旋毛虫成虫排泄-分泌蛋白对脓毒症急性肝损伤的保护作用及机制研究

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (2): 99-104.

旋毛虫成虫排泄-分泌蛋白对脓毒症急性肝损伤的保护作用及机制研究

李徽徽^1,², 贺文欣^1,², 蔡兆根³, 仇大鹏⁴, 褚亮⁴, 陈兴智¹, 方强^1,², 夏惠¹, 李楠³, 崔宁宁³, 徐岚松¹, 杨小迪^1,^2,^*()

蚌埠医学院 1 基础医学院
2 安徽省感染与免疫重点实验室
3 第一附属医院
4第二附属医院,蚌埠 233000

收稿日期:2016-12-16 出版日期:2017-04-20 发布日期:2017-05-02
通讯作者: 杨小迪
基金资助:
国家自然科学基金（No. 81441120）;高校科研创新平台团队项目（No. 2016-40）;安徽省自然科学基金（No. 1508085QH158）;安徽省教育厅资助项目（No. KJ2015B013, KJ2017A235, KJ2017A236）;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）

Protective effects of adult-worm excretory-secretory protein of Trichinella spiralis against sepsis-induced acute liver injury and the mechanisms

Hui-hui LI^1,², Wen-xin HE^1,², Zhao-gen CAI³, Da-peng QIU⁴, Liang CHU⁴, Xing-zhi CHEN¹, Qiang FANG^1,², Hui XIA¹, Nan LI³, Ning-ning CUI³, Lan-song XU¹, Xiao-di YANG^1,^2,^*()

1 Basic Medical College
2 Anhui Key Laboratory of Infection and Immunity
3 The First Affiliated Hospital
4 The Second Affiliated Hospital, Bengbu Medical College, Bengbu 233000, China

Received:2016-12-16 Online:2017-04-20 Published:2017-05-02
Contact: Xiao-di YANG
Supported by:
Supported by the National Natural Science Foundation of China (No. 81441120), Scientific Research Innovation Team Platform of University (No. 2016-40), the Natural Science Fund of Anhui (No. 1508085QH158), Educational Commission of Anhui Province （No. KJ2015B013, KJ2017A235, KJ2017A236）, the Key Supporting Project of Prominent Youth in Anhui University (No. gxyqZD2016159) and National University Students' Innovation and Entrepreneurship Training Program (No. 201610367009)

摘要/Abstract

摘要：

目的观察旋毛虫（Trichinella spiralis）成虫排泄-分泌蛋白（adult-worm excretory-secretory protein,AES）对小鼠脓毒症急性肝损伤的保护作用。方法 24只雄性BALB/c小鼠采用抽签法随机分为假手术组（Sham组）、盲肠结扎穿孔组（CLP组）、AES治疗组（AES + CLP组）和AES对照组（AES + Sham组）,每组6只。CLP组和AES + CLP组采用盲肠结扎穿孔术制备脓毒症急性肝损伤动物模型,Sham组和AES + Sham组不结扎、不穿孔盲肠,其余操作与CLP组相同。术后30 min,Sham组和CLP组腹腔注射PBS,AES + CLP组和AES + Sham组腹腔注射AES（25 μg/只）。术后12 h,检测小鼠血清中丙氨酸转氨酶（ALT）和天门冬氨酸转氨酶（AST）水平,观察小鼠肝脏组织病理改变,蛋白质印迹（Western blotting）测定肝脏组织中Toll样受体4（TLR4）、髓样分化因子88（MyD88）的表达水平,免疫组化法测定肝细胞核因子-κB（NF-κB）p65的表达水平并分析NF-κB p65核阳性率,ELISA检测小鼠肝脏组织中α肿瘤坏死因子（TNF-α）、白细胞介素-6（IL-6）和IL-1β的水平。结果术后12 h,CLP组小鼠血清中ALT和AST水平分别为（269.83 ± 77.60）和（712.00 ± 186.52）IU/L,明显高于Sham组的（45.00 ± 15.01）和（132.50 ± 36.95）IU/L,及AES + CLP组的（136.67 ± 30.27）和（419.00 ± 60.86）IU/L（P < 0.05）;AES + Sham组分别为（50.83 ± 13.24）和（134.67 ± 35.78）IU/L,与Sham组比较差异无统计学意义（P > 0.05）。HE染色显示：CLP组肝索紊乱,肝细胞水肿,有炎性细胞浸润;AES + CLP组肝脏结构损伤明显减轻。Western blotting检测显示：CLP组小鼠术后肝脏组织中TLR4/β-actin和MyD88/β-actin相对表达量分别为0.66 ± 0.12和0.69 ± 0.13,明显高于Sham组的0.31 ± 0.06和0.22 ± 0.04（P < 0.05）;AES + CLP组TLR4/β-actin相对表达量为0.56 ± 0.09,与CLP组的比较差异无统计学意义（P > 0.05）,而MyD88/β-actin相对表达量为0.47 ± 0.06,较CLP组降低（P < 0.05）。免疫组化分析结果显示：CLP组肝细胞中NF-κB p65核阳性率为（18.93 ± 2.91）%,明显高于Sham组的（0.50 ± 0.21）%及AES + CLP组的（9.60 ± 1.59）%（P < 0.05）。 ELISA检测结果显示：CLP组小鼠肝脏组织中TNF-α、IL-6和IL-1β水平分别是（26.21 ± 2.96）、（80.80 ± 9.94）和（28.99 ± 3.70）pg/mg,明显高于Sham组的（15.42 ± 1.21）、（47.70 ± 4.77）和（16.86 ± 1.68）pg/mg,及AES + CLP组的（18.93 ± 2.04）、（61.82 ± 7.24）和（23.18 ± 2.21）pg/mg（P < 0.05）。结论 AES可通过降低肝脏组织中MyD88表达和NF-κB p65核阳性率,从而降低肝脏组织中TNF-α、IL-6和IL-1β水平,减轻脓毒症对肝脏的损伤。

关键词: 旋毛虫, 脓毒症, 急性肝损伤, 排泄-分泌蛋白, Toll样受体4, 髓样分化因子88, 核因子-κB;

Abstract:

Objective To investigate the protective effect of adult-worm excretory-secretory protein (AES) of Trichinella spiralis on sepsis-induced acute liver injury. Methods Twenty-four male BALB/c mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture group (CLP group), AES treatment group (AES + CLP group), and AES control group (AES + Sham group) (n = 6 in each group). The mice in the CLP group and AES + CLP group received cecal ligation and puncture procedure to induce sepsis-associated acute liver injury. At 30 min after surgery, intraperitoneal injection of PBS or AES (25 μg) was performed. At 12 h after surgery, the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were determined and mice were sacrificed to prepare paraffin sections of the liver for HE staining. Protein levels of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in liver were measured by Western blotting. Expression of nuclear factor-κB (NF-κB) p65 in hepatocytes was detected by immunohistochemistry, and the nuclear positive rate was analyzed. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in liver were determined with ELISA. Results At 12 h after surgery, the serum levels of ALT and AST in the CLP group [(269.83 ± 77.60) and (712.00 ± 186.52) IU/L, respectively] were both significantly higher than those in the Sham group [(45.00 ± 15.01), (132.50 ± 36.95) IU/L] and the AES + CLP group [(136.67 ± 30.27), (419.00 ± 60.86) IU/L] (P < 0.05), and the serum levels of ALT and AST in the AES + Sham group [(50.83 ± 13.24), (134.67 ± 35.78) IU/L] had no significantly difference with those in the Sham group. HE staining revealed disordered hepatic cords, hepatocyte swelling, and infiltrations of inflammatory cells in the CLP group, but significantly ameliorated injury in the AES + CLP group. Results of Western blotting showed that the relative protein levels of TLR4 and MyD88 in the CLP group (0.66 ± 0.12, 0.69 ± 0.13, respectively) were significantly higher than those in the Sham group (0.31 ± 0.06, 0.22 ± 0.04) (P < 0.05). There was a significant difference between the AES + CLP and CLP groups in MyD88 level (0.47 ± 0.06 vs. 0.69 ± 0.13), but not for TLR4 (0.56 ± 0.09 vs. 0.66 ± 0.12). Immunohistochemistry revealed that the nuclear positive rate of NF-κB p65 was significantly higher in the CLP group [(18.93 ± 2.91)%] than in the Sham group [(0.5 ± 0.21)%] and the AES + CLP group [(9.6 ± 1.59)%] (P < 0.05). ELISA revealed that the levels of TNF-α, IL-6 and IL-1β in the CLP group [(26.21 ± 2.96), (80.80 ± 9.94) and (28.99 ± 3.70) pg/mg, respectively] were all significantly higher than those in the Sham group [(15.42 ± 1.21), (47.70 ± 4.77) and (16.86 ± 1.68) pg/mg] and the AES + CLP group [(18.93 ± 2.04), (61.82 ± 7.24) and (23.18 ± 2.21) pg/mg] (P < 0.05). Conclusion AES reduces the levels of TNF-α, IL-6 and IL-1β via decreasing MyD88 expression and the nuclear positive rate of NF-κB in liver, thereby relieving sepsis-induced acute liver injury in mice.

Key words: Trichinella spiralis, Sepsis, Acute liver injury, Excretory-secretory protein, Toll-like receptor 4, Myeloid differentiation factor 88, Nuclear factor-κB;

中图分类号:

R383.15

李徽徽, 贺文欣, 蔡兆根, 仇大鹏, 褚亮, 陈兴智, 方强, 夏惠, 李楠, 崔宁宁, 徐岚松, 杨小迪. 旋毛虫成虫排泄-分泌蛋白对脓毒症急性肝损伤的保护作用及机制研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 99-104.

Hui-hui LI, Wen-xin HE, Zhao-gen CAI, Da-peng QIU, Liang CHU, Xing-zhi CHEN, Qiang FANG, Hui XIA, Nan LI, Ning-ning CUI, Lan-song XU, Xiao-di YANG. Protective effects of adult-worm excretory-secretory protein of Trichinella spiralis against sepsis-induced acute liver injury and the mechanisms[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 99-104.

图/表 3

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