Antagonism Of Ganoderma Lucidum Polysaccharides On Immune Suppression Of Peritoneal Macrophages Caused By Supernatants Of Melanoma Cells

Objective:Melanoma is a malignant tumor of melanocytes origin. Because of its high degree and poor prognosis, it is a great threat to human health. The incidence and prognosis of the disease is closely related to the immune system. Macrophage is important in the innate immune system. It performs anti-tumor effects not only by killing tumor cells directly, but also by presenting antigen. Macrophages bind to tumor cells and release lysosomal enzyme to kill tumor cells directly, process and present tumor cell antigens to T cells as well as secrete cytokines to kill tumor cells indirectly. However, tumor cells can evade the attacks by the immune system and inhibit immune function through many ways. They secrete immunosuppressors which can restrain many kinds of immune effect cells such as lymphocytes, macrophages, LAK cells and NK cells. Ganoderma lucidum polysaccharides (Gl-PS), an important bioactive of Ganoderma lucidum, can activate macrophages and increase macrophages secrete NO, TNF-a et al. This study investigates the antagonism of the Ganoderma lucidum polysaccharides on immune suppression of peritoneal macrophages caused by supernatants of melanoma cells (B16F10).Methods:1. Cells:B16F10cell is a high metastastic melanoma cell line of C57BL/6j mouse origin. Peritoneal macrophages were freshly prepared from C57BL/6j. L929cell was a cell line derived from mouse fibroblasts.2. Preparation of supernatant of B16F10cell culture:2×104/ml B16F10cells were seeded into the culture bottles at10ml/bottle, and the media were replaced with fresh one when the cells grown to80%of the bottom and further 8h culture were continued. The supernatant of the cell culture were harvested, filtered with0.22μm filter, and stored at-20℃.3. Experimental sub-groups:B16F10supernatant, B16F10supernatant with different concerntration of Gl-PS (0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), and RPMI1640normal control group. Each group contains the same concentration of LPS (10μg/ml).4. MTT colorimetry for peritoneal macrophages:MTT colorimetry was applied to investigate the effects of the antagonism of Gl-PS on suppression of peritoneal macrophages’viability caused by supernatants of melanoma cells.5. Neutral red phagocytosis:Neutral red uptake assay was applied to investigate the effects of the antagonism of Gl-PS on suppression of peritoneal macrophages phagocytosing neutral red caused by supernatants of melanoma cells.6. Griess nitric oxide assay:Griess nitric oxide assay was applied to investigate the effects of the antagonism of Gl-PS on suppression of NO release in peritoneal macrophages caused by supernatants of melanoma cells.7. Immunocytochemistry:Immunocytochemistry was applied to investigate the effects of the antagonism of Gl-PS on suppression of TNF-a secretion in peritoneal macrophages caused by supernatants of melanoma cells.8. MTT colorimetry for L929cells:MTT colorimetry was applied on L929cells to investigate the effects of the antagonism of Gl-PS on suppression of TNF-a secretion in peritoneal macrophages caused by supernatants of melanoma cells.9. Western blot:Western blot was applied to investigate the effects of the antagonism of Gl-PS on suppression of TNF-a secretion in peritoneal macrophages caused by supernatants of melanoma cells as well.10. Statistical analysis:The statistical software of SPSS13.0was used. The data was analyzed with Turkey and Donnet t test after one way ANOVA. P values<0.05were considered significant.Result: 1. Suppressive effects of B16F10culture supernatants on peritoneal macrophages:It was shown that with the presence of supernatants of B16F10culture supernatants without Gl-PS, the viability, Neutral red phagocytosis and release of NO in peritoneal macrophages were0.411±0.035,0.596±0.100and4.830±1.384respectively, all much lower than those of0.861±0.208,1.275±0.102and11.455±0.175in normal control group respectively, with statistical differences between each groups after ANOVA (p<0.05). Detected by Western-blot assay and L929proliferation assay, with the presence of supernatants of B16F10culture supernatants without Gl-PS, the expression and activity of TNF-a in peritoneal macrophages were3.221±0.333and0.387±0.030respectively, all much lower than those of0.84±0.213and0.746±0.049in normal control group respectively, with statistical differences between each groups after ANOVA (p<0.05).2. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages after activation by LPS:It was shown by MTT assay that with the presence of supernatants of B16F10culture, different concerntration of Gl-PS acting on syngenic mouse peritoneal macrophages with the induction by LPS, detected24h after, the assays were0.439±0.041,0.492±0.037,0.679±0.051,0.818±0.123respectively, obviously higher than that of supernatants without Gl-PS,0.411±0.035, lower than that of RPMI1640control0.861±0.208, with remarkable statistical significance (F=21.339,p<0.05). Except for B16F10supernatant vs0.2μg/ml,0.8μg/ml;0.2μg/ml vs0.8μg/ml;3.2μg/ml vs12.8μg/ml, normal control group;12.8μg/ml vs normal control group, all differences among groups were statistically significant (p<0.05).3. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages phagocytose neutral red after activation by LPS:It was shown by Neutral red phagocytosis assay that with the presence of supernatants from B16F10culture, different concerntration of Gl-PS acting on syngenic mouse peritoneal macrophages with the induction by LPS, detected24h after, the assays were0.686±0.056,0.765±0.037, 0.778±0.046,0.787±0.057respectively, obviously higher than that of supernatants without Gl-PS0.596±0.100, lower than that of RPMI1640control1.275±0.102, with remarkable statistical significance (F=67.054, p<0.05). Except for B16F10supernatant vs0.2μg/ml;0.2μg/ml vs0.8μg/ml,3.2μg/ml,12.8μg/ml;0.8μg/ml vs3.2μg/ml,12.8μg/ml;3.2μg/ml vs12.8μg/ml group, all differences among groups were statistically significant (p<0.05).4. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages to release NO after activation by LPS:It was shown by Griess nitric oxide assay that with the presence of supernatants from B16F10culture, different concerntration of Gl-PS acting on syngenic mouse peritoneal macrophages with the induction by LPS, detected24h after, the assays were5.749±1.109,5.717±1.131,6.081±1.597,8.060±1.375respectively, obviously higher than that of supernatants without Gl-PS4.830±1.384, lower than that of RPMI1640control11.455±0.175, with remarkable statistical significance (F=12.032,p<0.05). Except for B16F10supernatant and every different concerntration of Gl-PS groups, all differences among RPMI1640control group were statistically significant (p<0.05).5. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages expression of TNF-a after activation by LPS, detected with immunocytochemistry:It was shown by immunocytochemistry that with the presence of supernatants from.B16F10culture, different concerntration of Gl-PS acting on peritoneal macrophages with the induction by LPS, detected24h after, the suppression were obviously reduced. The expression of TNF-a were obviously stronger than that of supernatants without Gl-PS, but lower than that of RPMI1640control group.6. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages secrete TNF-a after activation by LPS, detected with L929Proliferation:It was shown by L929proliferation assay that with the presence of supernatants from B16F10culture, different concerntration of Gl-PS acting on syngenic mouse peritoneal macrophages with the induction by LPS, detected48h after, the assays were0.600±0.032,0.599±0.084,0.550±0.076,0.496±0.045respectively, obviously lower than that of supernatants without Gl-PS0.746±0.049, higher than that of RPMI1640supernatant0.387±0.030, each lower than that of RPMI1640control0.897±0.038, with remarkable statistical significance (F=57.145, p<0.05). Except for0.2μg/ml vs0.8μg/ml,3.2μ.g/ml;0.8μg/ml vs3.2μg/ml;3.2μg/ml vs12.8μg/ml group, all differences among groups were statistically significant (p<0.05).1. Antagonism of the Gl-PS against suppressive effects of supernatants from B16F10culture on peritoneal macrophages secrete TNF-a after activation by LPS, detected with Western Blot:It was shown by Western Blot that with the presence of supernatants from B16F10culture, different concerntration of Gl-PS acting on syngenic mouse peritoneal macrophages with the induction by LPS, detected24h after, the relatively expression level of TNF-α were1.068±0.243,1.367±0.138,1.753±0.083,2.006±0.323respectively, obviously higher than that of supernatants without G/-PS (0.84±0.21), lower than that of RPMI1640control3.221±0.333. With remarkable statistical significance (F=38.296,p<0.05). Except for B16F10supernatant vs0.2μg/ml group;0.2μg/ml vs0.8μg/ml;0.8μg/ml vs3.2μg/ml;3.2μg/ml vs12.8μg/ml group, all differences among groups were statistically significant (p<0.05).Conclusion:1. Supernatant from B16F10melanoma cell showed the immune-suppressive effects on peritoneal macrophages of syngenic mouse.2. Gl-PS antagonised the immune-suppressive effects of supernatant from B16F10melanoma cell culture on peritoneal macrophages of syngenic mouse.