| Inflammatory bowel diseases(IBD) are chronic inflammatory disorders of the intestinal tract which is associated with an immunological imbalance in the intestinal mucosa. About 15% of the IBD patients develop into colorectal cancer eventually, which is also called colitis associated colon cancer(CAC). The risk of CAC increased significantly as the degree of severity, extent of injury and duration of the colitis elevated. Thus, IBD is closely associated with the tumorigenesis and progression of CAC. Strategies that effectively inhibit the course of IBD inflammatory development may decrease the risk and severity of CAC.Recent studies highlight that platelets have functions of potent pro-inflammatory cells in addition to their role in hemostasis, coagulation, and pathologic thrombosis. It can mediate the balance between innate and adaptive immune systems and are involved in occurrence and progression of multiple inflammations and cancers. It has been confirmed that many platelets changes occurred in IBD patients, including morphological alterations, over secretion of granular content, release of microparticles(MPs), etc. In IBD patients, Platelets are highly activated and have a unique spontaneously aggregated feature. Thromboembolism is one of the four leading causes of death in IBD patients, and it is correlated with clinical disease activity in IBD patients. The existing studies indicate that the state of platelets may play a crucial regulatory role in the progression of IBD. Inhibition of platelets activation and aggregation may be a potential cure for IBD patients. Recent clinical studies highlight that long term and low dose administration of aspirin could reduce the incidence and mortality of many inflammation-induced cancers. Platelets inhibition may be one of the reasons, while the specific mechanism is still unclear. Simultaneously, studies also discovered that elevated level of platelet counts and activity occurred in multiple malignant tumors. Malignant tumor induces reactively increased platelet counts. Productions of TXA2 and ADP from tumor result in platelet activation and aggregation. The process above is termed as tumor cell induced platelet aggregation(TCIPA). Eventually, platelets physically shield tumor cells at vascular endothelium leading to the escape of tumor cells from immunological surveillance and promoted survival of tumor cells. Platelet α-granule contains multiple growth factors including PDGF, VEGF, etc. Malignant tumor stimulates platelet degranulation and release a large amount of growth factors that promote tumorigenesis and angiogenesis. In addition, platelets also facilitate tumor invasion and metastasis. Therefore, platelets play an important role in promoting occurrence and progression of the tumor.According to the studies above, the level of platelets activation and aggregation is closely associated with IBD and tumorigenesis. Highly activated and aggregated platelets can promote the formation and development of IBD and tumor. Long-term repeated IBD has greatly increased the incidence of CAC. Therefore, we hypothesize that anti-platelet aggregation drugs may be adopted to relieve the development of IBD, thereby affecting the occurrence and development of CAC. To confirm the idea, we applied the anti-platelet aggregation agent clopidogrel to the experimental CAC mouse model in order to evaluate the effect of clopidogrel on chemically induced CAC mouse model and explored the possible mechanism.Methods: We established a mouse model of CAC through a single dose of chemical carcinogen azoxymethane(AOM) combined with three cycles of pro-inflammatory agent dextran sulfate sodium(DSS). Mice were divided into 5 groups: normal control group, AOM/DSS model group, 12.5 mg/kg clopidogrel treated group, 25 mg/kg clopidogrel treated group and 50 mg/kg clopidogrel treated group. The first day when mice obtained DSS water was defined as day 0. Mice were continuously received different dose of clopidogrel since then and their weight and clinical scores were recorded. At the 56 th day, mice were sacrificed and the samples were removed. Tumor number and size were recorded and analyzed. We evaluated colon epithelium hyperplasia degree of mice by Hematoxylin & eosin(HE) stain. In order to study the possible suppression mechanism of clopidogrel on CAC, we divided the progression of CAC mouse model into two periods: inflammatory period, tumorigenesis and progression period. A mouse model of CAC inflammatory period was established through a single dose of AOM combined with continuous DSS water for 8 days. Mice were continuously received different dose of clopidogrel(1.39 mg/kg、4.17 mg/kg、12.5 mg/kg) since the first day of DSS administration. We evaluated the body weight change, clinical score, colon length, histopathology structure, release of inflammatory cytokines, and the proliferation of colon epithelium in mice. Samples from the 35 th day and the 56 th day during the establishment of the model were obtained to study the effect of clopidogrel on CAC tumorgenesis and progression period. We observed the proliferation of colon epithelium cells(PCNA and Ki67 stain), the expression of cytokines and chemokines(TNF-α, CXCL2&CXCR2, CCL3&CCR1) of the mice colon tissue separately. As described above, we made a preliminary exploration of suppression mechanism of clopidogrel on CAC formation and progression.Results:1. Clopidogrel elevated mice body weight change compared to their initial body weight from 102.5% to 106.4% on the 9th day(P<0.05). Clopidogrel decreased mice clinical score from 2.5 and 4.5 to 0.85 and 2 separately on the 31 th day and the 50 th day(P<0.05).2. As for the formation of CAC tumor, clopidogrel had a tendency of decreasing tumor number and alleviating tumor load. It decreased tumor number from 8 to 5(P>0.05), and alleviated tumor load from 27.4 mm to 14.4 mm(P>0.05). Clopidogrel reduced tumor size(from 3 mm to 2.3 mm, P<0.05), maximum diameter of tumor(from 4.6 mm to 3.2 mm, P<0.05), percentage of >4 mm tumor(from 15.4% to 3.0%, P<0.05).3. Analysis of CAC mice colon tissue pathological section showed that clopidogrel reduced the level of colon epithelium hyperplasia in mice. The hyperplasia degree of each mouse in AOM/DSS model were moderate and severe, while that of the clopidogrel treated group were mild and moderate mostly.4. For analysis of the samples from CAC inflammatory period, we found that clopidogrel elevated mice body weight change compared to their initial body weight from 88.97% to 97.55%(P<0.01), alleviated mice clinical score from 4.4 to 2.5(P<0.01), decreased shrinkage of colon length induced by inflammation from 6.2 cm to 7.3 cm(P<0.001). Analysis of pathological section of colon tissue showed that clopidogrel significantly reduced infiltration of inflammatory cells, cut down number and extent of ulceration, decreased epithelium hyperplasia degree, P<0.05. Clopidogrel relieved extent of lesion in mice from 43.6% to 26.5%(P<0.001) and decreased extent of Ki67 stain on mice colon tissue, P<0.05.5. For analysis of the samples from CAC tumorigenesis and progression period, we found that on the 35 th day of the model, clopidogrel reduced TNF-α expression from triple to 1.5 times compared to normal control(P<0.01). Clopidogrel decreased level of Ki67 stain on mice colon tissue(P<0.05) and decreased m RNA and protein level of CXCL2&CXCR2 which is associated with neutrophils recruitment(P<0.05). As for samples from the 56 th day of the CAC mouse model establishment, though clopidogrel had little effect on Ki67 stain and TNF-α expression, it decreased m RNA and protein level of CXCL2&CXCR2(P<0.05). It also decreased m RNA level of CCL3&CCR1(P<0.05), which is associated with macrophages recruitment.Conclusions:1. During the development of colitis associated tumorigenesis, clopidogrel reduced tumor size and suppressed formation of large tumor, reduced hyperplasia level of tumor. On account of significant individual difference in each group, clopidogrel had little effect on tumor number and tumor load. The results above indicated that clopidogrel had an inhibitory effect on tumorigenesis and progression of CAC.2. During CAC inflammatory period, clopidogrel relieved mice body weight change, clinical score, infiltration of the inflammatory cells, degree of ulceration and hyperplasia, extent of lesion and suppressed cell proliferation of colon epithelium in mice. It indicated that clopidogrel significantly alleviated the progression of inflammation during CAC inflammatory period.3. During CAC tumorigenesis and progression period, analysis of the sample obtained from the 35 th day showed that clopidogrel reduced level of colon epithelium cell proliferation and TNF-α expression. Whereas analysis of the sample obtained from the 56 th day, clopidogrel had little effect on colon epithelium cell proliferation and TNF-α expression. It suggested that clopidogrel had a significant inhibitory effect on CAC tumorigenesis and progression at the 35 th day of CAC model, while at the 56 th day of CAC model clopidogrel could not inhibit further progression of the tumor.4. According to the analysis of the m RNA and protein level of chemokines during CAC tumorigenesis and progression period, we learned that the effect of clopidogrel on CAC tumorigenesis and progression may be associated with the recruitment of neutrophils at the tumor site.To sum up, we confirmed the retarding effect of clopidogrel on CAC tumorigenesis and progression by using an experimental CAC mouse model. The potential mechanisms could be associated with reducing the severity of inflammation, inhibiting colon epithelium cell proliferation, and decreasing the recruitment of neutrophils at the tumorsite. |
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