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

Role of defective intestinal mucosal barrier in the intestinal carcinogenesis induced by deoxycholic acid

目的持续暴露脱氧胆酸(deoxycholic acid,DOC)可诱导肠腺瘤癌变,机制尚不清楚。本研究旨在探讨肠黏膜屏障损伤在DOC诱导肠腺瘤癌变过程中的作用。方法将20只4周龄Apcmin/+小鼠均分为对照组和DOC组,对照组常规饮水,DOC组给予含质量分数0.2%DOC饮用水,给药12周后处死,观察两组小鼠肠道腺瘤大小、数目及癌变率。采用HE染色评价肠道腺瘤病理类型,采用异硫氰酸荧光素标记的葡聚糖(fluorescein isothiocyanate-conjuagted dextran,FITC-D)检测小鼠肠道通透性,采用实时荧光定量PCR及免疫组织化学染色法评价两组小鼠肠道组织各种紧密连接蛋白、杯状细胞及潘氏细胞相关分子的表达水平。结果 DOC组Apcmin/+小鼠肠道息肉数量为57.00±3.07,较对照组21.50±4.69明显增加,t=20.03,P<0.000 1。DOC组小鼠血清中FITC-D浓度为(1.17±0.12)μg/mL,明显高于对照组(0.51±0.07)μg/mL,t=4.30,P=0.004;DOC组小鼠肠道ZO-1(0.31±0.04)和Claudin 3 mRNA表达量(0.14±0.02)明显低于对照组的0.78±0.07(t=5.53,P=0.000 3)和0.92±0.08(t=9.80,P=0.000 6),而DOC组增加肠道通透性的Claudin 7mRNA表达量为5.79±0.53,明显高于对照组的1.61±0.30,t=6.83,P=0.002 4;DOC组小鼠肠道染色阳性细胞数PAS(20.88±0.79)和MUC2(13.10±1.16)均较对照组35.44±1.68(t=7.84,P<0.000 1)及28.50±0.96(t=10.24,P<0.000 1)明显减少;且DOC组小鼠肠道MUC2mRNA表达量为0.15±0.04,低于对照组的0.91±0.05,t=11.34,P<0.000 1;DOC组胃型黏蛋白MUC5mRNA表达量为2.40±0.26,高于对照组的0.96±0.02,t=4.21,P=0.005 6;DOC组小鼠肠道潘氏细胞溶菌酶染色阳性细胞数为5.19±0.26,较对照组7.49±0.33显著下降,t=5.49,P=0.000 6。同时DOC组潘氏细胞分泌防御素(0.12±0.27)及隐凹素(0.20±0.35)基因表达量均低于对照组的0.87±0.05(t=13.75,P<0.000 1)和0.87±0.05(t=10.95,P<0.000 1)。结论 DOC可通过增加肠道黏膜通透性、破坏肠道上皮间紧密连接蛋白、减少杯状细胞及潘氏细胞数量损伤肠黏膜屏障,从而促进Apcmin/+小鼠腺瘤生长及癌变。

OBJECTIVE Continued exposure to deoxycholic acid （DOC） can induce adenomas cancerous, but the mechanisms between DOC and Colorectal cancer （CRC） are not fully clear. We aim to investigate the role of defective in- testinal mucosal barrier on carcinogenesis of intestinal adenoma promoted by DOC in Apcmin/＋ mice. METHODS Twenty four-week-old mice were divided into two groups： control group and DOC treatment group （with 0.2% DOC in drinking water）, all mice were sacrificed after 12 weeks. Parameters of intestinal tumor development including size, number and canceration rate were determined. The pathological type of adenoma was assessed by hematcxylin-eosin （HE） staining.Intestinal permeability was assessed by concentration in serum of fluorescein isothiocyanate-conjuagted dextran（FITC-D）. Intestinal tight junctions related data about goblet cell and paneth cell were detected by real-time PCR, periodic acid-schiff （PAS） staining and immunohistochemistry. RESULTS Compared with control group （21.50 ± 4. 69）, DOC increased the number （57.00±3.07, t=20.03, P〈0. 000 1） and carcinogenesis of intestinal tumors. DOC increased the concen- tration of FITC-D in blood in DOC group （1.17±0.12） μg/mL, compared to control group （0.51±0.07） μg/mL （t= 4.30,P=0. 004）. DOC decreased the mRNA expression of tight junction ZO-1 （0.31±0.04） and Claudin 3 （0.14± 0. 02）, but increased the mRNA expression of Claudin 7 （5.79±0.53）, which contributes to intestinal leak paraeellular transport pathway, compared to the mRNA expression of ZO-1 （0.78±0.07, t= 5.53, P = 0. 000 3）, Claudin 3 （0.92±0.08, t=9.80, P=0. 000 6） and Claudin 7（1.61±0.30, t=6.83, P=0. 002 4） in the control group. The number of PAS-positive （20. 88±0.79）, MUC2-positive （13. 10±1.16） goblet cell and lysozyme-positive paneth cell （5.19±0.26） per villus were significantly decreased in DOC treatment group compared with that in control group （35.44±1.68, t= 7.84, P〈0. 000 1; 28.50±0.96, t=10.24, P〈0. 000 1;7.49±0.33, t=5.49, P=0. 000 6）. Besides, the mRNA ex- pression of defensin （0.12±0.27）, cryptdin （0.20±0.35） secreted by paneth cell and MUC2 （0.15±0.04） secreted by goblet cell were significantly decreased, while the mRNA expression of gastric MUG5 was increased （2. 40 ± 0.26） by DOC treatment compared to control group （0.87±0.05, t= 13.75, P〈0. 000 1; 0.87 ± 0.05, t= 10.95, P〈0. 000 1; 0. 91±0. 05, t=11.34, P〈0. 000 1; 0.96±0.02, t=4.21, P=0. 005 6）. CONCLUSIONS Defective Intestinal mueo- sal barrier, including increased mucosal permeability, decreased tight junction, decreased number of goblet cells and pan- eth cells, has important effect on carcinogenesis of intestinal polyps promoted by DOC.