水飞蓟宾抑制小鼠结肠炎相关肠癌发生机制研究

Inhibition effect of silibinin on colitis-associated cancer in mice

目的随着炎症性肠病发病率的逐年增高,作为其主要的致死病因——结肠炎症相关肠癌(colitis-associated cancer,CAC)也越来越受到关注。植物类药物水飞蓟宾(silibinin,SB)抗感染抗肿瘤作用已在多种细胞动物模型中应用。本研究利用氧化偶氮甲烷(azoxymethane,AOM)/葡聚糖硫酸钠(dextran sulfate sodium salt,DSS)诱导CAC小鼠模型评价水飞蓟宾的化学预防作用及其对白细胞介素-6/信号转导及转录激活因子3(interleukin-6,IL-6/Signal transducers and activators of transcription 3,STAT 3)通路的调节。方法 6周龄的C57BL/6小鼠随机分为空白对照组(5只)、AOM/DSS组(15只)和水飞蓟宾治疗组AOM/DSS/SB组(15只)。AOM/DSS(10mg/kg)组于实验第1天腹膜内注射,持续5d给予小鼠2%DSS(溶解于无菌饮用水),随后16d给予无菌水,后继续下一个循环,共3个循环。AOM/DSS/SB组全程给予水飞蓟宾灌胃750mg/(kg·d),实验第10周末处死小鼠。免疫组化染色测定细胞增殖情况,TUNEL方法判断细胞凋亡情况,Real-time PCR评价炎症因子IL-6mRNA的表达。免疫组化及蛋白质印迹方法评价水飞蓟宾对IL-6/STAT 3通路的抑制作用。结果AOM/DSS组与AOM/DSS/SB组的肿瘤数量及直径差异均有统计学意义,P<0.05;尤其是直径≥4mm的肿瘤,2组数量差异有统计学意义,t=4.12,P<0.001。AOM/DSS/SB组的肿瘤评分及炎症评分均低于AOM/DSS组。水飞蓟宾组小鼠肠道Ki-67阳性细胞百分比为(37.00±13.00)%,较AOM/DSS组(71.00±11.00)%减少,t=12.65,P<0.001。AOM/DSS/SB组TUNEL阳性细胞百分比为(28.40±13.60)%,较AOM/DSS组(15.00±9.00)%增加,t=2.41,P<0.05。AOM/DSS/SB组小鼠肠组织中炎症因子IL-6mRNA及磷酸化STAT 3表达水平低于AOM/DSS组,而STAT 3蛋白表达水平分别为(48.60±2.45)%和(56.80±3.15)%,2组差异无统计学意义,t=2.06,P>0.05。结论水飞蓟宾可下调IL-6/STAT 3信号通路从而抑制CAC的发生,提示水飞蓟宾对CAC的化学预防具有一定的潜力。

OBJECTIVE While the incidence of inflammatory bowel disease（IBD） is cumulatively increasing,colitis- associated cancer（CAC）, as its leading cause of death,is drawing more and more attention. Silibinin, a kind of herbal medi cine,has been demonstrated to be anti-inflammatory and anti-neoplastic in multiple cellular and animal models. This study aimed to evaluate the chemopreventive effect of silibinin utilizing a colitis-associated cancer（CAC） mice model induced by azoxymethane/dextran sulfate sodium （AOM/DSS）, and determine its modulation to IL-6/STAT3 signaling pathway. METHODS Six-week-old C5713L/6 mice were randomly divided into three groups： control group, AOM/DSS group and AOM/DSS/SB group. AOM （10 mg/kg） was injected intraperitoneally on the beginning day of our experiment. Two per- cent DSS dissolved in drinking water was administered to mice for the following 5 days,and then 16 days of aseptic water followed. This cycle was repeated two more times. Silibinin 750 mg/（kg · d） was continuously administered to mice of the AOM/DSS/SB group for 10 weeks,and the mice were sacrificed at the end of the 10th week of the experiment,immuno- histochemical staining for cell proliferation evaluation,TUNEL for apoptosis assessment. The production of IL-6 mRNAwas determined by Real-time PCR. The inhibition effect of silibinin on IL 6/STAT3 signaling pathway was evaluated by immunohistochemieal staining and Western blot. RESULTS The number and size of colonic tumors in the AOM/DSS/SB group was significantly reduced compared with that in the AOM/DSS group. Difference in lumor number between the two groups was especially apparent in tumors whose diameter was over than 4mrn （t=4.12, P〈0. 001）. Both the tumor score and the inflammation score were evidently lower in the AOM/DSS/SB group than that in the AOM/DSS group. The per centage of intestinal ki-67 positive cells in mice of the AOM/DSS/SB group [（37.00± 13.00）%] was significantly lower than that of the AOM/DSS group [（71.00±11.00）%;t= 12.65,P〈0. 001]. TUNEL stain showed a higher percentage of apoptotic cells in tumors of the AOM/DSS/SB group compared with that of the AOM/DSS group （t=2.41 ,P〈20.05）. The production of IL-6 mRNA and phosphorylated STAT3 in mice colon of the AOM/DSS/SB group showed a significant decrease compared with that of the AOM/DSS group. In contrast, total expression of STAT3 didn＇t show any statistically significant difference between the two groups （t=2.06,P〈0.05）. CONCLUSION Silibinin could protect against CAC in mice via inhibiting IL-6/STAT3 signaling,which showed promising chemopreventive potential against CAC.