| Part One Colitis-associated Colon Cancer Induced by Azoxymethane and Dextran Sodium Sulfate in Mouse ModelBackground:Chronic inflammation plays an important role in the pathogenesis of malignant tumors. Patients with inflammatory bowel disease (IBD) have higher risks to colorectal cancer (CRC). Colitis-associated colon cancer (CAC) is one of the hot research topics during these decades. However, as the long duration of disease and a variety of mechanically-unknown cytokines involved, the animal model of colitis-associated colon cancer is hard to be established.Objectives:To set the mouse model of colitis-associated colon cancer (CAC) with azoxymethane (AOM) and dextran sulfate sodium (DSS). The model could promote the follow-up researches about the immunologic mechanisms between intestinal inflammation and malignant colon cancer.Methods:30 male Balb/c mice were randomly divided into A-E groups:control group (Group A), AOM group (Group B), DSS group (Group C), AOM/DSS low dose group (Group D), AOM/DSS high dose group (Group E). Group A was given free access to pure drinking water. Group B was given a single intraperitoneal injection of AOM (10 mg/kg body weight) only and exposed to pure drinking water. Group C was given 3% DSS solution for 7 days and then change with pure drinking water for 14 days, altogether 4 cycles. Group D was given a single intraperitoneal injection of low dose AOM (10 mg/kg body weight). After that,4 cycles of "DSS-pure water" regimen (the same as Group C) was given. Group E was given a single intraperitoneal injection of high dose AOM (20 mg/kg body weight) and 4 cycles of "DSS-pure water" regimen (the same as Group C). All animals were sacrificed at the end of the study (week 12). The general status, body weight, disease activity index (DAI) and length of colon were observed. Pathological changes of colonic tissues were observed by hematoxylin and eosin (HE) stains and calculated by the injury score. The liver injury caused by drugs was measured by HE staining and reticular fiber staining for liver tissues. Serum liver function indicators (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, hydroxyproline and albumin) were also analyzed by automatic biochemical analyzer. The expression of NF-κB p65, TLR2, TLR4 in colonic tissues was determined by immunohistochemistry and semi-quantitative detection. Serum Thl phenotype cytokines (TNF-α, IL-6), Th2 phenotype cytokines (IL-4, IL-10), inflammation and angiogenesis indicators (TGF-β1, VEGF) concentrations were measured by enzyme linked immunosorbent assay (ELISA).Results:Compared with the control group, the mice in DSS group, AOM/DSS low dose group and AOM/DSS high dose group showed significant symptoms of colitis, mainly about poorer general status; more severe diarrhea, hemafecia and rectal prolapse; slower weight gain (P＜0.05); higher scores of disease activity index (P＜0.05); shorter colon length (P<0.05) and higher injury score of HE stained colonic tissues (P＜0.05). The two protrude lesions found in mice of AOM/DSS low dose group were pathologically confirmed as colon adenocarcinoma. Lesions of dysplasia were also found in this group. One confirmed adenocarcinoma was found in mice of AOM/DSS high dose group. No more malignant tumor was found in other groups. Different degrees of liver necrosis and fibrosis were found in Group B to Group E by HE staining and reticular fiber staining. By using automatic biochemical analyzer, the concentrations of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin and hydroxyproline significantly increased (P＜0.05), while the albumin significantly decreased (P＜0.05). The NF-κB p65, TLR2 and TLR4 expressions in colonic tissue of DSS group, AOM/DSS low dose group and high dose group were significantly stronger than that in control group by means of immunohistochemistry and semi-quantitative count (P＜0.05). Measured by ELISA, serum cytokines (TNF-α, IL-6, IL-4, IL-10, TGF-β1, VEGF) levels were significantly increased (P＜0.05) in Group B to Group E.Conclusions:The mouse model of colitis-associated colon cancer can be induced by AOM and DSS with a proper dose. However, liver injuries can be caused during the process. A variety of cytokines were involved in the pathogenesis between intestinal inflammation and malignant tumor. The activation of Toll-like receptors and NF-κB plays an important role in the pathogenesis of colitis-associated colon cancer.Part Two The Blocking Effect of TLR2mAb and TLR4mAb in the Pathogenesis of colitis-associated colon cancer induced by Azoxymethane and Dextran Sodium Sulfate in Mouse ModelBackground:The recurrence of inflammatory bowel diseases may cause colitis-associated colon cancer with a higher risk. We can conclude from Part One that NF-κB is a key modulator in the pathogenesis of colitis-associated colon cancer. Toll-like receptors (TLRs), a class of type I cross-membrane receptors, are the upstream of NF-κB signaling pathway. TLR2 and TLR4 in host intestinal membrane are closely associated with the complicated innate immune response. However, the transduction mechanism of TLRs/NF-κB signal pathway is still a mystery, which attracts us to investigate.Objectives:To investigate the mechanism and the potential probability as therapeutic targets of TLR2 and TLR4 in the pathogenesis of colitis-associated colon cancer.Methods:The mouse model of colitis-associated colon cancer was set by AOM and DSS (reference to AOM/DSS low dose group in Part One). Specially, TLR2 monoclonal antibody (TLR2mAb) and TLR4 monoclonal antibody (TLR4mAb) were intraperitoneally injected during the process. The general status, body weight, disease activity index (DAI) and length of colon were observed. Pathological changes of colonic tissues were observed by hematoxylin and eosin (HE) stains and calculated by the injury score. The liver reticular fiber stain was performed additionally to measure the protection effects of TLR2mAb and TLR4mAb. Serum liver function indicators (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, hydroxyproline and albumin) were also analyzed by automatic biochemical analyzer. The expression of NF-κB p65, TLR2 and TLR4 in colonic tissues was determined with immunohistochemistry and semi-quantitative detection. Serum Thl phenotype cytokines (TNF-a, IL-6), Th2 phenotype cytokines (IL-4, IL-10), inflammation and angiogenesis indicators (TGF-(31, VEGF) concentrations were measured by enzyme linked immunosorbent assay (ELISA).Results:Compared with mice in model group, the symptoms of colitis in TLR2/TLR4 mAb treatment group alleviated significantly, mainly about milder diarrhea, hemafecia and rectal prolapse; accelerated weight gain (P<0.05) and lower disease activity index (P<0.05). The average colon length of mice in treatment group was longer (P<0.05). The tissue injury score of mice in treatment group was lower by HE staining (P＜0.05). No pathological manifestation of dysplasia or malignancy was found. Milder liver necrosis and fibrosis was detected in treatment group by means of HE staining and reticular fiber staining. Additionally, some liver function indicators (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase) decreased significantly (P<0.05) while the others (total bilirubin, hydroxyproline, albumin) appeard no change (P>0.05). The expression of NF-κB p65, TLR2 and TLR4 in colonic tissues decreased significantly measured by immunohistochemistry and semi-quantitative count (P<0.05). Measured by ELISA, serum cytokine (TNF-α, IL-6, IL-4, IL-10, TGF-β1 and VEGF) concentrations were decreased dramatically (P＜0.05).Conclusions:TLRs/NF-κB is a key signal transduction pathway in the pathogenesis between chronic inflammation and malignant tumor. TLR2 mAb and TLR4 mAb can help to reduce the occurrence of the colitis-associated colon cancer by adjusting the imbalanced status of Th1/Th2 phenotype cytokines. TLR2mAb and TLR4mAb could protect the liver from AOM and DSS. |
PDF Full Text Request