环鸟苷酸腺苷酸促日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化

doi:10.12140/j.issn.1000-7423.2022.04.004

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (4): 441-445.doi: 10.12140/j.issn.1000-7423.2022.04.004

环鸟苷酸腺苷酸促日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化

梁乐¹^,²(), 张璟¹, 沈玉娟¹, 胡媛¹, 曹建平¹^,^*()

1.中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）,国家卫生健康委寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际联合研究中心,上海 200025
2.上海健康医学院,上海 201328

收稿日期:2022-05-18 修回日期:2022-06-11 出版日期:2022-08-30 发布日期:2022-09-07
通讯作者: 曹建平
作者简介:梁乐（1981-）,女,博士研究生,从事寄生虫感染与免疫研究。E-mail： lianglecdc@163.com
基金资助:
国家自然科学基金面上项目(81971969);国家自然科学基金面上项目(81772225);上海市公共卫生体系建设三年行动计划（2020—2022）重点学科项目(GWV-10.1-XK13)

Cyclic guanosine monophosphate-adenosine monophosphate promotes liver egg granuloma formation and fibrosis in mice infected with Schistosoma japonicum

LIANG Le¹^,²(), ZHANG Jing¹, SHEN Yu-juan¹, HU Yuan¹, CAO Jian-ping¹^,^*()

1. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology; WHO Collaborating Center for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, China
2. Shanghai University of Medicine & Health Sciences, Shanghai 201328, China

Received:2022-05-18 Revised:2022-06-11 Online:2022-08-30 Published:2022-09-07
Contact: CAO Jian-ping
Supported by:
National Nature Science Foundation of China(81971969);National Nature Science Foundation of China(81772225);Three-Year Public Health Action Plan (2020—2022) of Shanghai(GWV-10.1-XK13)

摘要/Abstract

摘要：

目的研究第二信使分子环鸟苷酸腺苷酸（cGAMP）对日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化的影响,并初步阐明其调控机制。方法将C57BL/6小鼠随机分为cGAMP处理组（8只）、感染对照组（28只）、感染cGAMP处理组（28只）。cGAMP处理组尾静脉单独注射cGAMP（2 μg,100 μl）,感染对照组和感染cGAMP处理组经腹部皮肤贴片感染日本血吸虫尾蚴[（20 ± 2）条/鼠]后,分别经尾静脉注射PBS（100 μl）和cGAMP（2 μg,100 μl）,每周1次。3组小鼠各8只分别注射10次,每次注射后观察小鼠死亡情况。感染对照组和感染cGAMP处理组小鼠（各5只）于感染后7周,取小鼠眼眶血,ELISA检测丙氨酸氨基转移酶（ALT）与天冬氨酸氨基转移酶（AST）的水平;取小鼠肝组织切片进行苏木精-伊红染色（HE）和Masson染色,计算单个虫卵肉芽肿面积和肝纤维化面积,并计数肝组织虫卵数。感染对照组和感染cGAMP处理组小鼠（各15只）于感染后0、4和7周,取小鼠肝组织,提取总RNA,实时荧光定量PCR（qRT-PCR）检测肝组织β干扰素（IFN-β）编码基因Ifnb1的表达水平;取小鼠眼眶血,ELISA检测血清中IFN-β水平。采用GraphPad Prism 8.0.2软件进行统计学分析,两组间比较采用t检验。结果 cGAMP处理组小鼠无死亡;感染后10周,感染对照组小鼠死亡2只,感染cGAMP处理组小鼠死亡5只,两组差异有统计学意义（χ² = 7.55,P < 0.01）。感染对照组和感染cGAMP处理组小鼠血清ALT分别为（394.80 ± 72.82）和（627.20 ± 93.58）U/L（t = 4.38,P < 0.01）,血清AST分别为（605.00 ± 148.90）和（871.80 ± 138.10）U/L（t = 2.94,P < 0.05）。感染对照组和感染cGAMP处理组小鼠肝组织虫卵数分别为50 600 ± 15 473和53 400 ± 16 273,差异无统计学意义（t = 0.28,P > 0.05）;HE染色结果显示,感染对照组和感染cGAMP处理组均出现明显的肝组织结构被破坏,并有炎性细胞浸润,肝组织病理损害比例分别为（35.32 ± 15.09）%和（64.98 ± 13.91）%（t = 3.23,P < 0.05）;肝组织虫卵肉芽肿面积分别为（0.57 ± 0.45）和（1.17 ± 0.74）mm²（t = 2.95,P < 0.01）;Masson染色结果显示,感染对照组和感染cGAMP处理组小鼠均发生肝纤维化,比例分别为（24.50 ± 6.39）%与（39.02 ± 6.79）%（t = 3.48,P < 0.01）。感染后4和7周,感染cGAMP处理组小鼠肝组织中Ifnb1 mRNA的相对表达量分别为16.58 ± 6.71和10.52 ± 2.88,较感染对照组的9.280 ± 4.50和6.62 ± 1.58均上调（t = 2.95、2.18,P < 0.05）;感染cGAMP处理组小鼠血清IFN-β的含量分别为（1 206.45 ± 211.87）和（960.62 ± 151.33）pg/ml,较感染对照组的（845.12 ± 284.11）和（685.20 ± 143.88）pg/ml均上调（t = 2.28、2.95,P < 0.05）。结论 cGAMP可促日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化,通过诱导IFN-Ⅰ发挥免疫调控作用。

关键词: 日本血吸虫, 环鸟苷酸腺苷酸, 干扰素基因刺激蛋白, Ⅰ型干扰素

Abstract:

Objective To investigate the effects of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a second messenger molecule, on liver egg granulomas and fibrosis in Schistosoma japonicum-infected mice and to preliminarily clarify its regulatory mechanism. Methods C57BL/6 mice were randomly divided into cGAMP-treated group (8 mice), infected control group (28 mice), and infected cGAMP-treated group (28 mice). For the cGAMP-treated group, cGAMP (2 μg, 100 μl) was injected via the tail vein, while the infected control group and the infected cGAMP-treated group were injected with PBS (100 μl) and cGAMP (2 μg, 100 μl) via the tail vein once a week, respectively, after being infected with S. japonicum cercariae [(20 ± 2)/mouse] via abdominal skin patch. From each group, 8 mice received 10 ingections to observe survivl state after each injection. After 7 weeks, mice orbital blood samples from infected control group and infected cGAMP-treated group (each 5) were collected to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by ELISA. The liver tissues sections were prepared and stained with hematoxylin eosin (HE) and Masson to estimate the area of individual egg granuloma and fibrosis, and count the number of eggs in the liver tissues. Mice liver tissues were collected from infected control group and infected cGAMP-treated group (15 each), on 0, 4, and 7 weeks post-infection, for extraction of total RNA, which was used for performing real time fluorescent quantitative PCR (qRT-PCR) to examine the expression level of IFN-β-encoding gene Ifnb1; the mice orbital blood was collected to detect the serum β-interferon (IFN-β) level by ELISA. GraphPad Prism 8.0.2 software was used for statistical analysis, and t-test was used for comparison between the two groups. Results There were no deaths in the cGAMP-treated group, and 10 weeks after infection, 2 mice died in the infected control group and 5 mice died in the infected cGAMP-treated group, with statistically significant differences between the two groups (χ² = 7.55, P < 0.01). The number of eggs in the liver of mice from the infected control group and the infected cGAMP-treated group was 50 600 ± 15 473 and 53 400 ± 16 273, respectively, with no statistically significant difference (t = 0.28, P > 0.05). HE staining showed significant liver histopathological damage in both the infected control and infected cGAMP-treated groups, with proportions of (35.32 ± (15.09)% and (64.98 ± 13.91)% (t = 3.23, P < 0.05), respectively. The area of liver egg granulomas was (0.57 ± 0.45) mm² and (1.17 ± 0.74) mm² in the infected control and infected cGAMP-treated groups, respectively (t = 2.95, P < 0.01). Masson staining showed that liver fibrosis occurred in both the infected control and infected cGAMP-treated mice, with proportions of (24.50 ± 6.39)% and (39.02 ± 6.79)%, respectively (t = 3.48, P < 0.01). At 4 and 7 weeks post-infection, the expression levels of Ifnb1 mRNA in liver tissues of mice from infected cGAMP-treated group were 16.58 ± 6.71 and 10.52 ± 2.88, respectively, which were upregulated compared with 9.280 ± 4.50 and 6.62 ± 1.58 in the infected control group (t = 2.95, 2.18, P < 0.05). The serum IFN-β levels in the infected cGAMP-treated group were (1 206.45 ± 211.87) pg/ml and (960.62 ± 151.33) pg/ml, respectively, which were upregulated compared to (845.12 ± 284.11) pg/ml and (685.20 ± 143.88) pg/ml in the infected control group (t = 2.28, 2.95, P < 0.05). ALT was (394.80 ± 72.82) U/L and (627.20 ± 93.58) U/L (t = 4.38, P < 0.01) and AST was (605.00 ± 148.90) U/L and (871.80 ± 138.10) U/L (t = 2.94, P < 0.05) in the mice of infected control and infected cGAMP-treated groups, respectively. Conclusion cGAMP promots liver egg granuloma formation and fibrosis in mice infected with S. japonicum, indicative of exerting the promotive effect through inducing type Ⅰ interferon immune response.

Key words: Schistosoma japonicum, Cyclic guanosine monophosphate-adenosine monophosphate, Stimulator of interferon genes, TypeⅠinterferon

中图分类号:

R383.24

梁乐, 张璟, 沈玉娟, 胡媛, 曹建平. 环鸟苷酸腺苷酸促日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 441-445.

LIANG Le, ZHANG Jing, SHEN Yu-juan, HU Yuan, CAO Jian-ping. Cyclic guanosine monophosphate-adenosine monophosphate promotes liver egg granuloma formation and fibrosis in mice infected with Schistosoma japonicum[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(4): 441-445.

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参考文献 26

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环鸟苷酸腺苷酸促日本血吸虫感染小鼠肝虫卵肉芽肿形成及纤维化

Cyclic guanosine monophosphate-adenosine monophosphate promotes liver egg granuloma formation and fibrosis in mice infected with Schistosoma japonicum

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