Immunomodulation And Its Mechanism Of Polysaccharide From Anoectochilus Roxburghii

Objectives:1.To investigate the immunomodulatory mechanism of Anoectochilus roxburghii polysaccharides(ARP)on murine splenic lymphocytes in vitro,splenocytes proliferation,the cell cycle,secretion and mRNA expression of cytokines IL-2,IL-4,IL-6 and IFN-? were measured.2.To investigate the mechanism of immunomodulatory effects of ARP on RAW264.7 cells in vitro,cell proliferation,secretion of nitric oxide(NO),secretion and mRNA expression of iNOS,TNF-?,IL-6,IL-1?,IL-10,the expression of protein I?B-p65 and NF-kB were measured.3.To investigate the immuno-enhancing activity of ARP in cyclophosphamide(CTX)-induced immunosuppressed mice in vivo,splenocytes proliferation,phagocytic activity of macrophages,the concentration of IL-2,IL-4,IL-6 and IFN-y in sera,the mRNA expresson of IL-2,IL-4,IL-6 and IFN-? in the spleen,the counts of CD4+ and CD8+ T lymphocytes in peripheral blood were measured.4.To further investigate the mechanism of immuno-enhancing activity of ARP in CTX induced immunosuppressed mice in vivo,morphological changes of spleen,the splenic cell cycle and the mRNA expression of BcL-2,Bax,Fas and FasL were measured.Methods:1.Mice spleen lymphocytes were treated with ARP at various concentration in vitro;the proliferation level of mice spleen lymphocytes was measured by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay;the cell cycle were determined by flow cytometry;the concentration of NO and IL-2,IL-4,IL-6,IFN-? in the supernatant were measured by Griess reaction and by ELISA,respectively;the mRNA levels of IL-2,IL-4,IL-6,IFN-?,T-bet and GATA-3 were measured by quantitative real-time polymerase chain reaction(qRT-PCR).2.RAW264.7 cells were treated with ARP at various concentration in vitro;the proliferation level of RAW264.7 cells were assessed by MTT;apoptosis of cells was determined by flow cytometry;phagocytic activity of RAW264.7 cells was determined with neutral red uptake assay;the concentration of NO in supernatant was performed by Griess method;the concentration of TNF-?,IL-1?,IL-6 and IL-10 in the supernatant were quantified by ELISA;the mRNA levels of inducible nitric oxide synthase(iNOS)and TNF-?,IL-6,IL-1? IL-10 were evaluated by qRT-PCR and the expression of protein I?B-p65 and NF-?B inRAW264.7 cells were established by western blot.3.One hundred and fifty KM mice(male)were randomly divided into five groups(30 mice/group).All animals were acclimatized to the experimental conditions for one week before the start of the experiment.From day 1 to 3,Normal control group mice were given physiological saline solution via intraperitoneal injection,the other four groups of mice were given 80mg/kg/d CTX via intraperitoneal injection.From day 4 to 10,normal control group and model group(CTX)were treated with distilled water;while low,medium and high doses of ARP groups were treated with 100mg/kg,200mg/kg and 400 mg/kg ARP via gavage in 0.2mL solution.Twenty four hours after the last ARP administration,animals were weighted and blood samples were collected,then animals were sacrificed by cervical dislocation.The thymus and spleen index were calculated;splenic lymphocyte proliferation were assessed by MTT;mononuclear phagocytes function were assessed by carbon clearance test;the WBC was detected by automatic biochemical analyzer;the concentration of IL-2,IL-4,IL-6 and IFN-? in sera were determined by ELISA;the mRNA expression of cytokines and regulatory factors in the spleen were detected by qRT-PCR;the counts of CD4+ and CD8+ T lymphocytes in peripheral blood were determined by flow cytometry.4.Experimental protocol was the same as described above.The animals were sacrificed by cervical dislocation and the spleen were excised.Morphological changes and cell apoptosis in the spleen tissues were observed by HE staining and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL),respectively;apoptosis of splenic lymphocyte were evaluated by Annexin V-FITC/PI double-staining methods with flow cytometry;DNA cycle of splenic lymphocyte were tested by PI staining with FCM;BcL-2,Bax,Fas,FasL mRNA expression were qualified by qRT-PCR.Results:1.Compared with the control group,ARP significantly increased the splenocyte proliferation ability in a concentration-dependent manner with the highest OD value at a concentration of 400?g/mL;compared to ARP only,ARP supplemented with ConA or LPS could significantly increase splenocyte proliferation(P<0.05).Compared with control group,the proportion of spleen lymphocytes in GO/G1 phase treated with ARP tended to be decreased,while increased in the S phase and G2/M phase(P<0.01);the secretion of NO and IL-2,IL-4,IL-6,IFN-y were higher and the mRNA levels of iNOS,IL-2,IL-4,IL-6,IFN-?,TNF-?,T-bet,GATA3 were significantly increased(P<0.01).2.Compared with RAW264.7 cell control group.the OD value treated with ARP was significantly increased at 25?g/mL;the apoptosis rate was declined;the phagocytosis activity was improved and the OD value was significantly increased at 50?g/mL;the secretion of NO,TNF-a,IL-1?,IL-6 and IL-10 secretion levels were increased at 25?g/mL(P<0.01);the mRNA level of iNOS,TNF-a,IL-6,IL-1? and IL-10 were promoted;the protein expression of IkB p65 in cytosolic was decreased and the expression of NF-kB in nucleuses was increased.3.Compared with model group,the body weight gain in ARP high group was increased(P<0.05);the spleen index in ARP treatment groups were decreased and the thymus index were increased(P<0.05);the phagocytic index in ARP low and medium groups were increased(P<0.05);the stimulating index(SI)of T lymphocyte proliferation in high ARP group was increased(P<0.05);the total number of WBC in ARP high dosage group was decreased(P<0.05);the content of cytokine IL-2 in sera of ARP low and high groups were increased(P<0.05),and IL-6 in sera of ARP high group was increased(P<0.05);the IL-4 mRNA expression level in ARP high group was decreased(P<0.05),the IL-6 and IFN-y mRNA expression level in ARP medium and high groups were decreased(P<0.05);the T-bet mRNA expression level in ARP groups were decreased(P<0.05),the GATA-3 mRNA expression level in ARP high group was increased(P<0.05);the percentage of CD4+,CD8+ T lymphocytes in ARP groups were increased(P<0.05),the ratio of CD4+/CD8+ was decreased(P<0.05).4.In model group,splenic nodule shrunk,germinal center were not obviously observed,lymphocytes number decreased and structure was fuzzy;compared with the model group,in the ARP-treated groups,cells number increased in the fringe area of spleen significantly,splenic nodule was larger and lymphocytes number increased,the boundary of red pulp and white pulp was obvious.The apoptotic ratio of splenic lymphocytes in model group mice was 37.3%,which was significantly higher than that in the control group(P<0.05);the apoptotic ratio of splenic lymphocytes in the ARP-treated groups were lower than that of in the model group,which were 22.9%,15.7%,and 18.7%respectively.The percentage of early apoptotic cells in model group was higher than that of model group,the percentages of early apoptotic cells in the ARP-treated groups were significantly lower than that of model group in a dose-dependent manner.The total percentage of apoptotic cells(the total number of FITC single positive cells and FITC/PI double positive cells)among the groups was significantly different.The percentage of splenic lymphocytes in the G1 phase in model group was significantly higher than that in the control group,and splenic lymphocytes percentage in the S phase was lower than that of control group.The percentages of splenic lymphocytes in the sub-G1 phase in the ARP-treated groups decreased obviously,while the percentages in S phase and G2 phase significantly increased.The Bax mRNA levels in ARP-treated groups were significantly lower than that in model group in dose-dependent manner,the Bax mRNA levels reduced gradually.However,The BcL-2 mRNA expression levels in all of ARP treatment groups were significantly higher than that in model group,the BcL-2 mRNA also increased gradually with the increasing concentration of ARP.The Fas mRNA levels in ARP treatment groups were markedly lower than that of model group;the expression of Fas and FasL mRNA reduced gradually with the upregulation of BcL-2 mRNA.Conclusions:1.The immuno-enhancing activities of ARP on murine splenic lymphocytes in vitro were related to prompting lymphocytes proliferation,regulating cell cycle and up-regulating transcription factor T-bet,GATA-3 mRNA expression,which mainly result from stimulating secretion and mRNA expression of Thl cytokines(IFN-y,TNF-a,IL-2)and Th2 cytokines(IL-4,IL-6).2.The immuno-enhancing activities of ARP on RAW264.7 cell in vitro were related to stimulating RAW264.7 cell proliferation,reducing the cell apoptosis rate,enhancing phagocytosis activity,increasing NO production and TNF-a,IL-6,IL-1? and IL-10 levels,increasing mRNA expression of iNOS,TNF-a,IL-6,IL-1? and IL-10,down-regulating I?B p65 protein expression in cytosolic and up-regulating NF-?B protein expression in nucleuses.These results suggested that APR could activate macrophage actvities via activating NF-?B pathway.3.Administration of ARP could significantly mitigate the enlargement of spleen and the atrophy of thymus,stimulate the phagocytosis of phagocyte,stimulate T-lymphocyte proliferation,regulate the secretion and expression of some cytokines mRNA in the immunosuppressed mice induced by CTX in vivo.It was concluded that ARP could improve the immune function in suppressive mice via regulating T-lymphocyte differeration to some extend.4.Administration of ARP could significantly restore splenic tissue lesions,reduce the apoptotic ratio of spleen cells,decrease the apoptotic ratio of splenic lymphocytes,promote DNA replication and mitosis process in the immunosuppressed mice induced by CTX in vivo.These results suggested that APR could inhibit splenocyte apoptosis induced by CTX via upregulating of rate of BcL-2/Bax and downregulating of the expression of Fas mRNA.