Neutral Polysaccharide From Panax Notoginseng Combined With Cyclophosphamide For The Treatment Of H22 Tumor-bearing Mice And Immunization Study After Re-inoculation Of Tumor Cells In Cured Mice

Objective:To investigate the treatment of neutral polysaccharide from panax notoginseng combined with cyclophosphamide on H22 tumor-bearing mice and immunization study after re-inoculation of H22 tumor cells in cured mice.Methods:NPPN combined with CTX for the treatment of the H22 tumor-bearing mice:The H22 solid tumor model was constructed to investigate the inhibitory rate,spleen index,thymus index,mononuclear-macrophage carbon clearance ability and the blood function of H22 tumor model treated with NPPN combined with CTX.The percentage of CD3+T lymphocytes,CD4+T lymphocytes,CD8+T lymphocytes,B220+B lymphocytes and CD49b+NK cells in peripheral blood of tumor-bearing mice was detected by flow cytometry.Serum levels of immunocytokines IL-2,TNF-?,IFN-y and immunoglobulin IgG in tumor-bearing mice were detected by double-antibody sandwich ELISA.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of tumor tissue after treatment.The expressions of CD3+,CD4+,CD8+,CD49b+and CD19+in mouse tumor tissues were detected by immunohistochemistry.The effects of NPPN on the immune function of mice bearing H22 tumor were evaluated.Immunization study after re-inoculation of H22 tumor cells in cured mice:The cured mice obtained by the experiment were subcutaneously injected with H22 tumor cell suspension in the right anterior axilla,and the tumor growth was observed at different time points after inoculation of H22 tumor cells.The percentage of CD3+T lymphocytes,CD4+T lymphocytes,CD8 T lymphocytes,B220+B lymphocytes and CD49b+NK cells in peripheral blood of mice after re-inoculation of H22 tumor cells at different time points was detected by flow cytometry.Serum levels of immunocytokines IL-2,TNF-?,IFN-? and immunoglobulin IgG were detected by Double-antigen sandwich ELISA.The histomorphological changes of the inoculated sites tissues were observed by HE staining.Results:NPPN combined with CTX for the treatment of H22 tumor-bearing mice:The results of tumor inhibition rate showed that the average tumor weight of the mice in the saline group was more than 1 g,and the transplanted tumor was successfully established.Compared with the saline group,the tumor weight of each group decreased significantly(P<0.01).Compared with the CTX group,the inhibition rate of the medium dose NPPN combined with CTX group was 83.23%,and the high dose NPPN combined with CTX group was 90.07%(P<0.05),which was higher than that of CTX group(68.8%).The results of NPPN combined with CTX on the splenic and thymic index of H22 tumor-bearing mice showed that compared with the saline group,the splenic and thymic index of the CTX group decreased,which was significantly different(P<0.01),compared with the CTX group,the splenic index of the low,middle and high dose NPPN combined with CTX group was increased,and the thymic index of the high dose NPPN combined with CTX group was increased,which was statistically significant(P<0.05).The carbon clearance test showed that the phagocytic index a of the CTX group was significantly lower than that of the normal group.The phagocytic index of the NPPN combined with CTX group in the low,medium and high dose groups was significantly higher than that of the CTX group(P<0.05).The results of hematological examination showed that the white blood cells,lymphocytes and reticulocytes of the CTX group was significantly lower than that of the saline group,which was significantly different(P<0.01).Compared with the CTX group,the white blood cells and reticulocytes of the low,medium and high doses of NPPN combined with CTX group increased,no statistical difference.The white blood cells of the high-dose NPPN combined with CTX group was significantly higher(P<0.01).Flow cytometry was used to detect the proportion of T,B lymphocytes and NK cells in the peripheral blood of tumor-bearing mice.The results showed that the immune cells(CD3+,CD4+,CD8+,B220+)in the peripheral blood of CTX group were significantly lower than the normal group,medium and high.Compared with the CTX group,the CD3+level of the medium and high dose NPPN combined with CTX group was significantly higher(P<0.05),and the CD3+CD4+,CD3+CD8+,and CD3-B220+levels were not significantly different.Cytokines(IL-2,TNF-?,IFN-?)and IgG were detected by ELISA.The results showed that the content of IL-2 in the CTX group was lower than that in the saline group.Compared with the CTX group,the medium and high dose NPPN combined with CTX group were statistically different(P<0.05;P<0.01).Compared with the saline group,the content of TNF-? in the CTX group was decreased(P<0.05).Compared with the CTX group,the levels of TNF-? in the low,medium and high doses of NPPN combined with CTX group were significantly higher(P<0.01).NPPN had no significant effect on serum levels of IFN-y in tumor-bearing mice.Compared with the saline group,the content of IgG in the CTX group was significantly lower(P<0.01).Compared with the CTX group,the IgG content in the low and high dose NPPN combined with CTX group was slightly increased,and there was no statistical difference.The pathological observation of HE staining showed that the tumor cells in the saline group had high density and less necrotic area.Compared with the saline group,the number of tumor cells in the low,medium and high dose NPPN group and CTX group was small and necrotic.The regional increase;the low,medium and high dose NPPN combined with CTX group compared with the CTX group,the necrotic area in the tumor tissue was larger,and the necrotic area from the low dose group to the high dose group gradually increased.The results of immunohistochemistry showed that compared with the saline group,the brown pigmented particles in the cytoplasm of the cyclophosphamide group were significantly reduced,and the color gradually decreased,indicating that the expression levels of CD3+,CD4+,CD8+,CD49b+and CD19+were significantly decreased(P<0.01);Compared with the CTX group,the cytoplasmic brown particles in the low,medium and high doses of NPPN combined with CTX group increased,and the color gradually increased,indicating that the expression of CD3+,CD8+,CD49b+and CD 19+were significantly increased(P<0.01).Immunization study after re-inoculation of H22 tumor cells:Flow cytometry was used to detect the number of immune cells at different time points after re-inoculation of the tumor in the curedd mice.The results showed that CD49b+NK cells of the peripheral blood were elevated on the 1st,4th,7th and 14th day after inoculation,compared with the one day before the inoculation.which were statistical differences on the 7th and 14th day after inoculation(P<0.05;P<0.01).The cytokines(IL-2,TNF-?,IFN?)were detected at different time points after re-inoculation of the tumors by ELISA.The results showed that the serum levels of IL-2 were not significantly affected after re-inoculation of the tumors in the recovered mice.Compared with the one day before inoculation,the serum levels of TNF-? were decreased at different time points after inoculation of H22 tumor cells,and there was significant difference on the first day,the fourth day and the 14th day(P<0.01).The serum level of IFN-? in the cured mice on day 14 after re-inoculation of H22 tumor cells was increased as compared with the one day before inoculation.Histomorphological changes in the inoculated site of HE staining:After the H22 tumor cells were inoculated again,the inoculated sites were stained with HE,one day before inoculation and on days 1,4,7,and 14 after inoculation.The results showed that the pathological observation of HE staining at different time points after H22 tumor showed that under the microscope,there was no tumor cell in the muscle tissue under the armpit of the healing day before the inoculation.With the increase of the inoculation time,the inoculation was 1 day,4 days,7 days,After 14 days,tumor cells were still present at the inoculation site,and the tumor did not grow;after the 200th day of inoculation,there were no tumor cells at the inoculation site.Conclusion:Cyclophosphamide is a cytotoxic chemotherapeutic drug and immunosuppressive agent.In the treatment of H22 tumor-bearing mice,both cellular immunity and humoral immunity reduce the immune function of mice.NPPN combined with CTX promotes the repair of immune organ damage caused by CTX in mice with H22 tumor,improves spleen index and thymus index;increases the number of white blood cells and lymphocytes in blood;enhances phagocytic ability of mouse phagocytic cells,improves immune function;The content of T lymphocytes showed an increase in the proportion of CD3+cells,indicating that NPPN can enhance the cellular immunity of mice and achieve anti-tumor effect.The serum levels of IL-2 and TNF-? were increased,indicating that NPPN regulated immune cytokines in vivo.increase the content of B lymphocytes,showing an increase in the proportion of B220 cells,and the increase of IgG secreted by B lymphocytes,indicating that NPPN can enhance humoral immunity in mice,and achieve anti-tumor effects.In summary,NPPN protects mouse immune organs from spleen and thymus,increases the number of white blood cells and lymphocytes,and increases the non-specific immunity,humoral immunity,and cellular immunity of H22 tumor-bearing mice.To achieve anti-tumor effect,NPPN combined with CTX treatment of H22 tumor-bearing mice is superior to CTX alone.After the mice were re-inoculated with H22 tumor cells,the tumors did not grow.After H22 tumor cells were inoculated again,the proportion of NK cells in the peripheral blood increased.It is suggested that the tumor growth in the cured mice after re-inoculation of the tumor may be related to the memory NK cells.