胞外-5'-核苷酸酶在克罗恩病小鼠结肠组织中的表达及意义

Expression and significance of ecto-5'-nucleotidase in colon tissue of Crohn's disease mice

目的探讨克罗恩病胞外-5'-核苷酸酶(ecto-5'-nucleotidase,CD73)在结肠组织中的表达情况及其意义和可能的作用机制。方法雄性BALB/c小鼠30只,随机分为正常对照组、模型组和干预组。对照组正常饲养,模型组采用2,4,6三硝基苯磺酸(trinitrobenzene sulfonic acid,TNBS)+40%乙醇灌肠剂诱导建立慢性炎症诱导的克罗恩病小鼠模型,干预组在模型组的基础上,于每次灌肠的第2天腹腔注射AB-680。记录小鼠体重、粪便性状、粪便潜血用于炎症性肠病疾病活动指数(disease activity index,DAI)评分。于第7周处死小鼠,取出结肠组织,称重并测量长度。采用标准苏木精-伊红染色法(hematoxylin-eosin staining,HE)染色观察组织炎症情况;马松(Masson)染色测量小鼠结肠组织中胶原蛋白所占的面积;利用荧光定量PCR、免疫组化检测小鼠结肠组织CD73分子表达;酶联免疫吸附试验(enzymelinked immunosorbent assay,ELISA)检测小鼠结肠组织转化生长因子(transforming growth factor-β,TGF-β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)表达水平。结果对照组DAI评分0.10±0.16,模型组DAI评分2.80±0.79,干预组DAI评分3.07±0.34;与对照组相比,模型组、干预组DAI评分明显升高(P<0.05);与模型组相比,干预组DAI评分明显升高(P<0.05)。HE染色观察对照组小鼠结肠未见炎症表现,模型组、干预组小鼠结肠可见水肿充血,出现炎性细胞浸润、粘膜溃疡等典型的炎症表现。对照组胶原蛋白占据面积比(4.95±0.82)%,模型组胶原蛋白占据面积比(24.62±1.46)%,干预组胶原蛋白占据面积比(54.47±2.75)%;与对照组相比,模型组、干预组胶原蛋白占据面积比明显升高(P<0.05);与模型组相比,干预组胶原蛋白占据面积比明显升高(P<0.05)。与对照组相比,模型组、干预组小鼠结肠组织CD73表达明显增加(P<0.05);与模型组相比,干预组小鼠结肠组织CD73表达降低(P<0.05)。与对照组相比,模型组、干预组TGF-β、TNF-α、IL-6表达明显升高(P<0.05);与模型组相比,干预组TGF-β、TNF-α、IL-6表达下降(P<0.05)。结论CD73在克罗恩病小鼠结肠组织中表达水平升高,通过上调TGF-β表达抑制炎症反应并改善纤维化。另一方面,CD73通过上调IL-6、TNF-α表达加重炎症反应。因此CD73在克罗恩病的肠道炎症和纤维化中可能起着双向调节作用。

Objective This study aimed to investigate the expression of CD73 in colonic tissues in Crohn's disease(CD)and its significance and possible mechanism of action.Methods Thirty male BALB/c mice were randomly divided into normal control group,model group and intervention group.The control group was fed normally,and the model group was treated with TNBS+40%alcohol enema to establish a mouse model of Crohn's disease induced by chronic inflammation.The intervention group was treated with AB-680 intraperitoneally on the second day of each enema based on the model group.Mice body weight,fecal traits and fecal occult blood were recorded for disease activity index(DAI)score of inflammatory bowel disease.The animals were sacrificed at 7th week,their colonic tissues were removed,weighed and measured.The tissue inflammation was observed by standard hematoxylin-eosin(HE)staining.Masson staining was used to measure the area of collagen in colon tissue of mice.CD73 was detected by fluorescence quantitative PCR and immunohistochemistry.The expression levels of TGF-β,TNF-αand IL-6 in colon tissue of mice were determined by ELISA.Results The DAI score was(0.10±0.16)in the control group,(2.80±0.79)in the model group,and(3.07±0.34)in the intervention group.Compared with the control group,the DAI scores of the model and intervention groups were increased significantly(P<0.05).Compared with the model group,the DAI score of the intervention group was significantly increased(P<0.05).HE staining showed that there was no inflammation in the colon of the control group,while the colon of the model group and the intervention group showed typical inflammatory manifestations such as edema and congestion,inflammatory cell infiltration,and mucosal ulcer.The area ratio of collagen in the control group was(4.95±0.82)%,in the model group was(24.62±1.46)%,and in the intervention group was(54.47±2.75)%.Compared with the control group,the area ratio of collagen in the model group and the intervention group was significantly increased(P<0.05).Compared with the model group,the area ratio of collagen in the intervention group was significantly increased(P<0.05).Compared with the control group,the expression of CD73 in colon tissue of the model group and the intervention group was significantly increased(P<0.05).Compared with the model group,the expression of CD73 in colon tissue of the intervention group was decreased(P<0.05).Compared with the control group,the expressions of TGF-β,TNF-αand IL-6 in the model group and the intervention group were significantly increased(P<0.05).Compared with the model group,the expression of TGF-β,TNF-αand IL-6 in the intervention group decreased(P<0.05).Conclusions CD73 is upregulated in colon tissue of CD mice,it can inhibit inflammatory reaction and improve fibrosis by up-regulating TGF-βexpression.On the other hand,CD73 can aggravate the inflammatory response in CD intestinal inflammation and fibrosis by upregulating the expression of IL-6 and TNF-α.Therefore,CD73 may play a bidirectional regulatory role in intestinal inflammation and fibrosis of CD.