been shown to yield significant clinical benefits in cancer treatment.

The maturation and migration of dendritic cells (DCs) activate T cells and significantly enhance the anti-tumor immune response (87). ZHANG et al. (88) found that Lonicera japonica thunb. polysaccharides (LJP)-exosomes significantly promoted polysaccharide delivery. LJP-exosomes not only enhanced the migration and maturation of DCs in 4T1 hormonal mice but also stimulated pro-inflammatory cytokine secretion and upregulated MHC-II expression in lymph nodes. Additionally, LJP-exosomes significantly inhibited 4T1 tumor growth, enhanced the infiltration of DCs, CD4⁺ T cells, and CD8⁺ T cells, while reducing the percentage of regulatory T cells (Tregs) within the tumor, thereby improving the immunosuppressive tumor microenvironment and enhancing LJP transport and immune system activation. LAN et al. (89) investigated the Fructus corni acidic polysaccharide (FCP-3) complex and successfully prepared polysaccharide-selenium nanoparticle composites (FCP-SeNPs). The results showed that both FCP-3 and FCP-SeNPs effectively promoted the secretion of NO, TNF-α, and IL-12 from macrophages and increased the CD4⁺/CD8⁺ T cell ratio. Additionally, FCP-3 and FCP-SeNPs inhibited tumor growth and proliferation and promoted apoptosis of hepatocellular carcinoma cells, with FCP-SeNPs being more effective than FCP-3 alone, thereby playing a significant role in hepatocellular carcinoma immunomodulation. DONG et al. (39) demonstrated that high-dose treatment with Lactarius deliciosus polysaccharides (LDPs) significantly inhibited tumor growth (tumor suppression rate, 51.61%) and elevated the percentages of CD3⁺, CD4⁺, and CD8⁺ T cells within the peripheral blood of mice bearing tumors. The findings suggest that the antitumor immune function of LDPs is related to T-cell activation. QIAN et al. (90) found that purified ginger polysaccharide (UGP1) markedly increased the expression levels of TNF-α, IL-2, IL-6, and other pro-inflammatory mediators, decreased levels of TGF-β and bFGF (basic fibroblast growth factor), and other pro-tumorigenic factors, and induced apoptosis by regulating the p53, caspase-3, and Bax/Bcl-2 ratio, thereby effectively suppressing the proliferation of human colon cancer cells.

Autoimmune diseases are conditions in which the immune system mounts a pathologic immune response against self-antigens, leading to tissue injury and impaired function. The pathogenesis of autoimmune diseases involves multiple factors, including abnormal function of immune organs, dysregulated activation of immune cells, and cytokine network imbalance. Studies have shown that Th17 cells promote inflammatory responses and enhance resistance to certain pathogens but are also closely associated with the development of diverse autoimmune disorders, such as psoriasis and inflammatory bowel disease. Treg cells regulate the activation and expansion of effector T cells and inhibit excessive immune responses, thereby preventing autoimmune diseases (91). Polysaccharide from Atractylodes macrocephala Koidz (PAMK) (92) significantly alleviated weight loss, disease activity index (DAI) scores, and colon shortening in mice, and down-regulated the expression of pro-inflammatory cytokines, thereby inhibiting inflammation and alleviating pathological damage to the colon. Additionally, PAMK reduced the proportion of Th17 cells and their manifestation of related cytokines in mice with colitis, increased the expression of Treg-related factors, and modulated the Th17/Treg equilibrium, thus exerting a therapeutic effect on ulcerative colitis (UC) in mice. HAO et al. (93) investigated the diverse pathways through which ginger polysaccharide (GP) mitigates ulcerative colitis (UC) symptoms in mice. GP protects the intestinal barrier integrity by repairing the DSS-induced decrease in the expression of tight junction proteins, zona occludens 1 (ZO-1) and occludin-1, and by decreasing apoptosis of colonic epithelial cells. Additionally, GP significantly down-regulated the expression of inflammatory cytokines (e. g., TNF-α, IL-6, IL-1β, IL-17A, IFN-γ) in colonic tissues, thereby suppressing the abnormal inflammatory reaction of UC. These results suggest that Tonifying polysaccharides can alleviate ulcerative colitis (UC) in mice, with mechanisms including immune cell regulation, inflammatory cytokine modulation, increased intestinal flora diversity, and maintenance of the intestinal wall’s immune barrier function (94). Psoriasis is a chronic inflammatory autoimmune skin disease characterized by an imbalance between innate and adaptive immunity, which interact to form an inflammatory cascade response, leading to excessive proliferation of skin keratinocytes, abnormal differentiation, and epidermal inflammation (95). Punica granatum peel polysaccharides (PPPs) (96) reduce the psoriasis area and severity index (PASI) and transepidermal water loss (TEWL) by significantly improving psoriasis-like skin lesions, downregulating the expression of Ki67 and CD3, inhibiting the levels of pro-inflammatory cytokines (e. g., TNF-α, IL-6, IL-1β, IL-17, IL-23) in serum, and upregulating the mRNA and protein expression of aquaporin-3 (AQP3) and filaggrin (FLG) in mouse skin. These actions regulate immune balance and skin barrier function, thereby contributing to psoriasis treatment. Allergic asthma is a type 2 immune-mediated chronic inflammatory condition characterized by airway inflammation, mucus hypersecretion, and epithelial barrier dysfunction (97). Lentinan (LTN) (98) markedly diminished the quantity of eosinophils and neutrophils in bronchoalveolar lavage fluid (BALF) and decreased the levels of type 2 cytokines, thereby breaking the vicious cycle of inflammation and barrier damage. Additionally, LTN re-established the expression levels of tight junction protein ZO-1 and repaired the damaged airway epithelial barrier, thereby improving allergic asthma symptoms. The study revealed that both PSAP-1 and PSAP-2 (99), two acidic polysaccharides from Pyrus sinkiangensis Yu, significantly increased macrophage vitality, promoted the release of NO, TNF-α, and IL-6, and exerted immunomodulatory effects. Additionally, PSAP-1 particularly improved airway inflammation in asthmatic mice, as manifested by lower expressions of IL-4, IL-5, and IL-13, fewer inflammatory cells, improved lung histopathology, decreased mucus secretion, and resistance to airway inflammation through activation of the NF-κB and MAPK pathways.

Tonifying polysaccharides show promise for multiple sclerosis (MS) prevention and treatment by inhibiting central nervous system (CNS) inflammation through immune cell activation (100, 101). Investigations have demonstrated that Astragalus polysaccharides (APS) (102) suppress the proliferation of MOG_35-55-specific T cells, upregulate the PD-1/PD-L1 pathway, and downregulate the levels of pro-inflammatory cytokines (e. g., IFN-γ, TNF-α, IL-2, IL-17), thereby suppressing Th1- and Th17-mediated immune responses and alleviating experimental autoimmune encephalomyelitis (EAE) symptoms. Acidic polysaccharide of Panax ginseng (APG) (103) significantly retarded the proliferation of PLP_139-151 peptide-specific T cells, decreased the release of IFN-γ, IL-17, and TNF-α in splenocytes, reduced the number of CD4⁺ T cells in the CNS and CD11b⁺ macrophages, attenuated demyelination and axonal damage, and effectively improved clinical symptoms and histopathological changes in relapsing-remitting experimental autoimmune encephalomyelitis (rr-EAE). The typical etiology of multiple sclerosis (MS) includes central nervous system (CNS) inflammation and myelin destruction. Tonifying polysaccharides may promote myelin repair by augmenting the expression of the anti-inflammatory M2 phenotype. Studies have shown that Lentinan (LNT) (104) promotes the phenotypic shift of microglia from M1 to M2 by activating the Dectin-1 receptor, downregulating the expression of the M1 marker iNOS, and upregulating the expression of the M2 marker Arg-1. Meanwhile, LNT also significantly promoted myelin regeneration and motor function recovery in demyelination model mice, thereby demonstrating its potential for treating multiple sclerosis. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by immune cell dysfunction and abnormal secretion of pro-inflammatory factors (105). Angelica Sinensis polysaccharide (ASP) (106) has demonstrated multiple therapeutic effects in the therapeutic intervention of RA, including marked reduction in joint swelling, inhibiting the release of anti-CII antibodies and pro-inflammatory factors, modulating the gut microbiota by augmenting the proportion of positive anti-inflammatory bacteria (e. g., Lactobacillus) and suppressing the proportion of harmful pro-inflammatory bacteria (e. g., Desulfovibrionaceae), enhancing the production of tight junction proteins (e. g., Cldn5) to repair the gastrointestinal mucosal barrier, and rebalancing the expression of Slit3 and Rgs18 to regulate osteoblast and osteoclast function and reduce bone destruction. Astragalus polysaccharide (ASP) (107) significantly reduced toe swelling, inhibited Th17 cell differentiation and the release of pro-inflammatory mediators (e. g., IL-1β, TNF-α), and blocked the NF-κB signaling pathway, thereby alleviating rheumatoid arthritis symptoms in rats. The pathological mechanism of type 1 diabetes mellitus (T1DM) involves the erroneous attack and disruption of pancreatic β-cells by the immune system, resulting in absolute insulin deficiency and subsequent hyperglycemia, and is classified as an autoimmune disease (108). Lentinan (LNT) (109) significantly reduced pancreatic islet inflammation scores, decreased β-cell damage, and attenuated pancreatic islet inflammation. Additionally, LNT induces Treg cell proliferation, inhibits the Th1-type immune response, blocks the PI3K/AKT/NF-κB pathway, and demonstrates therapeutic potential for type 1 diabetes mellitus (T1DM) through immunomodulation.

Tonifying polysaccharides exert antiviral effects by enhancing T cell regulation, B cell activation and antibody production, macrophage activity, intestinal mucosal immunity, and by directly inhibiting viral replication (110). Lentinan (LNT) (111) effectively alleviates diarrhea caused by rotavirus (RV) infection in weaned piglets, with its mechanism strongly correlated with the improvement of the intestinal immune response. LNT markedly elevated serum immunoglobulin expression, including IgA, IgG, and IgM, as well as intestinal secretory IgA (sIgA), strengthened the barrier function of the intestinal mucosal membrane, upregulated TLR3, retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) in the intestinal mucosal membrane, and enhanced intestinal immunity. TLR3, retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) expression activated the RIG-I/MDA5/MAVS antiviral pathway and promoted the secretion of host defense peptides (HDPs) to enhance intestinal antimicrobial capacity. Refined polysaccharide from Dendrobium devonianum (DVP-1) (112) inhibited H1N1 influenza virus infection and attenuated lung inflammation by triggering the TLR4/MyD88/NF-κB pathway, enhancing the release of Th1/Th2-type cytokines, significantly increasing intestinal mucosal IgA expression, and enhancing lymphocyte proliferation and intestinal mucosal immunity. Sophora flavescens polysaccharides (SFP-100) (113) reduced Concanavalin A (ConA)-induced hepatocyte necrosis and inflammatory cell infiltration, promoted IFN-γ and IL-6 secretion by splenocytes, and enhanced the Th1-type immune response. Meanwhile, neutral Sophora flavescens polysaccharide (SFP-100-A) inhibited hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in a concentration-dependent manner, with a more pronounced suppressive effect on HBsAg, thereby exerting hepatoprotective and hepatitis B virus (HBV)-inhibitory activities.

Additionally, owing to their natural derivation, minimal toxicity, and absence of residual substances, Tonifying polysaccharides hold potential as vaccine adjuvants in antiviral immunization strategies (114). Polysaccharide from Ganoderma lucidum (PS-G) (115), as a mucosal immunopotentiator, significantly enhanced EV-A71 vaccine-induced mucosal (IgA) and systemic (IgG) antibody responses, increased the secretion of IFN-γ and IL-17, and stimulated Th1/Th17-type cellular immunity, thereby providing protection against lethal viral infections. Astragalus polysaccharide (ASP) (116), as an adjuvant for inactivated SARS-CoV-2 vaccine (ISV) and recombinant SARS-CoV-2 vaccine (RSV), possesses bi-directional immunomodulatory functions. ASP activates both humoral and cellular immunity by enhancing the release of IgG1 (Th2) and IgG2a (Th1), as well as increasing the expression of cytokines like IL-2, IFN-γ, and IL-4. Meanwhile, ASP adaptively regulates immune responses based on antibody levels: it suppresses excessive inflammatory responses when antibody levels are high and enhances T/B cell proliferation and cytokine secretion when antibody levels are low, thereby maintaining immune homeostasis. Lentinan-CaCO₃ microparticles (CaCO₃-LNT) (117), as a vaccine adjuvant for H5N1, significantly upregulated the levels of MHC-II and CD86 in lymph node dendritic cells (DCs), promoted DC maturation and activation, elevated the CD4⁺/CD8⁺ T-cell ratio while augmenting the expression of IgG1 and IgG2, thereby activating Th cell immune responses. Studies have shown that TRIM29 and PARP9 are critical regulators of antiviral immunity. TRIM29 deficiency enhances antiviral innate immunity against DNA and RNA viruses, including influenza, by upregulating type I interferon (IFN-I) production in alveolar macrophages (118) and dendritic cells (119, 120). Additionally, TRIM29 deficiency has been shown to mitigate viral myocarditis by modulating PERK-mediated endoplasmic reticulum (ER) stress and immune responses (121). Recent evidence also suggests that TRIM29 deficiency reduces viral enteritis severity through enhanced NLRP6 and NLRP9 inflammasome activation (122). PARP9 is a non-classical RNA sensor that forms a complex with DTX3L (deltex E3 ubiquitin ligase 3L). Within the IFN signaling pathway, PARP9 participates in antiviral responses via ADP-ribosylation. Additionally, PARP9 induces type I IFN production via the PI3K/AKT3-IRF3/IRF7 pathway, thereby playing a pivotal role in antiviral immunity (123, 124). The aforementioned Sophora flavescens polysaccharide (SFP-100) (113), Polysaccharide from Ganoderma lucidum (PS-G) (115), and Astragalus membranaceus polysaccharide (ASP) (116) all promote cytokine production (including IFN-γ) and exhibit antiviral immune activity. Downregulating TRIM29 and upregulating PARP9 also promote IFN-γ production by modulating the IFN signaling pathway, thereby exerting antiviral immune activity. These two mechanisms are highly similar, suggesting that tonic polysaccharides may exert antiviral immune activity through the downregulation of TRIM29 and upregulation of PARP9.

Currently, the extraction, separation, purification, and structural analysis of polysaccharides have garnered considerable attention. There are various methods for extracting tonic polysaccharides, each of which significantly affects their molecular weight, monosaccharide composition, glycosidic bond type, and molecular conformation. Common extraction methods include traditional methods (water extraction, acid/alkali extraction, alcohol extraction) and modern auxiliary technologies (ultrasound-assisted extraction, microwave-assisted extraction, enzymatic hydrolysis, etc.) (12, 125). Different extraction methods are suitable for different types of polysaccharides, each with its own advantages and disadvantages ( Table 1 ). Extracted polysaccharides often contain impurities such as proteins, water-soluble small molecules, and pigments, necessitating separation and purification. The most commonly used methods for removing proteins include the Sevag method, freeze-thaw method, NaCl method, and CaCl₂ method. Additionally, ethanol fractionation precipitation and column chromatography are also frequently used purification methods (125). After extraction and purification, polysaccharides can be structurally characterized using various techniques to determine their chemical composition, molecular weight distribution, and glycosidic bond linkage patterns. High-Performance Gel Permeation Chromatography (HPGPC), Fourier Transform Infrared Spectroscopy (FT-IR), Gas Chromatography-Mass Spectrometry (GC-MS), High-Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR), and Ultraviolet-Visible Spectroscopy (UV–Vis) are the most commonly used methods for analyzing the composition and structure of polysaccharides. Structural characterization clarifies the relationship between structure and immunomodulatory activity, guiding the screening and modification of active polysaccharides. The commonly used structural characterization methods for tonic polysaccharides are summarized in Table 2 .

The structure of polysaccharides can be categorized into primary and higher-order structures. The primary structure serves as the foundation for the higher-order structure, which represents the specific spatial arrangement of the primary structure (126). Low-molecular-weight polysaccharides (<10 kDa) are mainly linear chains or short branched structures, predominantly distributed in the kidney and intestinal mucosa. These polysaccharides exhibit higher water solubility and stronger tissue barrier penetration capabilities, which enable them to penetrate tissues and exert immune-modulating effects. Medium-molecular-weight polysaccharides (10–100 kDa), which are commonly found in tonic polysaccharides, are primarily distributed in lymph nodes and blood. They exert immune-modulating activity by regulating lymphocytes and dendritic cells. High-molecular-weight polysaccharides (>100 kDa) have complex conformations and are predominantly distributed in the liver and spleen. They cannot easily penetrate the intestinal mucosal barrier and require binding to cell membrane surface receptors to mediate endocytosis or signal transduction, thereby exerting immune-modulating activity. YANG et al. (127) demonstrated that UV/H₂O₂ treatment of Moringa oleifera leaf polysaccharides (MOLP) produces the derivative DMOLP-3 (2494 Da). Compared with the original MOLP, DMOLP-3 has a lower molecular weight and broken glycosidic bonds (e.g., a decreased ratio of arabinose to glucose), leading to a simplified structure. This structural change facilitates recognition and fermentation by gut microbiota (such as Bifidobacterium and Bacteroides), thereby producing short-chain fatty acids (SCFAs). SCFAs can inhibit the NF-κB pathway, reduce pro-inflammatory factors (such as IL-6 and TNF-α), enhance intestinal barrier function, and promote Treg cell differentiation, thus balancing Th1/Th2 immune responses. Additionally, the surface morphology of DMOLP-3 changes from a coarse granular to a smooth plate-like structure, with a reduced particle size and enhanced stability. LIANG et al. (48) found that polysaccharides purified from fermented Lentinus edodes polysaccharide (F-LEP-2a) and non-fermented Lentinus edodes polysaccharide (NF-LEP-2a) exhibit distinct characteristics. Fermentation increased the molecular weight of F-LEP-2a from 1.16×10⁴ Da to 1.87×10⁴ Da and altered its monosaccharide composition, with an increased proportion of glucose. This enabled F-LEP-2a to activate macrophage phagocytosis and enhance the recognition ability of immune cell receptors. Compared with the control group, F-LEP-2a increased serum IgG and IgM levels by 42% and 38%, respectively, and outperformed NF-LEP-2a. Therefore, F-LEP-2a exhibits superior immunomodulatory activity. The types and sequence of monosaccharides determine the binding specificity of polysaccharides to immune receptors (e.g., TLR, MR, and Dectin-1). The type of glycosidic bond is crucial for immune activity. Different glycosidic bond structures influence the binding efficiency of immune receptors, signal transduction pathways, and immune cell function, thereby modulating innate and adaptive immune responses. β-glycosidic bonds (e.g., β-1,3, β-1,4, and β-1,6) are key structures for immune activation. Their linear or branched structures readily form helical conformations, enhancing surface receptor binding efficiency and thereby activating the innate immune system. α-glycosidic bonds (e.g., α-1,4 and α-1,6) can generate inhibitory signals to reduce excessive inflammatory responses and are more involved in immune tolerance and adaptive immune regulation. WANG et al. (25) isolated and purified four components (PSP1–PSP4) from Polygonatum sibiricum polysaccharides. Among these, PSP3 is mainly composed of rhamnose (57.69%) and galactose (37.17%). Galactose is linked via β-glycosidic bonds, whereas rhamnose contains methyl protons. These structural features enhance PSP3’s binding capacity to macrophage surface receptors, thereby restoring the Th1/Th2 cytokine imbalance in immunosuppressed mice. ZHANG et al. (73) isolated two heteropolysaccharides, CRPS-1 (193 kDa) and CRPS-2 (9 kDa), from Catharanthus roseus. CRPS-2, a low-molecular-weight polysaccharide primarily composed of galactose and arabinose, exhibits stronger cellular permeability and higher binding affinity with macrophage surface receptors (TLR2/4). CRPS-1 primarily adopts an α-configuration (e.g., α-1,3, α-1,5, etc.), whereas CRPS-2 primarily adopts an α/β-configuration (e.g., α-1,3, β-1,6, etc.), and CRPS-2 has a more complex branched structure, providing more recognition sites. This enables CRPS-2 to more effectively promote the activation of the MAPK/Akt/NF-κB signaling pathway, thereby enhancing NO and IL-6 secretion. In contrast, CRPS-1, due to its high molecular weight and simple branching structure, exhibits relatively weaker structure–activity relationships. Therefore, CRPS-2 demonstrates superior immunomodulatory efficacy. The degree of branching is a crucial structural feature of polysaccharide molecules. Polysaccharides with a moderate degree of branching can provide more receptor binding sites through their highly branched structures, enabling them to bind to multiple receptors on the surface of immune cells, thereby promoting the regulation of immune cells and modulating signaling pathways. However, excessive branching can lead to molecular conformational disorder and excessive steric hindrance, thereby reducing the binding efficiency between polysaccharides and receptors. Additionally, the branching position exhibits specific recognition functionality, with O-6 branching conferring immunological advantages over O-3 branching. FENG et al. (9) found that ginseng polysaccharides, after extraction and purification, yield three fractions: GPS-1a, GPS-1b, and GPS-2. Among these, the simple linear structures of GPS-1a and GPS-1b are less capable of triggering receptor clustering. GPS-2, however, possesses a multi-branched structure and a high aldose content, conferring a stronger binding capacity with the TLR2 receptor. It can activate the TLR2-NF-κB/TRAF6 signaling pathway, thereby significantly enhancing immune responses and repairing radiation damage. Additionally, GPS-2 has a moderate molecular weight (56.86 kDa), facilitating penetration of tissue barriers, while GPS-1a has a high molecular weight (1653 kDa), resulting in lower bioavailability. ZHANG et al. (128) extracted and identified two polysaccharides (CVPn and CVPa) from fruiting bodies of mushroom Coriolus versicolor, both of which have β-1,4 and β-1,3 glycosidic bonds as their main chains. CVPa has a linear main chain with fewer β-1,6 branches, connected via O-6 positions, resulting in stronger binding affinity with Dectin-1 and TLR. Additionally, CVPa has a lower molecular weight (29,783 Da) than CVPn (50,862 Da), making it more effective at promoting receptor dimerization, significantly activating the NF-κB signaling pathway, and inducing iNOS/TNF-α expression compared with CVPn. Therefore, CVPa exhibits significantly stronger immunomodulatory activity than CVPn.

Investigations have demonstrated that some tonifying polysaccharides lack significant immunomodulatory activity on their own, but their immunomodulatory activity is significantly enhanced after structural modification or functionalization (e. g., sulfation, phosphorylation, selenization, carboxymethylation, acetylation). These modifications also alter their properties, including molecular weight, solubility, and thermal stability (129). LONG et al. (130) reported that Codonopsis pilosula polysaccharide-modified selenium nanoparticles (CPP-SeNPs) significantly improved immune organ indices, inhibited tumor growth, and increased tumor cell apoptosis in immunosuppressed mice. Furthermore, CPP-SeNPs dose-dependently enhanced the splenic lymphocyte stimulation index, NK cell killing activity, and macrophage phagocytosis activity, and enhanced tumor-specific recognition and killing, thereby showing promise as an adjuvant for anti-tumor immunotherapy. LI et al. (131) extracted polysaccharides from Marsdenia tenacissima (Roxb.) and isolated MTP70-1, which has a relative molecular weight of 2738 Da and a main chain primarily composed of (1→6)-linked-D-Glcp and (1→4)-linked-D-Galp. MTP70-Zn was synthesized by introducing zinc into MTP70-1. Both MTP70–1 and MTP70-Zn enhanced macrophage phagocytic activity and promoted the release of cytokines (e. g., TNF-α, IL-6, IL-1β, IL-18) and NO synthesis, with MTP70-Zn showing a more pronounced effect. Additionally, both MTP70–1 and MTP70-Zn influenced TLR4-MyD88-NF-κB pathway-associated proteins by promoting macrophage cytokine expression, with MTP70-Zn exhibiting a higher binding strength for TLR4 proteins. Collectively, MTP70-Zn exhibited stronger immunoreactivity. The corresponding derivatives were obtained by chemically modifying Atractylodes lancea (Thunb.) DC polysaccharide (ALP-1) (132) through carboxymethylation (C-ALP-1), phosphorylation (P-ALP-1), and acetylation (A-ALP-1). These derivatives exhibited increased solubility (most pronounced for A-ALP-1), increased molecular weight and thermal stability, and decreased crystallinity. The chemical modifications significantly enhanced the immunomodulatory activity of ALP-1: P-ALP-1 was most effective in inducing NO synthesis by RAW264.7 macrophages, while A-ALP-1 was most effective in promoting TNF-α and IL-6 release. Poria cocos polysaccharides (PCP) (133) exhibit poor water solubility. By introducing hydrophilic carboxymethyl groups, two carboxymethyl pachymaran derivatives (CMP-1 and CMP-2) were prepared, which significantly improved their water solubility. Additionally, both CMP-1 and CMP-2 significantly promoted the proliferation and phagocytic capacity of RAW264.7 cells, with CMP-1 exhibiting a significantly greater effect on increasing NO, TNF-α, and IL-6 levels than CMP-2. Following sulfation and hydrolysis modifications of Polygonatum sibiricum polysaccharides, over-sulfated (OS) and hydrolyzed (HP) derivatives were obtained. Among these, HP significantly upregulated the expression of iNOS and IL-1β mRNA and activated the MR and TLR4 signaling pathways; whereas OS enhanced the cytotoxic activity of NK cells against HT-29 cells, upregulated the expression of IFN-γ and FasL genes, and promoted their interaction with CR3/TLR2 on the surface of NK cells, thereby enhancing immune regulatory activity (134).