中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (2): 127-136.doi: 10.12140/j.issn.1000-7423.2019.02.003

• 论著 • 上一篇    下一篇

日本血吸虫成虫可溶性蛋白和重组半胱氨酸蛋白酶抑制蛋白抑制小鼠结肠炎的研究

武艺1(), 李路1, 徐永伟1, 邢瑞欣1, 胡静1, 王书书2, 沈继龙2, 徐元宏3, 陈熙1,*()   

  1. 1 安徽医科大学第一附属医院消化内科,合肥 230022
    2 安徽病原生物学省级实验室和人兽共患病安徽省重点实验室,安徽医科大学病原生物学教研室,合肥 230032
    3 安徽医科大学第一附属医院检验科,合肥 230022
  • 收稿日期:2018-12-27 出版日期:2019-04-30 发布日期:2019-05-13
  • 通讯作者: 陈熙
  • 作者简介:

    作者简介:武艺(1993-),女,硕士研究生,主要从事寄生虫治疗炎症性肠病研究。E-mail: wy962047607@qq.com

  • 基金资助:
    国家自然科学基金(No. 81171606);安徽省高校自然科学研究重点项目(No. KJ2017A202)

Schistosoma japonicum soluble worm proteins and recombinant cystatin ameliorate experimental colitis in a murine model

Yi WU1(), Lu LI1, Yong-wei XU1, Rui-xin XING1, Jing HU1, Shu-shu WANG2, Ji-long SHEN2, Yuan-hong XU3, Xi CHEN1,*()   

  1. 1 Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
    2 Provincial Laboratories of Pathogen Biology and Zoonoses Anhui, Department of Pathogen Biology, Anhui Medical University, Hefei 230032, China
    3 Department of Clinical Laboratory, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
  • Received:2018-12-27 Online:2019-04-30 Published:2019-05-13
  • Contact: Xi CHEN
  • Supported by:
    Supported by the National Nature Science Foundation of China(No. 81171606);and the Key Project of Natural Science Researches in Universities of Anhui Province(No. KJ2017A202)

摘要:

目的 探讨日本血吸虫可溶性成虫蛋白(SjSWP)和重组半胱氨酸蛋白酶抑制蛋白(rSjcystatin)对模拟炎症性肠病的小鼠结肠炎的作用。 方法 24只BALB/c雌鼠分为健康对照组、磷酸盐缓冲液组(PBS组)、rSjcystatin治疗组(rSjcystatin组)、SjSWP治疗组(SjSWP组)。PBS、rSjcystatin、SjSWP组用0.1 ml 5% 2,4,6-三硝基苯磺酸(TNBS)灌肠诱导结肠炎,灌肠后6、24 h分别腹腔注射100 μl PBS、50 μg rSjcystatin、50 μg SjSWP;健康对照组在相应时间点用等量的PBS灌肠及腹腔注射。TNBS灌肠72 h后无痛处死各组小鼠,检测宏观评分、结肠长度、髓过氧化物酶活性和显微镜下微观评分来评估炎症参数和病理改变。流式细胞仪检测小鼠脾脏中Th1和Th2及T调节细胞(Tregs)的比例。ELISA检测小鼠结肠组织匀浆上清中γ干扰素(IFN-γ)、白细胞介素-4(IL-4)、IL-13、IL-10等炎性细胞因子的水平。多组计量资料分析采用单因素方差分析,多组均数间的两两比较用Student-Newman-Keuls检验。 结果 TNBS诱导的结肠炎小鼠表现出明显的炎症和病理学特征,表现为体质量下降,便血、腹泻、结肠缩短、溃疡,以及炎性细胞因子IFN-γ、炎性细胞浸润和髓过氧化物酶活性的增加。与PBS组相比,rSjcystatin和SjSWP的干预治疗可以显著改善TNBS诱导的结肠炎症,表现为宏观评分(改善结肠糜烂、黏连、溃疡和缩短)和微观评分(减少组织损伤和炎症)的降低。健康对照组、PBS组、rSjcystatin组和SjSWP组小鼠结肠的宏观评分分别为0、9.8 ± 0.8、4.3 ± 1.0和4.5 ± 1.4,微观评分分别为0、6.0 ± 0.4、2.2 ± 0.5和2.8 ± 0.6,rSjcystatin组、SjSWP组与PBS组相比差异均有统计学意义(F = 57.8、34.1,P < 0.01),rSjcystatin组与SjSWP组间差异均无统计学意义(P > 0.05)。健康对照组、PBS组、rSjcystatin组和SjSWP组髓过氧化物酶分别为0.3 ± 0.0、1.4 ± 0.1、0.6 ± 0.1和0.6 ± 0.1(F = 32.5,P < 0.01);脾脏Tregs比例分别为(4.4 ± 0.7)%、(8.2 ± 3.1)%、(18.4 ± 2.1)%和(13.4 ± 1.9)% (F = 8.3,P < 0.01);结肠组织匀浆上清中IFN-γ、IL-10水平分别为(952.0 ± 61.1)、(1 684.6 ± 158.3)、(1 092.0 ± 81.9)、(1 001.6 ± 111.2)pg/ml,(322.8 ± 27.4)、(399.3 ± 77.9)、(860.6 ± 153.6)、(892.0 ± 113.8)pg/ml(F = 9.6、8.3,P < 0.05);与PBS组相比,rSjcystatin与SjSWP组的INF-γ、髓过氧化物酶活性明显减低,Tregs应答增强,IL-10分泌增多,差异均有统计学意义(P < 0.01),rSjcystatin组与SjSWP组间差异均无统计学意义(P > 0.05)。 结论 SjSWP及rSjcystatin均可通过上调小鼠Treg和Th2介导的免疫应答,减轻小鼠实验性结肠炎,抑制导致结肠炎发病的Th1免疫应答。

关键词: 日本血吸虫, 可溶性成虫蛋白, 半胱氨酸蛋白酶抑制蛋白, 结肠炎, 免疫调节

Abstract:

Objective To explore the potential therapeutic effect of Schistosoma japonicum soluble worm protein (SjSWP) and recombinant cystatin (rSjcystatin) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in a mouse model that mimics human inflammatory bowel diseases(IBDs). Methods Twenty-four female BALB/c mice were divided into four groups: non-treated control group(control), phosphate buffer (PBS) treated group, rSjcystatin treated group (rSjcystatin) and SjSWP treated group(SjSWP). The PBS, rSjcystatin, SjSWP treated groups were respectively intraperitoneally injected with 100 μl PBS, 50 μg rSjcystatin and 50 μg SjSWP at 6 h and 24 h after being induced with 0.1 ml of 5% TNBS by enema for colitis, while the control group was injected with the same amount of PBS at corresponding time without colitis induced. After 72 hours of TNBS enema, mice in each group were sacrificed under anesthesia. The pathological and inflammatory parameters were determined by measuring macroscopic score, myeloperoxidase (MPO) activity, colon length and microscopic score. The Th1 to Th2 cells and T regulatory cells (Tregs) in the spleen of mice were measured by flow cytometry, and the immune responses induced by rSjcystatin and SjSWP were investigated. The levels of cytokines including interferon-gamma (IFN-γ), interleukin-4 (IL-4), IL-13, IL-10 in the colon tissue of mice were detected by enzyme-linked immunosorbent assay (ELISA). Multiple groups of measurement data were analyzed by one-way analysis of variance, and pairwise comparisons between multiple groups of mean were tested by Student-Newman-Keuls. Results The mice with TNBS-induced colitis showed significant inflammation and pathology characterized by the weight loss, rectal bleeding, diarrhea, colon shrinking and ulceration, as well as induced inflammatory cytokine INF-γ, inflammatory cell infiltration and increased MPO activity. In this study we identified that treatment with rSjcystatin and SjSWP significantly reduced the TNBS-induced colon inflammation characterized by reduced disease manifestations in macroscopic score (improved colon erosion, adhesion, ulceration and colon shrinking) and microscopic scores (less tissue damage and inflammation), compared to the group of mice received PBS only. The macroscopic scores of colon in the control group, PBS group, rSjcystatin group and SjSWP group were 0, 9.8 ± 0.8, 4.3 ± 1.0 and 4.5 ± 1.4; the mean microscopic scores of mice were 0, 6.00 ± 0.4, 2.2 ± 0.5 and 2.8 ± 0.6, respectively. Among the above results, rSjcystatin and SjSWP had significant differences compared with PBS group (F = 57.8, 34.1, P < 0.01). There was no significant difference between rSjcystatin group and SjSWP group(P > 0.05). MPO values in control group, PBS group, rSjcystatin group and SjSWP group were (0.3 ± 0.0), (1.4 ± 0.1), (0.6 ± 0.1) and (0.6 ± 0.1)(F = 32.5, P < 0.01); the Tregs in spleen were (4.4 ± 0.7), (8.2 ± 3.1), (18.4 ± 2.1) and (13.4 ± 1.9)% (F = 8.3, P < 0.01); the IFN-γ and IL-10 in colon tissue homogenate supernatants of colon tissue were (952.0 ± 61.1), (1 684.6 ± 158.3), (1 092.0 ± 81.9) and (1 001.6 ± 111.2) pg/ml; (322.8 ± 27.4), (399.3 ± 77.9), (860.6 ± 153.6) and (892.0 ± 113.8) pg/ml respectively (F = 9.6, 8.3, P < 0.05). The results showed that, in rSjcystatin group and SjSWP group, the inflammatory cytokine INF-γ, MPO activity in colon tissue decreased, combined with boosted Tregs response and the secretion of regulatory cytokine IL-10, compared to the group of mice received PBS only with statistical difference (P < 0.01). There was no significant difference for the therapeutic efficacy on inflammatory colitis between rSjcystatin group and SjSWP group(P > 0.05). Conclusions SjSWP and rSjcystatin could alleviate experimental colitis in mice via stimulating Tregs and Th2 mediated responses and, correspondingly, suppressing Th1 pro-inflammatory response related to the pathogenesis of colitis.

Key words: Schistosoma japonicum, Soluble worm protein, Recombinant cystatin, Colitis, Immunomodulation

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