水飞蓟宾抑制小鼠结肠炎相关肠癌发生机制研究
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摘要
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,colitisassociated cancer(CAC),as its leading cause of death,is drawing more and more attention.Silibinin,a kind of herbal medicine,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 C57 BL/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 percent 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 10 th week of the experiment,immunohistochemical staining for cell proliferation evaluation,TUNEL for apoptosis assessment.The production of IL-6 mRNA was determined by Real-time PCR.The inhibition effect of silibinin on IL-6/STAT3 signaling pathway was evaluated by immunohistochemical 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 tumor number between the two groups was especially apparent in tumors whose diameter was over than 4 mm(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 percentage 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<0.05).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.
OBJECTIVE While the incidence of inflammatory bowel disease(IBD)is cumulatively increasing,colitisassociated cancer(CAC),as its leading cause of death,is drawing more and more attention.Silibinin,a kind of herbal medicine,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 C57 BL/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 percent 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 10 th week of the experiment,immunohistochemical staining for cell proliferation evaluation,TUNEL for apoptosis assessment.The production of IL-6 mRNA was determined by Real-time PCR.The inhibition effect of silibinin on IL-6/STAT3 signaling pathway was evaluated by immunohistochemical 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 tumor number between the two groups was especially apparent in tumors whose diameter was over than 4 mm(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 percentage 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<0.05).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.
引文
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[8] Raina K,Kumar S,Dhar D,et al,Silibinin and colorectal cancer chemoprevention:a comprehensive review on mechanisms and efficacy[J].J Biomed Res,2016,30(6):452-465.
[9] Sati J,Mohanty BP,Garg ML,et al,Pro-oxidant role of silibinin in DMBA/TPA induced skin cancer:1H NMR metabolomic and biochemical study[J/CD],PLoS One,2016,11(7):e0158955.
[10] Huo X,Liu D,Gao L,et al,Flavonoids extracted from licorice prevents colitis-associated carcinogenesis in AOM/DSS mouse model[J].Int J Mol Sci,2016,17(9):1-14.
[11] Bosch-Barrera J,Queralt B,Menendez JA.Targeting STAT 3 with silibinin to improve cancer therapeutics[J].Cancer Treat Rev,2017,58:61-69.
[12] Liu LQ,Nie SP,Shen MY,et al.Tea polysaccharides inhibit colitis-associated colorectal cancer via interleukin-6/STAT3pathway[J].J Agric Food Chem,2018,66(17):4384-4393.
[13] Francescone R,Hou V,Grivennikov SI.Cytokines,IBD,and colitis-associated cancer[J].Inflamm Bowel Dis,2015,21(2):409-418.
[14] Bosch-Barrera J,Menendez JA.Silibinin and STAT3:A natural way of targeting transcription factors for cancer therapy[J].Cancer Treat Rev,2015,41(6):540-546.
[15] Grivennikov S,Karin E,Terzic J,et al.IL-6and Stat3are required for survival of intestinal epithelial cells and development of colitis-associated cancer[J].Cancer Cell,2009,15(2):103-113.
[2] Rajamanickam S,Velmurugan B,Kaur M,et al,Chemoprevention of intestinal tumorigenesis in APCmin/+mice by silibinin[J].Cancer Res,2010,70(6):2368-2378.
[3] Angelou A,Andreatos N,Antoniou E,et al.A novel modification of the AOM/DSS model for inducing intestinal adenomas in mice[J].Anticancer Res,2018,38(6):3467-3470.
[4] Kennedy RJ,Hoper M,Deodhar K,et al.Interleukin 10-deficient colitis:new similarities to human inflammatory bowel disease[J].Br J Surg,2000,87(10):1346-1351.
[5] DubéPE,Yan F,Punit S,et al.Epidermal growth factor receptor inhibits colitis-associated cancer in mice[J].J Clin Invest,2012,122(8):2780-2792.
[6]孔辛月,曹海龙,王姗,等,结肠炎相关肠癌的发病机制及化学防治的研究进展[J].肿瘤药学,2014,4(1):10-14.
[7]董文逍,曹海龙,许梦雀,等.肠黏膜屏障损伤在脱氧胆酸诱导肠腺瘤癌变过程中的作用研究[J].中华肿瘤防治杂志,2016,23(14):918-923.
[8] Raina K,Kumar S,Dhar D,et al,Silibinin and colorectal cancer chemoprevention:a comprehensive review on mechanisms and efficacy[J].J Biomed Res,2016,30(6):452-465.
[9] Sati J,Mohanty BP,Garg ML,et al,Pro-oxidant role of silibinin in DMBA/TPA induced skin cancer:1H NMR metabolomic and biochemical study[J/CD],PLoS One,2016,11(7):e0158955.
[10] Huo X,Liu D,Gao L,et al,Flavonoids extracted from licorice prevents colitis-associated carcinogenesis in AOM/DSS mouse model[J].Int J Mol Sci,2016,17(9):1-14.
[11] Bosch-Barrera J,Queralt B,Menendez JA.Targeting STAT 3 with silibinin to improve cancer therapeutics[J].Cancer Treat Rev,2017,58:61-69.
[12] Liu LQ,Nie SP,Shen MY,et al.Tea polysaccharides inhibit colitis-associated colorectal cancer via interleukin-6/STAT3pathway[J].J Agric Food Chem,2018,66(17):4384-4393.
[13] Francescone R,Hou V,Grivennikov SI.Cytokines,IBD,and colitis-associated cancer[J].Inflamm Bowel Dis,2015,21(2):409-418.
[14] Bosch-Barrera J,Menendez JA.Silibinin and STAT3:A natural way of targeting transcription factors for cancer therapy[J].Cancer Treat Rev,2015,41(6):540-546.
[15] Grivennikov S,Karin E,Terzic J,et al.IL-6and Stat3are required for survival of intestinal epithelial cells and development of colitis-associated cancer[J].Cancer Cell,2009,15(2):103-113.