中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (2): 110-114.

• 论著 • 上一篇    下一篇

两种蠕虫半胱氨酸蛋白酶抑制剂对小鼠腹腔渗出细胞一氧化氮产生及细胞因子分泌的影响

杨小迪1, 李徽徽1,2, 陶志勇1, 方强1, 程洋3, 徐岚松1, 薛仁敏1, 陈勇1, 夏惠1, 张慧1, 姜辉1, 刘涛1, 彭琨1, 陈兴智1,*()   

  1. 1蚌埠医学院病原生物学教研室,安徽省感染与免疫重点实验室,蚌埠 233030
    2蚌埠医学院组织学与胚胎学教研室,蚌埠 233030
    3江南大学无锡医学院病原感染与免疫组,无锡 214122
  • 收稿日期:2016-12-30 出版日期:2017-04-20 发布日期:2017-05-02
  • 通讯作者: 陈兴智
  • 基金资助:
    安徽省教育厅重点资助项目(No. KJ2017A235);安徽省自然科学基金(No. 1508085QH158);安徽高校科研创新平台团队项目(No. 2016-40);安徽省高校优秀青年人才支持计划重点项目(No. gxyqZD2016159);国家大学生创新创业训练计划项目(No. 201610367009)

Effects of cystatins derived from two species of helminths on the release of nitric oxide and secretion of cytokines from murine peritoneal exudate cells

Xiao-di YANG1, Hui-hui LI1,2, Zhi-yong TAO1, Qiang FANG1, Yang CHENG3, Lan-song XU1, Ren-min XUE1, Yong CHEN1, Hui XIA1, Hui ZHANG1, Hui JIANG1, Tao LIU1, Kun PENG1, Xing-zhi CHEN1,*()   

  1. 1 Department of Microbiology and Parasitology, Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu 233030, China
    2 Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, China
    3 Department of Patheogenic Infection and Immunity, Wuxi Medical School,Jiangnan University, Wuxi 214122, China
  • Received:2016-12-30 Online:2017-04-20 Published:2017-05-02
  • Contact: Xing-zhi CHEN
  • Supported by:
    Supported by the Education Commission of Anhui Province(No. KJ2017A235);The Natural Science Fund of Anhui Province(No. 1508085QH158);The Scientific Research Innovation Team Project of Anhui Colleges and Universities(No. 2016-40);A Key Supporting Project of Prominent Youth in University of Anhui(No. gxyqZD2016159);and a National University Students′ Innovation and Entrepreneurship Training Program(No. 201610367009)

摘要:

目的 观察两种蠕虫半胱氨酸蛋白酶抑制剂(cystatin)体外对小鼠腹腔渗出细胞(PEC)一氧化氮(NO)的产生及细胞因子分泌的影响。方法 无痛处死BALB/c小鼠10只,收集腹腔内液体,制备渗出细胞(PEC),主要为巨噬细胞。将源自旋毛虫(Trichinella spiralis)及广州管圆线虫(Angiostrongylus cantonensis)的cystatin(Tscystatin, Accystatin)与脂多糖(LPS)刺激的小鼠PEC共孵育,实验分为RPMI组、LPS组、Accystatin + LPS组和Tscystatin + LPS组,除RPMI组外,其余3组均提前2 h用LPS(2 μg/ml)刺激贴壁细胞制备炎症模型,Accystatin + LPS组和Tscystatin + LPS组分别用2 μg/ml Accystatin或Tscystatin与LPS刺激后的细胞共孵育,每组6孔,培养24 h后,收集上清,用ELISA和硝酸还原酶法检测上清中α肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、IL-10等细胞因子和NO的水平。采用SPSS11.5统计学软件处理实验数据。结果 ELISA检测结果显示:Accystatin + LPS组细胞上清中促炎因子TNF-α和IL-6分别为(507.50 ± 66.32)和(1 440.49 ± 77.25)pg/ml,较LPS组的(454.15 ± 53.11)和(1 016.2 ± 115.10)pg/ml明显升高(P < 0.05)。Tscystatin + LPS组分别为(296.35 ± 55.30)和(793.54 ± 86.61)pg/ml,较LPS组和Accystatin + LPS组明显降低(P < 0.05)。RPMI组和LPS组的IL-10分别为(38.80 ± 6.71)和(53.66 ± 7.72)pg/ml,差异无统计学意义(P > 0.05),Accystatin + LPS组和Tscystatin + LPS组的IL-10分别为(149.74 ± 26.01)和(158.76 ± 19.67)pg/ml,较LPS组均明显升高(P < 0.05)。Accystatin + LPS组和Tscystatin + LPS组NO水平分别为(12.54 ± 2.12)和(7.95 ± 1.40)μmol/L,较LPS组的(20.18 ± 3.99)μmol/L均明显降低(P < 0.05),且Tscystatin + LPS组NO水平低于Accystatin + LPS组(P < 0.05)。结论 旋毛虫和广州管圆线虫的cystatin均可抑制小鼠腹腔渗出细胞NO释放、上调IL-10的水平,Accystatin明显促进TNF-α及IL-6的分泌,Tscystatin则明显抑制两种细胞因子的水平。

关键词: 旋毛虫, 广州管圆线虫, 半胱氨酸蛋白酶抑制剂

Abstract:

Objective To investigate the effects of cysteine protease inhibitors(cystatins) from Trichinella spiralis (Tscystatin) and Angiostrongylus cantonensis (Accystatin) on the release of nitric oxide (NO) and secretion of cytokines from mouse peritoneal exudate cells (PECs). Methods Ten BALB/c mice were sacrificed under anesthesia to collect intraperitoneal fluid, from which PECs were prepared, consisting mostly of macrophages. The lipopolysaccharide(LPS)-stimulated PECs were incubated with Tscystatin or Accystatin. PECs were assigned into four groups, RPMI group, LPS group, Accystatin + LPS group and Tscystatin + LPS group. The cells in the latter three groups were stimulated with lipopolysaccharides (LPS 2 μg/ml) 2 hours prior to 2 μg/ml Accystatin or Tscystatin treatment for 24 hours. Each group had six replicates. The levels of tumor necrosis factor-α(TNF-α), interleukin IL-6(IL-6) and IL-10 in the culture supernatant were measured by ELISA. The concentration of nitric oxide(NO)was measured by nitrate reductase method. Statistic analysis was performed using SPSS11.5 software. Results The level of pro-inflammatory cytokines TNF-α and IL-6 was(507.50 ± 66.32) and (1 440.49 ± 77.25) pg/ml respectively in the Accystatin + LPS group, significantly higher than (454.15 ± 53.11) and (1 016.27 ± 115.10) pg/ml in LPS group (P < 0.05), but compared with LPS group and Accystatin + LPS group, the level of TNF-α [(296.35 ± 55.30) pg/ml] and IL-6 [(793.54 ± 86.61) pg/ml] in the Tscystatin + LPS group was significantly lower than those in the LPS group and Accystatin + LPS group (P < 0.05). The level of IL-10 was (38.80 ± 6.71) pg/ml in the RPMI group and (53.66 ± 7.72) pg/ml in the LPS group, with no significant difference(P > 0.05). The level of IL-10 in the Accystatin + LPS group [(149.74 ± 26.01) pg/ml] and the Tscystatin + LPS group [(158.76 ± 19.67) pg/ml] was significantly higher than that in the LPS group(P < 0.05). The level of NO in the Accystatin + LPS group and the Tscystatin + LPS group was(12.54 ± 2.12) μmol/L and (7.95 ± 1.40) μmol/L, respectively, significantly decreased compared to the LPS group (P < 0.05). In addition, the concentration NO in the of Tscystatin + LPS group was significantly lower than the Accystatin + LPS group (P < 0.05). Conclusion Cystatins derived from the two species of helminths can both inhibit the release of NO and up-regulate the level of IL-10. Accystatin remarkably promotes the secretion of TNF-α and IL-6, while Tscystatin has the opposite effects.

Key words: Trichinella spiralis, Angiostrongylus cantonensis, Cysteine protease inhibitor

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