The Effects Of Ganoderma Lucidum Polysaccharides On Intestinal Mucosal Immune In The Mice Of H22 Liver Cancer

Qi deficiency is the important foundation of tumor formation in traditional Chinese medicine,therefore supporting the healthy energy is the important principle in the tumor treatment.And the side reaction induced by the chemotherapy can be reduced by the drug of supporting the healthy energy,thus it is applied widely in the treatment of tumor and the side reaction induced by the tumor chemotherapy.The Ganoderma Lucidum has the effect of invigorating Qi and blood,calming the mind by nourishing the heart,supporting the healthy energy.The modern research indicate that the Ganoderma Lucidum polysaccharides (GLP) has the effect of anti-tumor and the adjusting the immunity function and was considered as the essential component of Ganoderma Lucidum.The study indicated that biological activity structure of GLP is the polyglucosan includingβ-(1,3) glucosan which is very difficult to be enzymolyzed in the gastro-intestinal tract from the existing reports, however.That is to say it is difficult to digest GLP.But long-term clinical practice and the animal experiments proved that there is the obvious immunity adjustment and the anti-tumor function by taking orally GLP.Therefore we supposed that the intestinal mucosal immunity function may play the role in the effect of anti-tumor.This study was to explore the mechanism through the in vivo and in vitro experiments. The results suggested as the followed.1 GLP could obviously inhibit the H22 cells cultured in vitro and promote the proliferation and secretion of intestinal mucosal lymphocytes.1.1 MTT methods were used to observe the effects of GLP on H22 cells growth cultured in vitro,and the results suggested that GLP could kill H22 cells directly,and have positive correlation with the GLP concentration and action time.500μg/ml GLP had a inhibit rate of 47.97%on H22 cells after GLP were administrated 3 days.1.2 ELISPOT methods were used to measure the influences of GLP on the cytokine secreted by peripheral blood mononuclear cells(PBMC) and lymphocytes in Peyer's patches lymphocytes(PPL).The results indicated that each GLP concentration could stimulate PBMC secret IL-2,but had no direct action on PPL.At the same time,with the synergistic action of ConA,GLP could stimulate the proliferation of PBMC,intestine epithelial lymphocytes(IEL) and PPL using MTT methods,and the supernatants were measured by ELISA and demonstrated that GLP could promote the IL-2 and IL-10 excretion by PBMC,IEL,PPL.The RT-PCR results suggested the expression of TNF-αand IL-10 mRNA increased.The gene and protein expression of IL-2,IL-10 and TNF-αby lymphocytes were the strongest when the GLP concentration were 62.5μg/ml,125μg/ml,but no dose dependence was found.2 GLP could adjust the intestinal mucosal immune function of H22 xenograft mice by in vivo experiments.2.1 The results of this study showed that the tumor sizes of the mice in the GLP group,CTX group and combined use of GLP and CTX group decreased significantly,and the tumor inhibition rate were 38.64%,78.12%,78.53%respectively.The body weight of the mice didn't have significant differences between the model group and normal control group,and compared with the mice in model group,the body weight of the mice in CTX group and CTX+GLP group decreased significantly,and no significant differences were found between the CTX group and CTX+GLP group.The results indicated that in this xenograft model,GLP had no synergism with CTX,and couldn't inhibit the decrease of the body weight of the mice caused by CTX.But through the observation of the color pattern of the mice,we found that the mice in CTX+GLP group had brighter hair and acted more promptly than the mice in CTX group,which suggested GLP can partly adjust the adverse effect of CTX,and inferred by the available results,GLP would inhibit the body weight decrease by CTX with the extension of the administration time.2.2 Flow cytometry methods were used to observe T lymphocytes subgroup changes of PBMC,IEL,LPL and PPL,The results demonstrated that the peripheral blood T lymphocytes(CD3~+T lymphocytes) of the tumor xenograft mice intended to decrease; CD4~+T lymphocytes decreased significantly,CD8~+T lymphocytes didn't change so much, which lead to the ratio of CD4/CD8 decreased significantly.Compared with the normal control group,the CD4~+T lymphocytes in IEL increased significantly and CD8~+T lymphocytes didn't change obviously,which lead to the ratio of CD4/CD8 increased significantly too.Both the CD4~+ T lymphocytes and CD8~+T lymphocytes in PPL hadn't significant differences with the normal control group.No significant differences of CD3~+T lymphocytes,CD4~+T lymphocytes changes in LPL were found between the tumor xenograft mice with the normal control mice,and CD8~+T lymphocytes in LPL of the tumor model mice increased significantly,which lead to the ratio of CD4/CD8 decreased significantly. The detection results of peripheral blood with ELISA methods showed that the IL-10 activity advanced significantly and IL-2 activity degraded significantly in the serum compared with the normal control group.The immunohistochemistry results demonstrated that IgA,IL-2 and TNF-αsecretion in the tumor model group diminished significantly and IL-10 secretion increased significantly.All the above results indicated that the body of H22 tumor xenograft mice was in a kind of immune suppression condition,and the intestinal mucosal lymphocytes changes(IEL,LPL,PPL) were different in different places.In different places of the intestine,the time that the lymphocytes approached to the antigens and the components of the antigens were different,which maybe were the reasons to form different phenotype and immune function.2.3 At present the reports about the study of the effect of GLP on intestinal mucosal immune system hadn't been found yet,this topic was to explore the mechanism.The Flow cytometry methods were used to detect the T lymphocytes subgroup,the results showed that CTX could increase CD3~+,CD4~+ and CD8~+T lymphocytes in PPL and decrease the ratio of CD4/CD8.GLP group had no significant differences with the model group,but could decrease the significant augmentation of CD3~+ and CD8~+T lymphocytes caused by CTX and inhibit the tendency of the increasing ration of CD4/CD8 by CTX.Compared with the model group,the CD3~+T lymphocytes and CD8+T lymphocytes in IEL of CTX,GLP, CTX+GLP group hadn't statistical differences.The major change was in CD4~+T lymphocytes.CTX made the CD4~+T lymphocytes and the ratio of CD4/CD8 in IEL decrease significantly.GLP had this function also.At the same time GLP could tend to improve the decrease of CD4~+T lymphocytes caused by CTX,but had no statistical meaning. The results were totally different with the peripheral blood,which maybe related to the immune function of IEL,the further mechanism need to be explored.CTX could decrease CD3~+,CD4~+ and CD8~+T lymphocytes in LPL significantly,and increase the ratio of CD4/CD8 compared with the tumor model group.There were no significant changes in CD3~+,CD4~+ and CD8~+T lymphocytes in LPL of GLP group,but for CD8~+T lymphocytes diminished,which lead to the significant raise of CD4/CD8.GLP could inhibit the decrease of CD3~+,CD4~+ and CD8~+T lymphocytes induced by CTX,which suggested that GLP could improve the immune function degrade caused by CTX.The major function of T lymphocytes in lamina propria was to assis the synthesis of B lymphocyte and secret IgA by secreting cytokines.This experiment showed that CTX diminished the IgA of tumor xenograft mice significantly,GLP improved the expression of IgA of tumor xenograft mice significantly compared with the normal control group,and at the same time,GLP could inhibit the decrease of IgA induced by CTX significantly.The expression of IL-2, IL-10 and TNF-αwere detected by IHC methods,and the results demonstrated that CTX and GLP could down regulated IL-10 levels and CTX could significantly improve the IL-2 and TNF-αlevels,GLP had this effect too and tended to have synergism with CTX,which indicates that GLP could strengthen the body immune function by adjust the ratio of Th1/Th2 cytokines of tumor bearing mice and tumor bearing mice treated by CTX,majored in improve the Th1 cytokines.The above results confirmed our hypothesis that GLP could stimulate the mucosal lymphocytes directly through intestine mucosal system to play the immune regulating role on the body.In summary,GLP extracted from Ganoderma Lucidum with the effect of supporting the healthy energy can suppress the growth of tumor and the side effect caused by CTX partly.One of its function mechanisms is that GLP stimulates the intestinal mucosal lymphocytes,and then activates the whole body immune system through the recirculation of lymphocytes to display the function of anti-tumor.This result indicated that GLP is one of efficacy material basis of Ganoderma Lucidum on supporting the healthy energy,and it prompts simultaneously that the mechanism of anti-tumor effect of Ganoderma Lucidum and the method of supporting the healthy energy may concern with the adjustment of intestinal mucosal immune function.