| Objective In this study,we built a well-established colitis-associated cancer model in CD1-ICR mice by the combined treatment with azoxymethane(AOM)and administrating dextran sulfate sodium(DSS),expected to clarify whether tocotrienol could regulate immune cell and cytokine in mice with colitis-associated cancer.Methods1.Male ICR mice(5 weeks old)were randomly divided into 8 groups(n=20/group),respectively Control group,AOM/DSS group,AOM/DSS+Solvent group,three different dosages δ-tocotrienol groups [AOM/DSS+T3-L(50 mg/kg b·w),AOM/DSS+T3-M(75 mg/kg b·w)and AOM/DSS+T3-H(100 mg/kg b·w)],oxaliplatin treatment group(AOM/DSS+OXA)and tocotrienol combined oxaliplatin treatment group(AOM/DSS+OXA+T3-H).They were housed in a specific pathogen-free(SPF)condition.The mice in AOM/DSS+T3-L,AOM/DSS+T3-M and AOM/DSS+T3-H groups were received administration with annatto tocotrienols at dose of 50mg/kg b·w,75 mg/kg b·w and 100 mg/kg b·w daily for twenty weeks,respectively.Similarly,the mice in AOM/DSS+Solvent and AOM/DSS+OXA group were received administration with 0.1mg/kg b·w vessel daily for twenty weeks,since the fourteenth week,the mice in AOM/DSS+OXA group were given 3mg/kg b·w oxaliplatin solution for 5 days,discontinued for 9 days,for three consecutive cycles.AOM/DSS+OXA+T3-H orally administered 100mg/kg b·w tocotrienol,and since the fourteenth week,the mice were given 3mg/kg b·w oxaliplatin solution for 5 days,discontinued for 9 days,for three consecutive cycles.The experiment lasted 20 weeks.2.During the course of the experiment,mice were monitored by continuous body weight measurement.At the end of experiment,recorded the body weight,weighted the spleen,colon and recorded the organ coefficient.Observed the pathology change in the colon and spleen by HE staining.3.Measure the T Lymphocyte subtype in peripheral blood by flow cytometry.Using flow cytometry and Luminex assay to determine the level of cytokine,such asIL-4,IL-6,IL-10,TNF-a.Using ELISA and Immunohistochemistry measured the level of My D88,NF-κB in colon tissue.Results1.During the experiment,the weight in each group showed an increasing state,and there were no significant difference between the groups(P>0.05).AOM/DSS,AOM/DSS+Solvent and AOM/DSS+T3-L group survival rate was significantly lower than that of Control group(P(27)0.05).There were no significant difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent and AOM/DSS+OXA groups,each intervention group survival rate increased,but the difference was not statistically significant(P>0.05).Since the sixth week,colon cancer model groups could be observed blood in the stool,and there were no significant difference between AOM/DSS and AOM/DSS+Solvent groups(P>0.05).In the ninth week,the rate of blood stools in tocotrienol treatment groups and AOM/DSS+OXA+T3-H group was significantly lower than AOM/DSS+Solvent and AOM/DSS+OXA groups(P(27)0.05).In the twentieth week,the rate of blood stools in AOM/DSS+OXA and AOM/DSS+OXA+T3-H groups was significantly lower than AOM/DSS+Solvent group(P(27)0.05).Compared with AOM/DSS+OXA,the rate of blood stools in AOM/DSS+T3-L and AOM/DSS+T3-M were significantly increased(P(27)0.05).The rate of colon tumor formation in AOM/DSS and AOM/DSS+Solvent groups were 100%,and the rate were decreased in each treatment groups,and there were no tumor nodules in some mice colon tissue,compared to AOM/DSS+Solvent group,the rate were markedly decreased in AOM/DSS+T3-H and AOM/DSS+OXA+T3 group(P(27)0.05).2.From morphological changes of spleen and colon,AOM/DSS mice showed splenic nodule number and red pulp decreased,had adenoma in the distal colon,the size and shape of cells showed different;in each treatment groups,the splenic nodule number,structure and colon tumor formation were improved.3.Compared with Control group,the percent of CD4+ and CD8+T lymphocyte was decreased in AOM/DSS and AOM/DSS+Solvent group(P(27)0.05),the percent of CD8+T lymphocyte in tocotrienol groups and combination group was decreased(P(27)0.05),however,AOM/DSS+Solvent,AOM/DSS+T3-L and AOM/DSS+T3-Mgroups showed CD4+/CD8+ increased(P(27)0.05).There were no significantly difference between CD4+ and CD8+T lymphocyte percent in AOM/DSS and AOM/DSS+Solvent group(P>0.05),compared with the ratio of CD4+/CD8+ in AOM/DSS,AOM/DSS+Solvent increased(P(27)0.05).Compared with AOM/DSS+Solvent group,AOM/DSS+T3-M group,AOM/DSS+T3-H CD4+ and CD8+ T lymphocytes were increased(P(27)0.05),the percentage of CD8+T lymphocyte in AOM/DSS+OXA and AOM/DSS+OXA+T3-H was elevated(P(27)0.05),and the ratio of CD4+/CD8+ was declined in AOM/DSS+T3-L and AOM/DSS+T3-M groups(P(27)0.05).4.The expression of IL-6 in AOM/DSS and AOM/DSS+Solvent was significantly higher than that of in Control(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent group,the expression of IL-6 in AOM/DSS+T3-M,AOM/DSS+T3-H,AOM/DSS+OXA and AOM/DSS+OXA+T3-H was significantly decreased(P(27)0.05).Compared with AOM/DSS+OXA group,AOM/DSS+T3-L showed higher IL-6 expression(P(27)0.05).Compared with the TNF-a expression in Control,each group were significantly increased(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).AOM/DSS+T3-H and AOM/DSS+OXA groups showed lower expression than that in AOM/DSS+Solvent group(P(27)0.05).Compared with AOM/DSS+OXA group,tocotrienol groups and combination group showed no significantly change(P>0.05).Compared with the IL-10 expression in Control group,AOM/DSS and AOM/DSS+OXA showed significantly decreased(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with IL-10 expression in AOM/DSS+Solvent,each treatment group showed no significant change(P>0.05).Compared with AOM/DSS+OXA,IL-10 expression was elevated in AOM/DSS+T3-M(P(27)0.05).5.Compared with the expression of IL-4 in Control group,each group was decreased except AOM/DSS+OXA+T3-H group(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent and AOM/DSS+OXA,the IL-4 expression inAOM/DSS+OXA+T3-H was elevated(P(27)0.05).Compared with the expression of IP-10 in Control group,AOM/DSS,AOM/DSS+Solvent,AOM/DSS+T3-L and AOM/DSS+OXA showed significantly increased(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent and AOM/DSS+OXA,the expression of IP-10 were decreased in tocotrienol groups and combination group(P>0.05).Compared with the expression of MIP-1β in Control group,AOM/DSS showed significantly increased(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent,the expression of MIP-1β were decreased in AOM/DSS+T3-H and AOM/DSS+OXA+T3-H(P(27)0.05).Compared with AOM/DSS+OXA group,tocotrienol groups and combination group showed no significantly change(P>0.05).Compared with the expression of G-CSF in Control group,each group was increased except AOM/DSS+T3-H group(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent,each group was decreased except AOM/DSS+T3-L group(P(27)0.05).Compared with AOM/DSS+OXA group,tocotrienol groups and combination group showed no significantly change(P>0.05).6.The results showed that the levels and the expressions of My D88 and NF-κB in AOM/DSS and AOM/DSS+Solvent groups were higher than those of Control(P(27)0.05).There were no significantly difference between AOM/DSS and AOM/DSS+Solvent group(P>0.05).Compared with AOM/DSS+Solvent group,the My D88 level in AOM/DSS+OXA group showed decreased(P<0.05),the My D88 expression in AOM/DSS+OXA+T3-H showed decreased(P<0.05).Compared with the level and expression of NF-κB,each treatment group was decreased except AOM/DSS+T3-L group(P(27)0.05).Compared with AOM/DSS+OXA,the expression of NF-κB in AOM/DSS+T3-L group showed significantly increased(P<0.05),the level of NF-κB in tocotrienol groups showed significantly increased(P<0.05).Conclusions1.Tocotrienol treatments could reduce the mortality rate,hemafecia and tumor formation rate.2.Tocotrienol could regulate lymphocyte subsets and the expression of cytokines in peripheral blood serum,such as TNF,IL-6,IP-10,MIP-1β,G-CSF.3.Tocotrienol can inhibit abnormal expression of My D88 and NF-κB in colon carcinoma suggested that tocotrienol may regulate the immune function of mice with CAC by inhibiting the persistent abnormal expression of TLR4/My D88/NF-κB signaling pathway. |
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