| It had been proved that polysaccharides possess immunomodulatory, anti-inflammatory, anti-cancer, lowering blood sugar, lowering blood pressure and other functions, which immune regulation is a basic role of many polysaccharides, and this regulation effect through the following ways mainly: activate the macrophages, NK cells and T, B lymphocytes directly or indirectly; promote the formation and secretion of cytokines; activate the complement system; promote antibody production and so on. Recent studies had found that polysaccharides activate immune cells play a role in immune regulation mainly mediated by the recognition of cell surface receptor. Currently found receptors included Toll-like receptors(TLRs), mannose receptor(MR), dendritic cell-associated C-type lectin-1(Dectin-1), complement receptor 3(CR3), scavenger receptor(SR) and so on. After the polysaccharide binding to these receptors could activate intracellular signaling pathways, which results in activation of immune cells, promoting the release of cytokines to regulate the immune response. MR may recognize many sugar molecules containing mannose or fucose residues, which is an important pattern recognition receptors and endocytosis receptors in the innate immune system, mainly present in the cell surface of macrophages and dendritic cells. It is play an important role in maintain homeostasis, identification of pathogens, inducing cytokines, antigen presentation and other processes. TLRs are currently found have the closest relationship with polysaccharides among all the receptors.Ganoderma atrum is a traditional Chinese medicinal fungi and folk food ingredients, it has many biological activities. Our laboratory separate and purified a water-soluble polysaccharide from Ganoderma atrum(PSG-1), which using modern chemical analysis method to identify its structural characteristics, the results showed that PSG-1 was composed of mannose, galactose and glucose and the molar ratio is 1:1.28:4.91. The previous study has confirmed PSG-1 can enhance the immune function of the host. On the basis of this research, it has important significance to explore MR in order to understand the activity function and immune mechanism of PSG-1 deeply, and the depth development of Ganoderma atrum based on targeting MR. Therefore, our study discussed the relationship between the immune regulation mechanism of PSG-1 and MR and the potential relationship between TLRs signaling pathway and MR activate by PSG-1 in LPS-induced macrophage. The main research contents are as follows:1. Effect of PSG-1 on MR of mouse peritoneal macrophages: in order to study the effect of PSG-1 on MR of normal mouse peritoneal macrophages, our experiment use 20~160 μg/mL PSG-1 act on macrophages and then the MR expression on the macrophage surface was analyzed by flow cytometry, while the mRNA expression level of MR was evaluated by reverse transcription polymerase chain reaction(RT-PCR); after the cells were pretreated with mannan(an MR inhibitor) at 30 minutes and then exposed to PSG-1, the phagocytosis of macrophages was assayed by flow cytometric method, the contents of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell culture supernatant were measured by enzyme-linked immunosorbent assay(ELISA), and the mRNA expression levels of TNF-α and IL-1β were determined by RT-PCR. The results showed that PSG-1 can upregulate the expression and the mRNA level of MR on macrophage; compared with the group treated with PSG-1 alone, the phagocytosis of macrophages and the secretion of IL-1β in cells pretreated with mannan were significantly inhibited and no effect on the secretion of TNF-α was observed in the presence of PSG-1. Conclusion: MR can partly mediate the immunoregulatory effect of PSG-1 on peritoneal macrophages in mice.2. Effect of PSG-1 on MR of LPS-stimulated mouse peritoneal macrophages: in order to investigate the effect of PSG-1 on MR of mouse peritoneal macrophages stimulated by LPS, we used the flow cytometry to detect the expression of MR and the RT-PCR to examine the mRNA expression level of MR for explore the effect of PSG-1 in LPS-stimulated macrophages, the results proved that the stimulation of LPS and then down-regulation of MR was up-regulated by PSG-1in inflammation model of mouse peritoneal macrophages stimulated by LPS. Further treatment with anti-MR antibody to the macrophages of the cooperation effect of LPS and PSG-1 after 30 minutes, the results showed that anti-MR antibody can significantly increase the ability of phagocytosis, the secretion of IL-1β and the mRNA level of IL-1β on which inhibited by PSG-1 in LPS-induced macrophage, but decrease the secretion of IL-10 and the mRNA level of IL-10 on which promoted by PSG-1 in LPS-induced macrophage. In addition, it did not show any effect to the secretion of TNF-α and the mRNA level of TNF-α on which inhibited by PSG-1 in LPS-induced macrophage. Conclusion: The immune regulation effect of PSG-1 on cell depends on the participation of MR in LPS-stimulated mouse peritoneal macrophages.3. Effect of PSG-1 on TLR2 and TLR4 of LPS-stimulated mouse peritoneal macrophages and the relationship between them and MR in the immune regulation effect mediated by PSG-1: our experiment used Western Blot to detect the expression of TLR2, TLR4 and the RT-PCR to examine the mRNA expression level of TLR2, TLR4 for study the effect of PSG-1, the experimental results showed that PSG-1 can regulate the function of cell by acting on TLR4 in LPS-induced macrophage. Based on the above experimental results and considering the effect of PSG-1 on MR in the previous two chapters, we further studied that after inhibition of MR whether can affect the signal pathway mediated by TLR4 by using the anti-MR antibody to block the function of MR. The results suggested that the MyD88-dependent pathway of TLR4 was activated and the expression of NF-κB p65 in nucleus, the phosphorylation of MAPKs were also increased in LPS-stimulated macrophages; while adding high dose of PSG-1(160 g/m L) together affect the cell, the expression of TLR4 was down regulated, the high expression of NF-κB p65 in nucleus decreased and the phosphorylation of MAPKs was also inhibited, thus it is ensure PSG-1 was indeed affect the signal transduction pathway mediated by TLR4; when the cells pretreated with anti-MR antibody, the results showed that only have the callback effect to the expression of NF-κB p65 in nucleus due to the reduced expression of PSG-1’s effcet, while had no obvious influence on protein expression of the other molecular compared with the group which has combined effect of LPS and PSG-1.Conclusion: The effect of PSG-1 on the expression of NF-κB p65 protein in nucleus may be regulated by TLR4 and MR in LPS-stimulated mouse peritoneal macrophages.In summary, PSG-1 can affect the immune regulating function of mouse peritoneal macrophages through MR. TLR4 and MR were involved in immune regulation mediated by PSG-1 and there were existed a certain interaction relationship between them in LPS-stimulated mouse peritoneal macrophages. Our experimental not only had provided a theoretical basis for improving the molecular mechanism of immune regulation related to the polysaccharides, but also provided some ideas to expand the further research directions of polysaccharides. |
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