Establishment Of Colitis-associated Colon Cancer Mice Model And The Effects Of Annatto Tocotrienol On Peripheral T Lymphocyte And Cytokines

ObjectiveThis study aims to research the effects of tocotrienols purified from annatto on inhibiting proliferation of colon cancer and peripheral blood T lymphocytes, IL-6, IFN-γ in mice which are the the animal model of orthotopic colon cancer induced by azoxymethane(AOM) and dextran sulfate sodium salt(DSS).Methods 1. The colitis-associated colon cancer mice model induced by AOM-DSSThe experimental animals were randomly assigned to four groups, including one solvent control group、LDSS group、HDSS group and LDSS+HDSS group. The mice in LDSS and HDSS groups received intraperitoneal injection with AOM(10mg/kg)for one time. After a week, The mice in model group received 3% inflammatic agent DSS for a week, and then distilled water for two weeks. After that, the mice in model group still received 2%DSS for five days, then distilled water for sixteen days, then 2%DSS for five days and then distilled water until the end. The mice in solvent control group received intraperitoneal injection with normal saline which had the same volume with AOM, and then distilled water until the twentieth week.We observed and recorded the mice hair,fecal character and bloody stool which can be visible by naked eyes. The mice were weighted each week. All the mice were picked the eyeball and drawed blood at the twentieth. After executing the mice by cervical dislocation, we weighed the spleen and recorded organ coefficient. All the colorectum from anus to the end of cecum were vertical dissected along the colic bands and douched with saline. We observed the intestinal lesions and counted the number of tumor. Observed the pathology change in the colon by HE staining.2. Effects of annatto tocotrienol on peripheral T lymphocyte and cytokines in colitis-associated colon cancer mice model induced by AOM-DSSThe experimental animals were randomly assigned to five groups and each group had fifteen mice, including one solvent control group and four colon cancergroups which were model group, annatto tocotrienols group with low dose, annatto tocotrienols group with middle dose and annatto tocotrienols group with high dose. The mice in solvent control group and model group received gavage with 0.1mg/kg.BW soybean oil through mouth daily for a week. The other groups received gavage with annatto tocotrienols at different dose of 5mg/kg.BW, 10 mg/kg.BW and 20 mg/kg.BW for a week. The mice in model group received intraperitoneal injection with AOM(10mg/kg)for one time. After a week, the mice in model group received 3% inflammatic agent DSS distilled water for a week, and then distilled water for two weeks. After that, the mice in model group still received 2%DSS distilled water for five days, then distilled water for sixteen days, then 2%DSS distilled water for five days and then distilled water until the end. The mice in solvent control group received intraperitoneal injection with normal saline which had the same volume with AOM, and then distilled water until the 20 th week. We observed and recorded the mice hair, fecal character and bloody stool which can be visible by naked eyes. The mice were weighted each week. All the mice were picked the eyeball and drawed blood at the twentieth. After executing the mice by cervical dislocation, we weighed the mice tissue(thymus, liver, double kidney and spleen) and recorded organ coefficient. All the colorectum from anus to the end of cecum were vertical dissected along the colic bands and douched with saline. We observed the intestinal lesions and counted the number of tumor. Observed the pathology change in the colon and kidney tissue by HE staining. Measured whole blood hematology indexes by fully automatic hematology analyzer, the level of IL-6 and IFN-γ in peripheral blood by ELISA, the CD4+T cells, CD8+T cells and CD4+T /CD8+T ratio in peripheral blood by flow cytometry.ResultsThe establishment of colitis-associated colon cancer mice model induced by AOM/DSS1. Low-dose, single intraperitoneal injection of AOM(10mg/ kg.BW) and 2-3% DSS(molecular weight 36000-50000MW) drinking water for three cycles can induced CD-1(ICR) mice mucosa thickening, folds disorder and distal colon tumor growth after 20 weeks. The tumor formation rate was 100%.2. The intestinal mucosa of the mice induced by AOM/DSS appeared different degree of thickening, fold disorders and colorectal carcinoma which had different sizes and numbers. The induced carcinoma occered in the intestines mucous membrane surface and protruded to the lumen. HE staining showed the mucosa and submucosa appeared widespread inflammation cells. The structure of the glandular tissue was in the mess and damaged. The cryptae-like structure was observed and had organization atypia. some extent. The pathology changes included low-grade of intraepithelial neoplasia(LGIN) and high-grade of intraepithelial neoplasia(HGIN). The All carcinoma were in adenoma phase and there were no invasive carcinoma.3. AOM/DSS mouse model showed splenomegaly and spleen coefficient increased. HE staining showed multinucleated giant cells increased, red pulp disappeared and extramedullary hematopoiesis.The effects of annatto tocotrienol on peripheral T lymphocyte and cytokines in colitis-associated colon cancer mice model induced by AOM-DSS1. The average weight of the mice in model group was lower than the solvent control group, and after giving DSS water, the average weight decreased comparing to the previous week. The average weight of the mice in the tocotrienols group higher than model group but lower than the solvent control group.2. Compared with the solvent control group, white blood cell count, red blood cell count, hemoglobin, hematoerit, blood platelet, eosinophils decreased in the cancer model control group. While white blood cell count, neutrophils decreased in 5mg/kg.BW annatto tocotrienol group and blood cell count, red blood cell count decreased in 10mg/kg.BW、20mg/kg.BW annatto tocotrienol groups. The differences were statistically significant(P<0.05)3. Compared with the model group, the annatto tocotrienols with 10mg/kg.BW、20mg/kg.BW dose could increase CD4+/CD8+ratio; 20mg/kg.BW annatto tocotrienols could increase the level of IFN-γ in peripheral blood and 10mg/kg.BW、20mg/kg.BW dose of annatto tocotrienols could decrease IL-6 in peripheral blood. The differences were statistically significant(P<0.05).Conclusions1. Low-dose, single intraperitoneal injection of AOM and DSS(molecular weight 36000-50000MW) drinking water for three cycles can induced CD-1(ICR) mice mucosa thickening, folds disorder and distal colon tumor growth after 20 weeks.The tumor formation rate was 100%.2. The tocotrienols can improve abnormal immune status of the mice which are ill with colon cancer induced by agent azoxymethane(AOM) and dextran sulfate sodium salt(DSS) and regulate immune imbalance.