壳寡糖抑制炎症相关性结直肠癌的研究

Inhibitory effects of chitooligosaccharides on colitis-associated colorectal cancer

目的观察壳寡糖对炎症相关性结直肠癌(CAC)的防治效果和对肠道细菌的影响。方法 32只8~10周龄雄性C57BL/6小鼠随机分为健康对照组(CK,正常饮食)、壳寡糖对照组(COS,每日灌胃壳寡糖300mg/kg)、炎症相关性结直肠癌模型组(CAC,氧化偶氮甲烷AOM/葡聚糖硫酸钠DSS诱导炎症相关性结直肠癌)和壳寡糖治疗组(CAC+COS,每日灌胃壳寡糖300mg/kg的CAC处理组),每组8只。每周称量小鼠体重并测定疾病活动指数(DAI),造模结束时(第10周末)取小鼠结肠组织,进行病理组织学检查和结肠上皮细胞因子COX-2、IL-1β、IL-6、IL-10和TNF-α丰度的RT-qPCR检测,以及小鼠粪便中总细菌、乳酸杆菌、肠球菌、具核梭杆菌、丁酸盐产生菌丰度的Real-time PCR检测,评估壳寡糖对CAC的防治效果和对肠道细菌的影响。结果 CAC+COS组小鼠成瘤率从CAC组的100%下降至71.4%,且小鼠体重下降得到明显改善,实验结束时CAC组小鼠体重为(22.14±1.18)g,CAC+COS组为(24.84±0.60)g,差异有统计学意义(P<0.05);CAC组小鼠出现肛门脱垂,CAC+COS组小鼠未出现明显脱肛。CK和COS组小鼠DAI为0;CAC和CAC+COS组小鼠在实验第1周即出现腹泻等疾病活动特征,但从第4周开始,CAC+COS组小鼠DAI显著降低至实验结束时分别为1.57±0.23和2.67±0.47,与CAC组比较差异有统计学意义(P<0.05)。CAC组小鼠结肠上皮细胞因子COX-2、IL-1β、IL-6、IL-10和TNF-αmRNA较CK和COS组均显著升高,壳寡糖的应用可显著下调上述5种因子的丰度(P<0.05),ΔΔCT值CAC+COS组分别为3.57±0.90、5.25±0.82、1.26±0.64、1.32±0.25和1.50±0.25,CAC组分别为5.56±0.94,7.99±0.94,3.53±0.48,3.63±0.30和2.78±0.40。CAC组小鼠肠道的肠球菌和具核梭杆菌较CK组显著增加(Lg值分别为3.52±0.18vs.0.97±0.22,2.06±0.07vs.1.21±0.06),丁酸产生菌较CK组显著减少(Lg值为1.00±0.21vs.2.85±0.09)(P<0.05);而壳寡糖治疗后的CAC+COS组中肠球菌和具核梭杆菌丰度均较CAC组小鼠显著降低(Lg值分别为1.16±0.15vs.3.52±0.18,1.69±0.20vs.2.06±0.07),丁酸盐产生菌显著增加(Lg值为2.11±0.22vs.1.00±0.21)。结论壳寡糖可通过显著降低小鼠疾病活动指数、成瘤率和改变结肠上皮细胞因子丰度控制小鼠炎症相关性结直肠癌的发生发展,且壳寡糖可显著增加肠道总细菌和丁酸盐产生菌丰度,降低肠球菌和具核梭杆菌的丰度,是预防和治疗炎症相关性结直肠癌的潜在有效药物。

Objective To evaluate the effects of chitooligosaccharides on colitis-associated colorectal cancer（CAC）and microbiota. Methods Thirty-two male C57 BL/6 mice aged 8-10 weeks were randomly divided into a healthy control group（CK,normal diet）,a control group treated with chitooligosaccharides（COS,daily intragastric administration of chitooligosaccharides at a dose of 300 mg/kg）,a CAC group（CAC was chemically induced with azoxymethane[AOM]and dextran sodium sulfate[DSS]）,and a CAC group treated with chitooligosaccharides（CAC＋COS,daily intragastric administration of chitooligosaccharides 300 mg/kg in mice with AOM/DSS treatment）.Each group consisted of 8 animals.The experiment was terminated and mice were sacrificed at the end of 10 weeks.To evaluate the effect of chitooligosaccharides on the prevention of CAC,changes in body weight and the disease activity index（DAI）were analyzed weekly,pathological features in colon tissue were assessed using HE staining,and levels of COX-2,IL-1β,IL-6,IL-10,and TNF-αmRNA in colon epithelial tissue were detected using real-time-qPCR.Real-time PCR was used to detect the total amount of bacteria in feces and the amount of Lactobacillus,Enterococcus,Fusobacterium nucleatum,and butyrateproducing bacteria at the end of 10 weeks. Results Results indicated that CAC was successfully induced with AOM and DSS.Intragastric administration of chitooligosaccharides reduced the incidence of tumors from 100% in the CAC group to 75.0%in the CAC＋COS group.Weight loss in the CAC group was also significantly ameliorated after treatment with chitooligosaccharides.At the end of the experiment,the average body weight was 22.14±1.18 g in the CAC group and 24.84±0.60 g in the CAC＋COS group（P〈0.05）.The DAI for mice in the CK and COS groups was 0 over the course of the experiment.Mice in the CAC and CAC＋COS groups displayed similar weight loss,diarrhea,and hematochezia in the first week of the experiment.After week 4,however,the DAI for the CAC＋COS group was significantly lower than that for the CAC group（P〈0.05）.At the end of the experiment（the end of 10 weeks）,the DAI was 1.57±0.23 for the CAC group and 2.67±0.47 for the CAC＋COS group.Levels of the colonic epithelial cytokines COX-2,IL-1β,IL-6,IL-10,and TNF-αin the CAC group were significantly higher than those in the CK,COS,and CAC＋COS groups（P〈0.05）.ΔΔCT analysis indicated that COX-2 was 5.56±0.94 in the CAC group,IL-1βwas 7.99±0.94,IL-6 was 3.53±0.48,IL-10 was 3.63±0.30,and TNF-αwas 2.78±0.40.In the CAC＋COS group,COX-2 was 3.57±0.90,IL-1β was 5.25±0.82,IL-6 was 1.26±0.64,IL-10 was 1.32±0.25,and TNF-αwas 1.50±0.25.Treatment with chitooligosaccharides significantly decreased the levels of cytokines（P〈0.05）.The amount of fecal Enterococcus（Lg：3.52±0.18 vs.0.97±0.22）and Fusobacterium nucleatum（2.06±0.07 vs.1.21±0.06）in the CAC group was significantly higher than that in the CK group,while the amount of butyrate-producing bacteria was significantly lower than in the CK group（1.00±0.21 vs.2.85±0.09）.Treatment with chitooligosaccharides significantly reduced the amount of Enterococcus（1.16±0.15 vs.3.52±0.18）and Fusobacterium nucleatum（1.69±0.20 vs.2.06±0.07）and increased the amount of butyrate-producing bacteria（2.11±0.22 vs.1.00±0.21）. Conclusion Treatment with chitooligosaccharides significantly reduced the DAI and the incidence of tumors in mice,and it down-regulated the levels of inflammatory cytokines during the development of CAC.Treatment with chitooligosaccharides increased the total amount of intestinal bacteria and butyrate-producing bacteria and it reduced the amount of Enterococcus and Fusobacterium nucleatum.Therefore,chitooligosaccharides are potential agents with which to prevent and treat CAC.