甲氨蝶呤通过抑制Wnt/β-catenin信号通路诱导小鼠肠道损伤

Methotrexate induces intestinal injury by inhibiting Wnt/β-catenin signaling pathway in mice

研究旨在探讨甲氨蝶呤(methotrexate, MTX)对小鼠肠上皮更新的影响及其机理。选用体重相近的5～6周龄的昆明小鼠64只,随机分为两组,每组设32个重复,每个重复1只小鼠,连续2 d分别腹腔注射生理盐水和50 mg/kg BW MTX溶液,并于注射后72 h处死小鼠;同时用0.025、0.05、0.10μM和0.20μM MTX处理小鼠结肠癌细胞系CMT-93。结果表明,MTX可显著降低小鼠平均日采食量、日饮水量和日增重(P<0.05);显著降低十二指肠、空肠和回肠肠重(P<0.05),其中空肠肠重降低幅度最大,达到28.14%;MTX导致小鼠空肠绒毛顶部上皮细胞脱落,绒毛高度显著降低(P<0.05),固有层裸露,隐窝增生明显,隐窝深度显著增加(P<0.05),绒毛高度/隐窝深度比值显著降低(P<0.05);空肠中ZO-1、Occludin、β-catenin和PCNA蛋白质表达显著降低(P<0.05),Caspase-3蛋白质表达显著增加(P<0.05);0.025、0.05、0.10μM和0.20μM MTX处理CMT-93细胞24 h即可显著抑制其增殖活性(P<0.05),且抑制效果呈剂量依赖性;0.025μM和0.05μM MTX显著降低CMT-93细胞跨膜电阻值(P<0.05)和ZO-1、Occludin、β-catenin、PCNA蛋白质表达(P<0.05),增加Caspase-3蛋白质表达。结果提示,MTX降低Wnt/β-catenin信号通路活性,抑制肠上皮细胞增殖,促进其凋亡,破坏肠道屏障功能,诱导肠道结构损伤。

This experiment was conducted to study the effect of methotrexate(MTX) on intestinal epithe-lial regeneration of KM mice. Sixty-four similar age and body weight healthy KM mice were selected and randomly assigned to two groups with thirty-two replicates and one mouse with each pen. The mice of two groups were injected with the normal saline and MTX solution with 50 mg/kg BW respec-tively, and were killed at 72 h after injection. Meanwhile, CMT-93 cells were treated with 0.025, 0.05, 0.10 μM and 0.20 μM MTX. The results showed that ① MTX decreased the average daily feed intake (ADFI), average daily water intake (ADWI) and average daily gain (ADG) significantly (P<0.05);② MTX reduced weight of the intestine (duodenum, jejunum and ileum) significantly (P<0.05), and the weight of jejunum reduced most significantly (28.14%);③ MTX decreased (P<0.05) villus height (VH), increased (P<0.05) crypt depth (CD), reduce (P<0.05) ratio of villus height/crypt depth (V/C);④ MTX significantly decreased the expression of ZO-1, Occludin,β-catenin and PCNA (P<0.05), and in-creased the expression of Caspase-3 in jejunum (P<0.05);⑤ CMT-93 cells proliferation activity significantly decreased (P<0.05) at 24 h after treated with 0.025, 0.05, 0.10 μM and 0.20 μM MTX;⑥ MTX significantly decreased the teer value (P<0.05) and the expression of ZO-1, Occludin,β-catenin and PCNA (P<0.05), and increased the expression of Caspase-3 protein in CMT-93 cells (P<0.05). These results indicated that MTX could decrease the activity of Wnt/β-catenin signaling pathway, inhibit the proliferation of intestinal epithelial cells, promote their apoptosis, destroy the intestinal barrier function, and induce intestinal structural injury.