南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (5): 664-670.doi: 10.12122/j.issn.1673-4254.2021.05.05

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华支睾吸虫来源的分子伴侣rCsHscB对小鼠慢性溃疡性结肠炎有治疗作用

华 慧,董 昕,张雨钊,方 凡,张蓓蓓,李向阳,于 倩,郑葵阳,颜 超   

  • 出版日期:2021-05-20 发布日期:2021-06-11

rCsHscB derived from Clonorchis sinensis has therapeutic effect on dextran sodium sulfate-induced chronic ulcerative colitis in mice

  • Online:2021-05-20 Published:2021-06-11

摘要: 目的 研究华支睾吸虫来源的分子伴侣rCsHscB对葡聚糖硫酸钠(DSS)诱导的小鼠慢性溃疡性结肠炎的作用。方法 诱导大肠杆菌表达体外获得高纯度重组rCsHscB蛋白。将C57BL/6雄性小鼠随机分为NC组(n=10)、rCsHscB组(n=10)、DSS组 (n=15)和DSS+rCsHscB组(n=15),采用2%的DSS常规建立小鼠慢性溃疡性结肠炎模型。rCsHscB组和DSS+rCsHscB组在给予DSS同一周的第4、7天腹腔注射125 μg/mL rCsHscB,NC组和DSS组注射无内毒素PBS。第84天处死小鼠后对结肠组织进行HE和Masson染色,并采用流式细胞术检测外周血和小肠固有层淋巴细胞中CD4+T细胞、CD8+T细胞的表达,ELISA检测结肠匀浆上清中IL-6、IL-10和单核细胞趋化蛋白1(MCP-1)含量,Western blot 检测结肠组织中MAPK信号通路相关蛋白ERK1/2、JNK和P38的磷酸化和非磷酸化水平。结果 与NC组相比,rCsHscB组小鼠未出现不良反应。DSS组小鼠结肠组织病理损伤显著,胶原纤维沉积加重,外周血及小肠固有层淋巴细胞中CD4+T细胞比例升高、CD4+ T/CD8+T细胞比值明显上升。结肠匀浆上清中IL-6和MCP-1水平显著升高,IL-10分泌没有明显变化(P=0.11),ERK1/2、JNK和P38的磷酸化水平显著升高;与DSS组比较,DSS+rCsHscB组小鼠结肠组织病理损伤缓解、胶原纤维沉积减少,外周血及小肠固有层淋巴细胞中CD4+T/CD8+T细胞比值明显降低,结肠匀浆上清中IL-6及MCP-1水平显著下降,IL-10水平显著上升(P=0.003),ERK1/2、JNK和P38磷酸化水平显著降低。结论 rCsHscB对小鼠慢性溃疡性结肠炎具有一定的预防改善作用,其可能是通过MAPK抑制促炎因子产生并调控 CD4+T/CD8+T细胞平衡来发挥作用的。

关键词: 华支睾吸虫;rCsHscB;溃疡性结肠炎;炎症反应

Abstract: Objective To investigate the therapeutic effect of rCsHscB derived from Clonorchis sinensis on dextran sodium sulfate (DSS)-induced chronic ulcerative colitis in mice. Methods C57BL/6 mice were randomized into negative control (NC) group (n=10), rCsHscB group (n=10), DSS group (n=15), and DSS+rCsHscB group (n=15), and in the latter two groups, chronic ulcerative colitis was induced in the mice using 2% DSS. In rCsHscB and DSS + rCsHscB groups, the mice received intraperitoneal injections of 125 μg/mL rCsHscB on the 4th and 7th day following DSS administration, and PBS was injected in the other two groups. The mice were euthanized on the 84th day, and pathological changes of the colon were evaluated by HE and Masson staining. The levels of CD4+ and CD8+ T cells in the peripheral blood and lamina propria gastric lymphocytes (LPL) were analyzed by flow cytometer; the levels of IL-6, MCP-1 and IL-10 in colon homogenate were determined using ELISA, and the phosphorylation of ERK1/2, JNK and P38 was detected with Western blotting. Results Compared with those in NC group, the mice in rCsHscB group exhibited no adverse responses to the treatment. The mice in DSS group had severe pathologies in the colon with significantly increased ratios of CD4+ and CD4+/CD8+ T cells in peripheral blood and LPL, increased levels of IL-6 and MCP-1 but no obvious changes in IL-10 in colon homogenate, and significantly augmented phosphorylation levels of ERK1/2, JNK and P38. Compared with those in DSS group, the mice in DSS + rCsHscB group showed ameliorated colon pathologies with decreased CD4+T/CD8+T cell ratio in the peripheral blood and LPL, significantly decreased IL-6 and MCP-1 levels and increased IL-10 in colon homogenate, and lowered phosphorylation levels of ERK1/2, JNK and P38. Conclusion rCsHscB can produce therapeutic effect on DSS-induced chronic ulcerative colitis in mice possibly by inhibiting the production of pro-inflammatory factors and regulating the balance of CD4+/CD8+T cells through the MAPK pathway

Key words: Clonorchis sinensis; rCsHscB; ulcerative colitis; inflammatory responses