The Effect On The Immunity Of Mice Bearing MFC Tumor Treated With Periplaneta Americana Peptide

ObjectiveTumor is one of the most health-threatening diseases in the world.It is the main tasks of scientific workers that to find effective and low toxicity treatment drugs.Periplaneta americana extact CⅡ-3 is a peptide-based substances extract from Periplaneta americana by Yunnan Key Laboratory for Biomedical Research and Development of Insects at Dali University,and has been found to inhibit a variety of human tumor cells.But anti-tumor efffect of the peptide and its mechanism is not clear in vivo.In this study,we established model of tumor-bearing BALB/c mice used mice gastric cancer cells(MFC).After treated with CⅡ-3 and the related artificial synthesis peptide(HFDT1),the effects of CⅡ-3 on the tumor and immune function in the mice were analyzed to clarified the mechanism of anti-tumor immune regulation of Periplaneta americana peptide,and provided a theoretical basis for the drug development.MethodsWe selected BALB/c mice with age of 6~8 weeks and weight in about 20 g,and inoculated logarithmic growth of MFC cells in the right armpit of the mice.We established the subcutaneous tumor model in BALB/c mouse,and divided them into 5 groups,model group,CTX group,the Periplaneta americana peptide CⅡ-3 high and low dose groups,synthetic peptide HFDT1 group.In addition,the normal control group was set up.The model group and the normal group mice were given normal saline by gavage(20m L/kg,once a day).The CTX group mice were injected with CTX by abdominal cavity(45mg/kg,10mL/kg,every other day).The High and low doses of CⅡ-3 groups mice were given with CⅡ-3 by gavage(400mg/kg and 200mg/kg,20 m L/kg,once a day).The HFDT1 group was injected with synthetic peptide HFDT1 by abdominal cavity(7.8mg/kg,10mL/kg,once a day).After 10 day treatment,the associate data were analyzed in the mice.At 24 hours after the last treatment,the tumor,spleen and thymus of the mice were separated and the effect of tumor inhibition,spleen and thymus index were calculated.The numbers of the immune cells were detected in the peripheral blood from the mouse inner canthus vein of mice.The levels of IgG,IgM and IgA in the serum were detected by ELISA.The peritoneal cells(PEC)were obtained from the mice and macrophages were purified from PEC.After the macrophages were cultured with LPS for 24 h,the culture supernatants were collected for measuring TNF-α levels.The tumor tissue’s lymphocytes were isolated by Percoll for Th1/Th2 reactions detection.The splenic single cell suspension were prepared and staining with fluorescencelabeled specific monoclonal antibodies to analyze the ratio of T cell,B cell,Treg,Th17,T and B cell subsets,NK cell,dendritic cell,macrophage and Th1/Th2 reaction through FACS Calibur.The splenic lymphocyte were cultured with ConA for 48 h,then collected the supernatants for detecting the levels of IL-2,IL-4,IL-10,IL-17,IFN-γ and TNF-α by ELISA.The splenic lymphocytes were labeled with CFSE and the proliferation of B and T cells were analyzed after cultured with LPS or ConA for 4d by FACS Calibur.Results1.Periplaneta americana extract CⅡ-3 effectively inhibited tumor growth in vivo.The results shown that the inhibition ratios to the tumor of CⅡ-3 high dose and low dose groups,HFDT1 and CTX groups are 33.11%,42.88%,46.20% and 54.59%,seperately.The inhibition ratio of CⅡ-3 low dose group was higher than that of CⅡ-3 high dose group.And The inhibition ratio of HFDT1 group was higher than that of CⅡ-3 low dose group.With the administration time longer,the weight of CTX mice decreased significantly,while CⅡ-3 and HFDT1 groups were not obvious change compared with normal group and model group.2.Compared with CTX group,Periplaneta americana extract C Ⅱ-3 and HFDT1 increased the spleen and thymus index(P<0.05),increased the number s of leukocyte,monocyte,lymphocyte and granulocyte in peripheral blood(P<0.05).The levels of IgA,IgM,IgG in the serum were also increased in CⅡ-3 and HFDT1 groups(P<0.05).3.Compared with CTX group,NK cells,macrophages and DC cells in spleen(P<0.05)were up-regulated,but Th17 cell was down-regulated in CⅡ-3 high and low does groups and HFDT1 group.Compared with normal control group,TNF-α which was secreted by Macrophages from spleen and abdominal cavity was increased in CⅡ-3 high and low does group and HFDT1 group.IL-17 which was secreted by Th17 cells was decreased in spleen compared with CTX group.4.Compared with CTX group,C Ⅱ-3 high and low dose and HFDT1 upregulated splenic lymphocyte.Compared with the model group,the splenic T lymphocytes in CⅡ-3 high and low dose and HFDT1 group were increased(P<0.05).Compared with CTX group,CⅡ-3 high and low dose and HFDT1 increased CTL cells(P<0.05)and reduced Treg cells(P<0.05),CⅡ-3 low dose group and HFDT1 group decreased naive T cells(P<0.05),and up-regulated the matured splenic B cells(P<0.05).5.Compared with the model group,C Ⅱ-3 and HFDT1 enhanced the proliferation of T cells,and the proliferation of CTL cells were more obviously(P<0.05).But the stimulative effect on B cells was weak(P>0.05).In addition,B cells and T cells proliferation ratio in CⅡ-3 low dose group were higher than CⅡ-3 high dose group.6.Th1 and Th2 reaction in different tissue Th cell shown different effect after treated with CⅡ-3 and HFDT1.In the spleen,the effect of CⅡ-3 low dose on the differentiation of Th cells to Th1 was stronger than that of CⅡ-3 high dose.With samilar effect,the level of IFN-γ and IL-2 secreted by Th1 cells in CⅡ-3 low dose group were higher than that of CⅡ-3 high dose group.But in the tumor tissue,the effect of CⅡ-3 high dose on the differentiation of Th cells to Th1 was stronger than CⅡ-3 low dose,and the level of IFN-γ secreted by Th1 cells in CⅡ-3 high dose group was higher than that of CⅡ-3 low dose group.Conclusions1.Periplaneta americana extract CⅡ-3 and synthetic peptide HFDT1 shown an effective anti-tumor and a low side effect in vivo.2.The anti-tumor effect of Periplaneta americana extract CⅡ-3 and synthetic peptide HFDT1 could be done through regulating immunity.It included that they increased the number of immune cells in peripheral blood;up-regulated the mouse spleen index and thymus index;improved the composition of lymphocyte subsets;stimulated CTL proliferation;up-regulated macrophage,NK cell,Dendritic cell,CTL,and Th1 reaction;improved and increased the secretion of immunoglobulin and down-regulated Treg.3.Periplaneta americana extract CⅡ-3 and synthetic peptide HFDT1 may be developed an effective and low side-effective anti-tumor drug.