收稿日期: 2021-07-06
修回日期: 2021-07-26
网络出版日期: 2021-11-10
基金资助
国家自然科学基金(81871677);南通市科技项目(JC2020021);南通大学医学院项目(TDYX2021006)
Expression and function of triggering receptor expressed on myeloid cells 1 in the liver of mice infected with Schistosoma japonicum
Received date: 2021-07-06
Revised date: 2021-07-26
Online published: 2021-11-10
Supported by
National Natural Science Foundation of China(81871677);Scientific Research Project of Nantong City(JC2020021);Scientific Research Project of Medical School of Nantong University(TDYX2021006)
目的 观察日本血吸虫感染小鼠肝组织髓样细胞触发受体-1（TREM-1）的动态表达,分析TREM-1与巨噬细胞M1极化的相关性。 方法 34只C57BL/6小鼠随机分为对照组（10只）和感染组（24只）,对照组小鼠不作任何处理,取肝组织;感染组每鼠经腹部皮肤接种日本血吸虫尾蚴（15 ± 2）条,感染后第3、6、9和12周,各取6只小鼠的肝组织。实时荧光定量PCR检测对照组、各时间点感染组小鼠肝组织中TREM-1和白细胞介素-1β（IL-1β）mRNA的相对转录水平;蛋白质免疫印迹（Western blotting）检测各鼠肝组织中TREM-1和诱导型一氧化氮合酶（iNOS）的蛋白相对表达水平;制备各鼠肝组织冰冻切片,免疫荧光染色技术检测肝组织巨噬细胞中TREM-1的表达情况。合成TREM-1 siRNA和阴性siRNA（NC siRNA）,分别转染RAW264.7巨噬细胞,培养6 h后分为TREM-1 siRNA、NC siRNA、TREM-1 siRNA + 日本血吸虫成虫抗原（SWA）和NC siRNA + SWA组,后两组加入终浓度为20 μg/ml的SWA作用48 h,收集细胞,Western blotting检测细胞中TREM-1和iNOS蛋白的相对表达水平。多组之间TREM-1、IL-1β mRNA相对转录水平,TREM-1和iNOS蛋白相对表达水平的比较使用单因素方差分析,两组之间的比较采用独立样本t检验。 结果 实时荧光定量PCR检测结果显示,感染后第3、6、9和12周,感染组小鼠肝组织中TREM-1 mRNA相对转录水平分别为14.28 ± 6.26、183.41 ± 37.37、68.17 ± 16.19和106.91 ± 45.70（F = 6.668,P < 0.01）,其中感染后第6周感染组与对照组（1.00）比较差异有统计学意义（t = -4.881,P < 0.01）;IL-1β mRNA的相对转录水平分别为8.16 ± 1.91、56.12 ± 10.68、24.41 ± 3.54和24.28 ± 2.98（F = 16.943,P < 0.01）,各时间点感染组与对照组（1.00）比较差异均有统计学意义（t = -3.740,P < 0.05;t = -5.159、-6.606和-7.799,P < 0.01）。Western blotting检测结果显示,感染后第3、6、9和12周,感染组小鼠肝组织中TREM-1蛋白相对表达水平分别为1.24 ± 0.38、1.50 ± 0.13、1.13 ± 0.28和1.17 ± 0.60（F = 1.547,P > 0.05）,其中感染后第6周感染组与对照组（1.00）比较差异有统计学意义（t = -6.011,P < 0.01）;iNOS蛋白相对表达水平分别为5.27 ± 3.66、23.27 ± 14.72、10.16 ± 4.97和2.69 ± 1.65（F = 9.384,P < 0.01）,各时间点感染组与对照组（1.00）比较差异均有统计学意义（t = -2.893、-3.716、-4.537和-2.571,P < 0.05）。免疫荧光染色结果显示,对照组小鼠肝组织中TREM-1（绿色）和F4/80（红色）的表达较少,感染组小鼠肝组织肉芽肿周围TREM-1和F4/80的表达增加,TREM-1和 F4/80共定位的细胞（黄色）增多。Western blotting检测结果显示,NC siRNA + SWA、TREM-1 siRNA、TREM-1 siRNA + SWA组TREM-1蛋白相对表达水平分别为1.35 ± 0.13、0.58 ± 0.09和1.09 ± 0.03（F = 46.689,P < 0.01）,其中NC siRNA + SWA、TREM-1 siRNA组与NC siRNA组（1.00）比较差异有统计学意义（t = -4.716、7.858,P < 0.05）,TREM-1 siRNA + SWA组与TREM-1 siRNA组比较差异有统计学意义（t = 9.000,P < 0.01）。NC siRNA + SWA、TREM-1 siRNA、TREM-1 siRNA + SWA 组iNOS蛋白相对表达水平分别为3.69 ± 1.04、0.77 ± 0.12和2.74 ± 0.86（F = 12.714,P < 0.01）,其中NC siRNA + SWA、TREM-1 siRNA组与NC siRNA组（1.00）比较差异有统计学意义（t = -4.451、 3.254,P < 0.05）,TREM-1 siRNA + SWA组与TREM-1 siRNA组差异无统计学意义（t = 3.913, P > 0.05）。 结论 日本血吸虫感染小鼠肝组织中TREM-1表达显著上调,抑制TREM-1的表达能够抑制SWA作用的巨噬细胞iNOS的表达。
关键词： 日本血吸虫; 髓样细胞触发受体-1; 巨噬细胞
黄爱龙 , 张蓓 , 沈函宇 , 陈果 , 李静 , 朱丹丹 , 段义农 . 日本血吸虫感染小鼠肝髓样细胞触发受体-1的表达及其功能[J]. 中国寄生虫学与寄生虫病杂志, 2021 , 39(5) : 621 -626 . DOI: 10.12140/j.issn.1000-7423.2021.05.010
Objective This study aimed to observe the dynamic expression of triggering receptor expressed on myeloid cells 1 (TREM-1) in the liver of mice infected with Schistosoma japonicum. The correlation between TREM-1 and M1 macrophage polarization was analyzed. Methods A total of 34 female C57BL/6 mice were randomly assigned to infection group (24 mice) and control group (10 mice). The mice in the control group were left untreated and were sacrificed for collection of liver tissues. The mice in the infection group were infected with (15 ± 2) S. japonicum cercaria per mouse; at 3, 6, 9 and 12 weeks after infection, six mice were randomly selected and sacrificed, and liver tissues were collected. The relative liver mRNA transcription levels of TREM-1 and IL-1β were examined with real-time quantitative PCR. Western blotting was used to observe the TREM-1 and inducible NO synthase (iNOS) protein expression in liver tissues. Frozen mouse liver sections were prepared. Immunofluorescence staining was used to observe TREM-1 expression in hepatic macrophages. The following four groups of RAW264.7 macrophage cells were examined: NC siRNA, TREM-1 siRNA, NC siRNA + SWA; and TREM-1 siRNA + SWA. The cells were transfected with synthetic TREM-1 siRNA or NC siRNA and cultured for 6 h, schistosome worm antigen (SWA, 20 μg/ml) was then added to the indicated groups for 48 h. The TREM-1 and iNOS protein expression levels were detected via Western blotting. One-way ANOVA was used to analyze the relative mRNA transcription of TREM-1 and IL-1β, and the relative protein expression of TREM-1 and iNOS. Independent samples t test was used for comparisons between two groups. Results Real-time quantitative PCR analysis indicated that the relative mRNA levels of TREM-1 in the liver in infection mice were 14.28 ± 6.26, 183.41 ± 37.37, 68.17 ± 16.19 and 106.91 ± 45.70 at 3, 6, 9 and 12 weeks after infection with respect to the control group level (1.00). A significant difference was observed among these groups (F = 6.668, P < 0.01) and between the control and infection groups 6 weeks after infection (t = -4.881, P < 0.01). The relative mRNA levels of IL-1β in the liver in infected mice were 8.16 ± 1.91, 56.12 ± 10.68, 24.41 ± 3.54 and 24.28 ± 2.98 at 3, 6, 9 and 12 weeks after infection with respect to the control (1.00). A significant difference was observed among these groups (F = 16.943, P < 0.01) and between the control and infection groups (t = -3.740, P < 0.05; t = -5.159, -6.606, -7.799, P < 0.01). Western blotting revealed that the relative expression levels of TREM-1 protein in the liver in infected mice were 1.24 ± 0.38, 1.50 ± 0.13, 1.13 ± 0.28 and 1.17 ± 0.60 at 3, 6, 9 and 12 weeks after infection. No significant difference was observed among these groups (F = 1.547, P > 0.05), and between the control and infection groups at 3, 9, 12 weeks after infection (t = -1.247, -0.745, -0.559, P > 0.05). However, a significant difference was found between the control and infection groups 6 weeks after infection (t = -6.011, P < 0.01). The relative expression of iNOS protein in liver tissues in infected mice were 5.27 ± 3.66, 23.27 ± 14.72, 10.16 ± 4.97 and 2.69 ± 1.65 at 3, 6, 9 and 12 weeks after infection. Significant differences were found among these groups (F = 9.384, P < 0.01) and between the control and infection groups (t = -2.893, -3.716, -4.537 and -2.571, P < 0.05). Immunofluorescence staining indicated that TREM-1 (green) and F4/80 (red) expression was low in the control group but was elevated around hepatic granulomas in infected mice 6 weeks after infection. The number of cells with co-localization (yellow) of TREM-1 and F4/80 increased around the mouse hepatic granulomas. Western blotting revealed that the relative expression levels of TREM-1 protein in the NC siRNA + SWA, TREM-1 siRNA and TREM-1 siRNA + SWA groups were 1.35 ± 0.13, 0.58 ± 0.09 and 1.09 ± 0.03 (F = 46.689, P < 0.01). A significant difference was found between the NC siRNA + SWA, TREM-1 siRNA and NC siRNA (1.00) groups (t = -4.716, 7.858, P < 0.05), and between the TREM-1 siRNA + SWA and TREM-1 siRNA groups (t = 9.000, P < 0.01). The relative expression levels of of iNOS in the NC siRNA + SWA, TREM-1 siRNA and TREM-1 siRNA + SWA groups were 3.69 ± 1.04, 0.77 ± 0.12 and 2.74 ± 0.86 (F = 12.714, P < 0.01). A significant difference was observed between the NC siRNA + SWA, TREM-1 siRNA and NC siRNA (1.00) groups (t = -4.451, 3.254, P < 0.05). No significant difference was observed between the TREM-1 siRNA + SWA and TREM-1 siRNA groups (t = 3.913, P > 0.05). Conclusion The expression of TREM-1 in the liver of mice infected with S. japonicum is significantly up-regulated. Inhibition of TREM-1 expression may suppress iNOS expression in SWA-treated macrophages.
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