| Background:Patients with colorectal cancer are responsible for about 10%of global cancer cases and cancer-related deaths each year.In fact,it is estimated that the global incidence of colorectal cancer will increase to 2.5 million by 2035.Traditional cancer treatments mainly include surgery,radiation therapy and chemotherapy.However,treatment is accompanied by side effects and recurrence in approximately 50%of colon cancer patients after treatment,while often leading to increased drug resistance.Therefore,there is an urgent need for new options with few or no side effects for the treatment of colon cancer.The immune system plays an important role in tumor development,and modulating the host immune system may bring new ideas for the treatment of colorectal cancer.IL-28B is a new cytokine discovered in 2003,which belongs to the Type Ⅲ interferon(IFN-Ⅲ,IFN-λ)family.As research has revealed that IFN-Ⅲ has a variety of biological effects,including antiviral,immunomodulatory and antitumor,as well as suppression of autoimmune diseases and allergic croup.Among them,antitumor activity has been demonstrated in a variety of tumor models,and by acting directly on tumor cells or modulating host immunity is the main mechanism by which IFN-Ⅲ acts against tumors.It has been shown that IL-28B inhibits the progression of colitis in mice,but the role of IL-28B in colon cancer and the specific mechanism is unknown.Therefore,exploring the role of IL-28B on colon cancer and its mechanism is expected to provide new ideas for cancer treatment.Objective:To investigate the effect of IL-28B on colon cancer in mice and its mechanism of action,and to provide a theoretical basis for basic research and clinical treatment of colon cancer.Method:1.Exploring the role of IL-28B in a mouse model of colon cancer.Subcutaneous tumor model of colon cancer in mice:C57BL/6 right dorsal skin was inoculated with MC38 tumor cells to establish a mouse subcutaneous tumor model.After one week,the tumor volume of mice was measured every two days with vernier calipers.Meanwhile,IL-28B protein(125 μg/kg)was injected around the subcutaneous tumor daily.Mice were executed 24 days after inoculation,and tumor tissues were collected for corresponding assays to observe the effect of IL-28B on colon cancer.(1)Collecting tumor tissues from mice and weighing the tumors.(2)HE staining of tumor tissues and observation.Colon cancer model in mice associated with colitis:C57BL/6 male mice were injected intraperitoneally with azoxymethane oxide(AOM,10μg/kg)for 1 week,and then given drinking water containing 1.75%dextran sodium sulfate(DSS)for 1 week,followed by normal drinking water for 2 weeks.This cycle was repeated for 3 cycles to establish the AOM/DSS-induced colon cancer model in mice.1 week after the end of establishment,IL28B protein(125 μg/kg,250 μg/kg)was injected intraperitoneally daily for treatment.After 5 weeks of treatment,the mice were decollated and executed,and colorectal tissues were collected for the corresponding experiments to observe the effect of IL-28B on the AOM/DSS-induced colon cancer model.2.To investigate the effect of MC38 and CT26 cells on cell viability after being treated with different concentrations of IL-28B protein for 48h and 72h by the cell viability assay kit.3.MC38 and CT26 cells were treated with IL-28B protein,and the apoptosis of tumor cells was detected by flow cytometry at 48h and 72h.4.Investigating the effect of IL-28B on immune cells in a subcutaneous tumor model of colon cancer in mice.As in study method 1,a mouse colon cancer subcutaneous tumor model was established,and spleen and tumor tissues were collected for corresponding assays after treatment with IL28B protein.(1)Mice spleens were collected and the proportion of NK cells,dendritic cells,macrophages and bone marrow-derived suppressor cells in them were examined by flow assay.(2)Tumor tissues from mice were collected and the ratio of M1 macrophages to M2 macrophages in tumors was detected by flow cytometry.(3)Mouse tumor tissues were collected and sectioned for immunofluorescence staining.5.Mice with established tumor models were treated with IL-28B neutralizing antibody and injected intraperitoneally with neutralizing antibody(10 μg/each)every three days,and tumor volumes were measured every two days.The mice were decollated and executed on day 21 of inoculation,and tumor tissues were collected for appropriate assays.6.To investigate the effect of IL-28 on M2 macrophages(1)M2 macrophages were cultured in vitro and treated with different concentrations of IL-28B protein,and the percentage of M2 macrophages was measured by flow cytometry on day 8.(2)M2 were cultured as above,and the levels of Arg-1 and TGF-β mRNA in M2 were detected by Quantitative Real-time PCR after the addition of IL-4 and IL-13 stimulating factors for 6 h.The protein of M2 was also extracted and the culture supernatant was collected for ELISA to detect the expression of Arg-1 and TGF-β,respectively.(3)M2 macrophages were cultured in vitro and treated with different concentrations of IL-28B protein,and the relative expression of STAT3,JNK,p-JNK and p-STAT3 in M2 was detected by Western blot after the addition of IL-4 and IL-13 stimulating factors for 1h.RESULTS:1.IL-28B inhibited the development of colon cancer and slowed tumor growth in mice.In the subcutaneous tumor model(1)IL-28B treatment group,tumor volume and weight of mice were significantly decreased.(2)HE staining showed increased tumor tissue necrosis.In the AOM/DSS-induced colon model,IL-28B similarly inhibited the number and growth of tumors in the colorectum of mice.2.IL-28B protein had no significant effect on apoptosis and cell viability in MC38 and CT26 cells.3.(1)IL-28B had no significant effect on immune cells in the spleen.(2)IL-28B reduced the proportion of M2 macrophages in tumor tissue and had no significant effect on Ml macrophages.(3)Immunofluorescence staining of tumor tissue showed that M2 macrophages were reduced in the IL-28B treated group.4.In the IL-28B neutralizing antibody treated group,tumor growth was accelerated,tumor weight was increased,and the proportion of M2 macrophages in tumor tissues was increased.5.IL-28B regulates the mechanism of M2 macrophages:(1)IL-28B inhibits the polarization of M2 macrophages.(2)EL-28B decreases the expression of Arg-1 and TGF-β in M2 macrophages.(3)IL-28B inhibits the polarization of M2 macrophages probably by inhibiting STAT3 and JNK phosphorylation.Conclusion:1.IL-28B inhibited the progression of colon cancer.2.In vitro IL-28B has no direct effect on colon cancer cells.3.IL-28B inhibits the polarization of M2 in tumors.4.IL-28B may affect M2 polarization through STAT3 and JNK... |
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