Ginsenoside Rg1 Effects On Dendritic Cells And Molecular Mechanisms

In Chinese medicine, ginseng has long been used as a general tonic to promote longevity and enhance bodily functions. It has also been claimed to be effective in combating stress, fatigue, oxidants and cancer.Ginsenoside Rgl is one of ginseng's active ingredients and has anti-tumor, angiomodulating and steroid-like activities through investigating its pharmacogenomics from the perspective of the Yin/Yang therory. Though celluar immunity mediated by T cells,NK cells and macrophages is predominant in tumor immunity, humoral immunity plays an important role in the virus-induced tumors. However, dendritic cells (DC), which is the most powerful professional antigen presenting cells, can capture soluble antigens secreted by tumor cells, present them to be recongnized by CD4+T cells by the MHCⅠor MHCⅡmolecules restricted manner and finally activate CD8+T or CD4+T cells which play anti-tumor effect. In addition, CD4+T cells also involved in activation of B cells, macrophages, NK cells and CTL, synergistic antitumor effects. It is reported that ginsenoside Rgl as the main component of ginsenoside improved the non-specific immune function in mice, however, wheather this enhancement is mediated by regulating the functions of DCs remains unknown. Gene chip technology has advantages in studying the regulation of the immune system and finding out the key molecules on the immune cells influence by drugs. This study, is focused on the influence which ginsenoside Rgl has on the specific immune responses in BALB/c mice as well as the key molecules on DCs which ginsenoside Rgl targets by gene chip technology, in order to find out the target signal passway of ginsenoside Rgl on DCs and provide the experimental evidence for the therapeutic principles of strenghthening body resistance and eliminating evil in TCM, the two-direction regulation of ginseng as well as ginsenoside Rg1 as the main composition of new anti-tumor Chinese medicines.Method1. At first, the influence of ginsenoside Rg1 on immune responses immunized by ovalbumin (OVA) in BALB/c mice was investigated.50μg OVA mixed ginsenoside Rgl or aluminum hydroxide (Al (OH) 3) adjuvant, were given to immunize BALB/c mice three times at a two-week interval.10 days after three immunizations, lymphocyte proliferation, specific total IgG and IgG subclass, cytokines IL-4 and IFN-y secreted by splenocytes from culture supernatants were measured respectively by MTT assay and ELISA.2. The influence of ginsenoside Rg1 on the surface molecules of bone marrow derived dendritic cells. After culturing bone marrow derived cells stimulated by Granulocyte macrophage colony stimulating factor (GM-CSF) and Interleukin-4 for 7 days, bone marrow derived dendritic cells (BM-DCs) were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours. Finally, the expression of the surface molecule I-A /I-E, CD40 and CD86 were detected by flow cytometry in order to investigate whether ginsenoside Rg1 could promote immature DCs to mature.3. In this study, the impacts of ginsenoside Rgl on the secretion of cytokines, phagocytosis and antigen-presenting of murine bone marrow dendritic cells were analyzed. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours or 48 hours. Then culture supernatant were collected and the sectretion of cytokines IL-12, IL-4, IL-10 levels by an indirect ELISA assay. In addition, after BM-DCs were treated by ginsenoside Rgl and then FITC-OVA for 1 hour, the phagocytosis was detected by flow cytometry. Finally, the influence of ginsenoside Rg1 on antige-presenting of BM-DCs were detected by IL-2 secreted from active B3Z T cells.4. The influence of ginsenoside Rg1 on the expression of functional gene from mouse bone marrow-derived DC was analyzed by gene chip technique. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours and then the total RNA was extracted. Oligo dendritic &antigen presenting cell gene chips were utilized to find out the difference genes.5.With GAPDH as the housekeeping gene, was extracted RNA, the same amount of RNA as template, the gene specific primers, using semi-quantitative RT-PCR and semi-quantitative detection of p44 mRNA expression levels EGFR; DC lysis cell extracts after incubation of protein, total protein run SDS-PAGE after the transfer film by Western bolt analysis EGFR and p44 protein secretion, and whether the aforementioned cytokine ELISA assay consistent with the results, thus confirming whether ginsenoside Rg1 by EGFR-p44 MAPK pathway on the regulation of DC generation.Result1. Among the different groups, body weights did not differ significantly. OD values of OVA-specific IgG antibodies responses are ranging in descending order:OVA<(OVA+ Rgl)<(OVA+Al). Among them, those from (OVA+Rg1) group were significantly higher than those from OVA group (p<0.01). In addition, IgG1, IgG2a, IgG2b, IgG3 antibody levels from(OVA+Rg1) group were significantly higher than those from OVA group (p<0.05); IgG1, IgG3, IgG2b levels from (Al+OVA) group were obviously higher while its IgG2a responses didn't increase significantly compared with the NS group. What's more, IgG1 and IgG3 responses from OVA-immunized alone group were evidently higher than those from NS group. In cellular immunity, the stimulation index of splenocytes from (OVA+Rg1) group was significantly enhanced compared with that from OVA group. Finally, the ELISA results showed that IFN-γlevels from (OVA+Rg1) group were the highest, and significant difference existed between that from (OVA+Rgl) group and OVA group (p=0.03) while (OVA+Al) groups secreted significantly higher levels of IL-4 compared with OVA group.2. After cultivating bone marrow derived hematopoietic stem cells for 7 days, more than 85% of the dendritic cells were collected. The surface molecule I-A/I-E, CD40 and CD86 on DCs did not change significantly after DCs were treated with ginsenoside Rgl at a concentration of 40μg/ml. However, after adding LPS, those were significantly higher (p <0.05, compared with the media control group). In addition, the cell surface I-A/I-E molecules on DCs from ginsenoside Rgl plus LPS group was significantly increased in (p <0.05, compared with LPS group) while CD40 and CD86 remained unchanged.3. Ginsenoside Rgl not only promoted DC in vitro to secrete higher levels of IL-12 but also strengthened this promotion if added with LPS simultaneously. Secondly, ginsenoside Rgl promoted immature dendritic cells to secrete IL-4 and IL-10 molecules. What's more, ginsenoside Rgl had an obvious influence on the phagocytosis of FITC-OVA antigen for immature DCs.Finally, ginsenoside Rgl promoted mature DCs to process and present peptides which activated B3Z T cells to secrete obviously higher levels of IL-2.4. A260/A280 ratios were about 2.0 for RNA extraction from dendritic cells. In denaturing agarose gel electrophoresis, bands were clear and 28s rRNA band intensity is nearly twice as much as that for 18s rRNA.There were 23 genes whose ratios of Rgl group to media group (Ⅱ/Ⅰ) were above 2, and 45 genes below 0.5. As for those of LPS group to media group (Ⅲ/Ⅰ),there were 15 genes above 2 and 47 below 0.5. In addition, there were 35 above 2 and 15 below 0.5 for the ratios of Rgl+LPS to Rgl (Ⅳ/Ⅱ) while there were 37 above 2 and 10 below 0.5 for (Ⅳ/Ⅲ). Among those differently expressing genes, Erbb2 and Ifi44 showed obviously expressed in DCs stimulated by ginsenoside Rg1.5. The results of semi-quantitative RT-PCR and Western Blot indicated that in DCs stimulated by ginsenoside Rgl, both EGFR and p44 which is also called ERK2, assumed obviously higher transcription and expression levels, compared with the media group.Conclusion1. Ginsenoside Rgl can enhance the specific immune responses in OVA-immunized mice. 2. Though ginsenoside Rgl can't promote the maturation of imDCs, it can up-reguate the expression of I-A/I-E molecules in mDCs. In addition, it can strengthen the phagocytosis of imDCs. Finally, it may enhance the antigen-presenting capability of mDCs.3. The differently expressing genes in DCs stimulated by ginsenoside Rgl are involved in multiple processing including the migration, phagocytosis, antigen-presenting and secreting cytokines in DCs. It is worthwhile that ginsenoside Rgl may up-regulate the EGFR-MAPK passway in order to secret IL-10 which influences the Th polarization and regulate the immune response.In conclusion, though ginsenoside Rgl can't promote the maturation of imDCs, it can strenghthen the photocytosis, antigen-presenting and the secretion of cytokines in murine DCs. These might be involed in multiple targets. Microarray and Western blot results indicated that ginsenoside Rgl might up-regulate the EGFR-MAPK passway to aid in secreting cytokines, which regulates the immune responses. However, whether the effect is direct or not need to be proved by further experiments such as biosensors or iRNA technology. What's more, ginsenoside Rgl enhances the specific immunity in OVA-immunized mice.This study proves that ginsenoside Rg1 has two-diection immune-regulation and up-regulate the EGFR-MAPK passway, which provides the experimental evidence for the therapeutic principles of strenghthening the Vital and Dispenlling the Pathogen in TCM, the two-direction regulation of ginseng as well as ginsenoside Rgl as the main composition of new anti-tumor Chinese medicines.