Hong-Xiang Sun

Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms

The polysaccharide from the roots of Actinidia eriantha (AEPS), a potent antitumor agent and immunological adjuvant, was investigated for the immunomodulatory effects on RAW264.7 macrophages and its molecular mechanisms. AEPS could significantly enhance the pinocytic and phagocytic activity, induce the production of NO, TNF-α, IL-10, IL-1β and IL-6, and promote the expression of accessory and costimulatory molecules in RAW264.7 cells. PCR array assay revealed that AEPS up-regulated 28 genes including TLRs (TLR2, TLR8, TLR9), proinflammatory factors (IL-1β, G-CSF, IL-1α, GM-CSF, IL-6, COX-2, TNF-α, IFN-β, CXCL10, CCL2, TNF-β, IL-10), and the genes involved in NF-κB signaling pathway, and down-regulated 6 genes such as TLR3, TLR4, PGLYRP1, EIF2αK2, MAP3K1 and IRF1. AEPS was further showed to promote cytoplasmic IκB-α degradation and increase nuclear NF-κB p65 levels in RAW264.7 cells. These results suggested that AEPS activated RAW264.7 macrophages and elicited a M1 and M2 response through TLRs/NF-κB signaling pathway.

Structure–function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity

To assess the contribution of the aglycone and sugar chain to the biological activity of saponins from Platycodon grandiflorum, seven structurally consecutive saponins, platycodin D (PD), D2 (PD2), D3 (PD3), platycoside A (PA), E (PE), deapioplatycoside E (DPE), and polygalacin D2 (PGD) were compared for their hemolytic activities and adjuvant potentials on the immune responses to Newcastle disease virus-based recombinant avian influenza vaccine (rL-H5) in mice. Among seven compounds, the order of the hemolytic activity was PGD ≈ PD > PD2 > PA > PD3 > PE > DPE. PD, PD2, PA, and PGD significantly not only promoted concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-induced splenocyte proliferation, but enhanced the NK cell activity in mice immunized with rL-H5. PD and PD2 increased the antigen specific IgG, IgG1, IgG2a, and IgG2b antibody titers, while PA and PGD only induce the IgG and IgG1 antibody responses in the immunized mice. However, the other three saponins were not observed for adjuvant activity. The results suggested that the sugar chains attached to C-3, the glycidic moiety at C-28 of aglycone, as well as aglycone affect their biological activities. Interestingly, their hemolytic and adjuvant activities increased with the retention time by reverse phase HPLC analysis. The retention time may be useful for primary estimation of fundamental adjuvanticity of saponin with the same aglycone.

Anti-tumor and immunomodulatory activity of peptide fraction from the larvae of Musca domestica.

ETHNOPHARMACOLOGICAL RELEVANCE The larvae of Musca domestica (Diptera: Muscidae) have been used traditionally for malnutritional stagnation, decubital necrosis, osteomyelitis, ecthyma and lip scald and also to treat coma and gastric cancer in the traditional Chinese medicine. Its in vitro antitumor activity and immunomodulatory effect in naïve mice in relation to the traditional uses were also reported. However, the in vivo antitumor effect of this insect and its mechanism of action have not yet been well studied. The objectives of this study were to evaluate the in vivo antitumor potential of the peptide fraction from Musca domestica larvae (MDPF) and to elucidate its immunological mechanisms. MATERIALS AND METHODS The mice inoculated with sarcoma S180 cells were orally administered with MDPF at three doses for 10 days. The effects of MDPF on the growth of mouse S180 sarcoma, splenocyte proliferation, the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), production and mRNA expression of cytokines from splenocytes, and serum antigen-specific antibody levels in tumor-bearing mice were measured. RESULTS MDPF could significantly not only inhibit the growth of mouse transplanted S180 sarcoma, but also promote splenocytes proliferation, NK cell and CTL activity from splenocytes, and enhance serum antigen-specific IgG, IgG2a and IgG2b antibody levels in S180-bearing mice. MDPF also significantly promoted the production of IFN-γ and up-regulated the mRNA expression levels of IFN-γ and Th1 transcription factors T-bet and STAT-4 in splenocytes from the S180-bearing mice. However, Th2 cytokine IL-10 and transcription factors GATA-3 and STAT-6 were not significantly changed both at transcriptional and protein levels following MDPF treatment. CONCLUSIONS MDPF significantly inhibit the growth of transplantable tumor in mice and its in vivo antitumor activity might be achieved by switching-on of Th1-based protective cell-mediated immunity. MDPF could act as antitumor agent with immunomodulatory activity.

Platycodin D Improves the Immunogenicity of Newcastle Disease Virus-Based Recombinant Avian Influenza Vaccine in Mice

The development of an effective influenza vaccine is urgently important for controlling outbreaks of the highly pathogenic avian influenza virus (HPAIV) and reducing the impact of pandemics. The use of an adjuvant in such a vaccine can significantly contribute to improve the immunogenicity. To explore a novel and safe adjuvant for improving the potency of influenza vaccines, platycodin D (1), a saponin from the root of Platycodon grandiflorum, was evaluated for the adjuvant potentials on the specific cellular and humoral immune responses to Newcastle disease virus‐based recombinant avian influenza vaccine (rL‐H5) in mice. Compound 1 significantly promoted the concanavalin A (Con A)‐, the lipopolysaccharide (LPS)‐, and the antigen‐induced splenocyte proliferation and enhanced the serum antigen‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers (P<0.05, P<0.01, or P<0.001) in mice immunized with rL‐H5. The mRNA expressions of Th1/Th2 cytokines (IFN‐γ and IL‐10) and transcription factors (T‐bet and GATA‐3) in splenocytes were also markedly up‐regulated by 1, compared with the control group immunized with rL‐H5 alone (P<0.01 or P<0.001). In addition, 1 remarkably increased the killing activities of natural killer (NK) cells from splenocytes in the immunized mice (P<0.05), which may have important implications for the vaccination against the avian influenza virus. We concluded that 1 could improve the immunogenicity of the rL‐H5 vaccine by enhancing both humoral and cellular immune responses in mice, and that 1 is a promising adjuvant for influenza vaccines.

Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L.

With manganese superoxide dismutase expressed in silkworm larvae, Bomby mori L, we investigate the effects of silkworm larvae powder containing SOD on the antioxidation and the immune system of mouse. The contents of MDA both in mice plasma or liver organ treated with silkworm larvae powder containing manganese superoxide dismutase were reduced compare to control. The superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) activities both in plasma or liver organ of the treated mice were significantly higher than that of both control and bromobenzene treated mice (group‐BM), suggesting the silkworm larvae powder containing SOD play a positive role in anti‐oxidation in mice. This experiment was also designed to investigate the effects of silkworm larvae powder containing SOD on the immune system of mouse, focused on hemolysin response, hemagglutination against SRBC and the activity of natural killer (NK) cells. All treated mice showed significant increase in hemolysin response to SRBC and demonstrated an activation of NK cell function by the SOD‐contained silkworm larvae powder, which suggest a promotion in humoral immunity. The results suggested the SOD expressed in silkworm maybe have potential application in medicine.

Adjuvant-active fraction from Albizia julibrissin saponins improves immune responses by inducing cytokine and chemokine at the site of injection.

The total saponin from the stem bark of Albizia julibrissin (AJSt) has previously showed the adjuvant potentials in mice. In this study, AJSt was subjected to resin column chromatography to afford four fractions (AJS30, AJS50, AJS75 and AJS95), and these fractions were further compared for the hemolytic activities and adjuvant potentials on the immune response to ovalbumin (OVA) and recombinant fowl pox virus vector-based avian influenza vaccine (rFPV). AJSt, AJS50, AJS75 and AJS95 showed a slight hemolytic effect. AJSt, AJS50 and AJS75 significantly enhanced not only the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-stimulated splenocyte proliferation, but also serum antigen-specific IgG, IgG1, IgG2a and IgG2b antibody titers in the mice immunized with OVA and rFPV. AJSt, AJS50 and AJS75 also significantly promoted the NK cell activity and delayed-type hypersensitivity (DTH) in the OVA-immunized mice. Furthermore, the mechanisms of adjuvant action were explored by determining the effects of AJS75 on cytokines and chemokines at the site of injection using antibody array. AJS75 induced or up-regulated the protein expression of 12 cytokines (IL-12p40, IL-12p40/p70, IFN-γ, IL-13, IL-1β, IL-6, IL-10, TNF-α, sTNFR I, sTNFR III, IL-3 and IL-9) and 10 chemokines (Eotaxin, I-TAC, MIG, MIP-1α, RANTES, TECK, Fracatlkine, FasL, M-CSF and GM-CSF) in the injected muscles. The results suggested that AJS75, the most adjuvant-active fraction of AJSt, could improve antigen-specific both cellular and humoral immune responses and simultaneously elicit a Th1/Th2 response by inducing cytokine and chemokine at the site of injection.

Immunomodulatory activity of 3β,6β-dihydroxyolean-12-en-27-oic acid in tumor-bearing mice

3β,6β‐Dihydroxyolean‐12‐en‐27‐oic acid (1) is a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis. To evaluate the in vivo antitumor potential and to elucidate its immunological mechanisms, effect of 1 on the growth of mouse‐transplantable tumors, and the immune response in naive and tumor‐bearing mice were investigated. The mice inoculated with mouse tumor cell lines were orally treated with 1 at the doses of 40, 60, and 80 mg/kg for 10 days. The effects of 1 on the growth of mouse‐transplantable S180 sarcoma and H22 hepatoma, splenocyte proliferation, cytotoxic T lymphocyte (CTL) activity, natural killer (NK) cell activity, and production of interleukin‐2 (IL‐2) from splenocytes in S180‐bearing mice were measured. Furthermore, the effect of 1 on 2,4‐dinitrofluorobenzene (DNFB)‐induced delayed‐type hypersensitivity (DTH) reactions and the sheep red blood cell (SRBC)‐induced antibody response in naive mice were also studied. Compound 1 could not only significantly inhibit the growth of mouse transplantable S180 sarcoma and H22 hepatoma, increase splenocytes proliferation, CTL and NK cell activity, and the level of IL‐2 secreted by splenocytes in tumor‐bearing mice, but also remarkably promote the DTH reaction and enhance anti‐SRBC antibody titers in naive mice. These results suggested that 1 could improve both cellular and humoral immune response, and could act as antitumor agent with immunomodulatory activity.

Comparison of immunosuppressive activity of Stephanoside E and its aglycone from Stephanotis mucronata in vitro

Stephanoside E (STE) and its aglycone Stephanthraniline A (STA) isolated from the stems of Stephanotis mucronata (Blanco) Merr. were evaluated for their suppressive potentials on Th1 and Th2 immune responses in vitro, and the effects of the C-3 sugar chains on their activities were discussed. STE and STA significantly inhibited Con A and LPS-induced splenocyte proliferation, and reduced the production of Th1/Th2 cytokines (IL-2, IFN-γ, IL-4, and IL-10) from Con A-stimulated splenocytes in a concentration-dependent manner. The mRNA expression levels of Th1/Th2 cytokines (IL-2, IFN-γ, IL-4, and IL-10) and transcription factors (T-bet and GATA-3) in Con A-stimulated splenocytes were also suppressed by STE and STA. The suppressive effect of STA on Th1 and Th2 immune responses in vitro was stronger than that of STE. These findings suggested that both STE and STA could simultaneously inhibit Th1/Th2 immune responses, and the glycosyl formation at the position C-3 of STA could decrease the immunosuppressive activity in vitro.

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 (1), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D (2), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and HBsAg‐induced splenocyte proliferation in HBsAg‐immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL‐2 and IFN‐γ) and Th2 (IL‐4 and IL‐10) cytokines from splenocytes in the HBsAg‐immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg‐specific IgG2a and IgG2b antibody response, as well as Th1‐cytokine secretion from splenocytes in the HBsAg‐immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1‐ and Th2‐potentiating activity.

Stephanthraniline A suppressed CD4(+) T cell-mediated immunological hepatitis through impairing PKCθ function.

Stephanthraniline A (STA), a C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., was previously shown to inhibit T cells activation and proliferation in vitro and in vivo. The purpose of this study was to further evaluate the in vivo immunosuppressive activity of STA and to elucidate its potential mechanisms. The results showed that pretreatment with STA significantly attenuated concanavalin A (Con A)-induced hepatitis and reduced CD4(+) T cells activation and aggregation in hepatic tissue in mice. STA directly suppressed the activation and proliferation of Con A-induced CD4(+) T cells, and inhibited NFAT, NFκB and MAPK signaling cascades in activated CD4(+) T cells in vitro. Moreover, it was proved that STA inhibited T cells activation and proliferation through proximal T cell-receptor (TCR) signaling- and Ca(2+) signaling-independent way. The molecular docking studies predicted that STA could tight bind to PKCθ via five hydrogen. The further findings indicated STA directly inhibited PKCθ kinase activity, and its phosphorylation in activated CD4(+) T cells in vitro. Collectively, the present study indicated that STA could protect against CD4(+) T cell-mediated immunological hepatitis in mice through PKCθ and its downstream NFAT, NFκB and MAPK signaling cascades. These results highlight the potential of STA as an effective leading compound for use in the treatment of CD4(+) T cell-mediated inflammatory and autoimmune diseases.