The First Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China.
Department of Gastroenterology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, Zhejiang Province, China.
World J Gastroenterol. 2018 Apr 28;24(16):1766-1778. doi: 10.3748/wjg.v24.i16.1766.
AIM: To explore the significance of corticotropin-releasing hormone (CRH)-receptor (R)2 in mucosal healing of dextran sulfate sodium (DSS)-induced colitis and the effect of Tong-Xie-Yao-Fang (TXYF) on CRH-R2 expression and regulation. METHODS: Ulcerative colitis was induced in mice by administration of 3% (w/v) DSS for 7 d. Once the model was established, mice were administered urocortin-2 (30 μg/kg), a peptide which binds exclusively to CRH-R2, or various doses of aqueous TXYF extracts (2.8-11.2 g/kg), a CRH-R2 antagonist Astressin (Ast)2B (20 μg/kg), Ast2B + Ucn2, or Ast2B with various doses of aqueous TXYF extracts for 9 d. Colonic mucosal permeability was then evaluated by measuring the fluorescence intensity in serum. The colitis disease activity index (DAI), histology, body weight loss and colon length were assessed to evaluate the condition of colitis. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was used to detect apoptosis of the intestinal epithelial cells. The expression level of Ki-67 represented the proliferation of colonic epithelial cells and was detected by immunohistochemistry. The expression levels of inflammation cytokines IL-6, TNF-α and CXCL-1 were examined in colon tissues using real-time PCR and ELISA kits. RESULTS: Compared with the DSS group, mice treated with the CRH-R2 antagonist Ast2B showed greater loss of body weight, shorter colon lengths (4.90 ± 0.32 6.21 ± 0.34 cm, < 0.05), and higher DAI (3.61 ± 0.53 2.42 ± 0.32, < 0.05) and histological scores (11.50 ± 1.05 8.33 ± 1.03, < 0.05). Additionally, the Ast2B group showed increased intestinal permeability (2.76 ± 0.11 μg/mL 1.47 ± 0.11 μg/mL, < 0.001), improved secretion of inflammatory cytokines in colon tissue, and reduced colonic epithelial cell proliferation (4.97 ± 4.25 22.51 ± 8.22, < 0.05). Increased apoptosis (1422.39 ± 90.71 983.01 ± 98.17, < 0.001) was also demonstrated. The Ucn2 group demonstrated lower DAI (0.87 ± 0.55 2.42 ± 0.32, < 0.001) and histological scores (4.33 ± 1.50 8.33 ± 1.03, < 0.05). Diminished weight loss, longer colon length (9.58 ± 0.62 6.21 ± 0.34 cm, < 0.001), reduced intestinal permeability (0.75 ± 0.07 1.47 ± 0.11 μg/mL, < 0.001), inhibited secretion of inflammatory cytokines in colon tissue and increased colonic epithelial cell proliferation (90.04 ± 15.50 22.51 ± 8.22, < 0.01) were all observed. Reduced apoptosis (149.55 ± 21.68 983.01 ± 98.17, < 0.05) was also observed. However, significant statistical differences in the results of the Ast2B group and Ast2B + Ucn2 group were observed. TXYF was also found to ameliorate symptoms of DSS-induced colitis in mice and to promote mucosal repair like Ucn2. There were significant differences between the Ast2B + TXYF groups and the TXYF groups. CONCLUSION: CRH-R2 activates the intestinal mucosal antiinflammatory response by regulating migration, proliferation and apoptosis of intestinal epithelial cells in colitis-induced mice, and plays an important antiinflammatory role. TXYF promotes mucosal repair in colitis mice by regulating CRH-R2.
目的：探讨促肾上腺皮质激素释放激素（CRH）-受体（R）2 在葡聚糖硫酸钠（DSS）诱导的结肠炎黏膜愈合中的意义，以及痛泻要方（TXYF）对 CRH-R2 表达和调节的影响。
方法：通过给予 3%（w/v）DSS 诱导溃疡性结肠炎小鼠模型，然后给予 Urocortin-2（30 μg/kg），一种与 CRH-R2 特异性结合的肽，或不同剂量的水提 TXYF 提取物（2.8-11.2 g/kg），CRH-R2 拮抗剂 Astressin（Ast）2B（20 μg/kg），Ast2B+Ucn2，或 Ast2B 与不同剂量的水提 TXYF 提取物共处理 9 天。通过测量血清中的荧光强度来评估肠黏膜通透性。通过疾病活动指数（DAI）、组织学、体重减轻和结肠长度评估结肠炎的情况。末端脱氧核苷酸转移酶 dUTP 缺口末端标记法（TUNEL）检测肠上皮细胞凋亡。免疫组织化学法检测 Ki-67 代表结肠上皮细胞的增殖水平。使用实时 PCR 和 ELISA 试剂盒检测结肠组织中炎症细胞因子 IL-6、TNF-α 和 CXCL-1 的表达水平。
结果：与 DSS 组相比，CRH-R2 拮抗剂 Ast2B 处理的小鼠体重减轻更明显，结肠长度更短（4.90±0.32 vs. 6.21±0.34 cm， < 0.05），DAI（3.61±0.53 vs. 2.42±0.32， < 0.05）和组织学评分（11.50±1.05 vs. 8.33±1.03， < 0.05）更高。此外，Ast2B 组的肠通透性增加（2.76±0.11 μg/mL vs. 1.47±0.11 μg/mL， < 0.001），结肠组织中炎症细胞因子的分泌增加，结肠上皮细胞增殖减少（4.97±4.25 vs. 22.51±8.22， < 0.05）。同时还观察到细胞凋亡增加（1422.39±90.71 vs. 983.01±98.17， < 0.001）。Ucn2 组的 DAI（0.87±0.55 vs. 2.42±0.32， < 0.001）和组织学评分（4.33±1.50 vs. 8.33±1.03， < 0.05）较低。体重减轻减轻、结肠长度更长（9.58±0.62 vs. 6.21±0.34 cm， < 0.001），肠通透性降低（0.75±0.07 vs. 1.47±0.11 μg/mL， < 0.001），结肠组织中炎症细胞因子的分泌减少，结肠上皮细胞增殖增加（90.04±15.50 vs. 22.51±8.22， < 0.01）。同时还观察到细胞凋亡减少（149.55±21.68 vs. 983.01±98.17， < 0.05）。然而，Ast2B 组和 Ast2B+Ucn2 组的结果存在显著的统计学差异。TXYF 也被发现可以改善 DSS 诱导的结肠炎小鼠的症状，并促进黏膜修复，与 Ucn2 类似。Ast2B+TXYF 组与 TXYF 组之间存在显著差异。
结论：CRH-R2 通过调节肠上皮细胞的迁移、增殖和凋亡，激活结肠炎小鼠的肠道黏膜抗炎反应，发挥重要的抗炎作用。TXYF 通过调节 CRH-R2 促进结肠炎小鼠的黏膜修复。
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