Andrographolide as an anti-H1N1 drug and the mechanism related to retinoic acid-inducible gene-I-like receptors signaling pathway.

OBJECTIVE: To observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1. METHODS: Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR). RESULTS: The optimal concentration of AD for anti-virus effect was 250 µg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-ß promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05). CONCLUSIONS: The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Immunologic mechanism of Patchouli alcohol anti-H1N1 influenza virus may through regulation of the RLH signal pathway in vitro.

Patchouli alcohol (PA) is a kind of methanol extracted from traditional Chinese medicine Pogostemonis Herba. Our research aimed to observe the anti-influenza virus role of PA in vitro. 16HBE (human respiratory epithelial cell) was infected by H1N1 (A/FM1/1/47) to set the cell model. Then the 16HBE was co-cultivated with three kinds of immune cells: dendritic cells, macrophages, and monocytes, PA (the concentration is 10 µg/mL) was added as a treatment intervention for 24 h. The immune cells and the supernate were collected for RT-PCR and ELISA detection related to RLH (RIG-1-like helicases) pathway. Results showed that the IL-4 and IFN-γ in supernate were increased after H1N1 infection, and the PA treatment suppressed the expression of cytokines and the mRNA of RLH pathway. PA anti-influenza virus may through regulate the RLH singal pathway.

The inducible effect of LBP on maturation of dendritic cells and the related immune signaling pathways in hepatocellular carcinoma (HCC).

OBJECTIVE: To investigate the effect of LBP on differentiation and maturation of healthy human peripheral blood-derived dendritic cells cultured in different tumor microenvironment in vitro, and discuss the molecular and immunological mechanisms of LBP in treatment of tumor. METHODS: In this study, we procured the peripheral blood-derived dendritic cells precursor cell by the Density gradient centrifugation method, and used the tumor-cell supernatant to prepare conditioned medium. The GM-CSF and IL-4 induced DCs precursor cell differentiation to DCs, the TNF-α promoted the immature DCs developed to mature DCs. In this way, we detected the influence of LBP on the expressions of surface molecules of DCs cultured in different environments, and especially on the role of related-immunity and NF-κB activity. RESULTS: In LBP-treated group, the molecular phenotype of DCs, its capacity to stimulate allogeneic lymphocyte proliferation, and the levels of IL-12p70 and IFN-γ secretion were higher than the untreated group (p < 0.05), with statistical significance. Meanwhile the expression of NF-κB of the DCs in the medium treated by the LBP was higher than the untreated group (p < 0.05), also with statistical significance. Between the two different tumor microenvironment groups, the cell nucleus protein NF-κB expression is obviously different, the hepG2.2.15 group higher than the hepG2 group. CONCLUSION: LBP could increase the expression of the phenotype of DCs, the secretion of IL-12p70 and IFN-γ in MLR, and enhance the NF-κB expression, especially in the virus-related group, suggesting LBP plays the anti-tumor role stronger in the virus-related environment and this phenomenon correlates with the NF-κB signaling pathway.

Carboxymethylpachymaran enhances immunologic function of dendritic cells cultured in two kinds of hepatoma carcinoma cell line's supernatant via nuclear factor κ B/Rel pathway.

OBJECTIVE: To study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs. METHODS: DCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis. RESULTS: The proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05). CONCLUSIONS: Two kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.