中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (1): 41-46.doi: 10.12140/j.issn.1000-7423.2020.01.007

• 论著 • 上一篇    下一篇

血吸虫病肝纤维化过程中Beclin1调节线粒体功能的初步研究

沈双1,*, 罗军涛2, 叶建平1   

  1. 1 上海健康医学院附属第六人民医院东院,上海 201306;
    2 上海海洋大学,上海 201306
  • 收稿日期:2019-08-21 出版日期:2020-02-28 发布日期:2020-03-19
  • 通讯作者: 沈双,E-mail: shuangshen.no.1@163.com
  • 作者简介:沈双(1985-),男,硕士,助理研究员,从事血吸虫肝纤维化机制的研究。E-mail:shuangshen.no.1@163.com
  • 基金资助:
    上海市卫生和计划生育委员会青年项目(No. 20174Y0124); 浦东新区科技发展基金(No. PKJ2018-Y55); 上海市第六人民医院医疗集团科研基金; 上海健康医学院种子基金(No. SFP-18-22-14-009)

Mitochondrial function regulated by Beclin1 in liver fibrosis in schistosomiasis

SHEN Shuang1,*, LUO Jun-tao2, YE Jian-ping1   

  1. 1 Shanghai Sixth People’s Hospital East Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai 201306, China;;
    2 Shanghai Ocean University, Shanghai 201306, China
  • Received:2019-08-21 Online:2020-02-28 Published:2020-03-19
  • Contact: E-mail: shuangshen.no.1@163.com
  • Supported by:
    Supported by Youth Project of Shanghai Municipal Commission of Health and Family Planning(No. 20174Y0124), Science and Technology Development Fund of Shanghai Pudong New Area(No. PKJ2018-Y55), Scientific Research Fund of Shanghai the Sixth People’s Hospital Medical Group and Seed Fund of Shanghai University of Medicine and Health Sciences(No. SFP-18-22-14-009)

摘要: 目的 探索Beclin1在血吸虫病肝纤维化中与线粒体功能的关系。 方法 将40只C57BL/6雌性小鼠随机分为感染组和健康对照组,每组20只。感染组小鼠采用腹部贴片法感染日本血吸虫尾蚴20条/鼠。感染后10周,取两组小鼠新鲜肝组织,苏木精-伊红(HE)和Masson染色观察肝组织病变情况。蛋白质印迹(Western blotting)分析肝组织中Beclin1、p-Beclin1及电压依赖型阴离子孔道蛋白1(VDAC1)的相对表达水平。荧光定量PCR检测肝组织中胶原蛋白Col 1a1、Col 3a1,以及肝组织中参与线粒体柠檬酸循环反应的柠檬酸合酶、异柠檬酸脱氢酶、α酮戊二酸脱氢酶和苹果酸脱氢酶mRNA的相对转录水平。取两组小鼠肝组织制备切片,电镜下观察线粒体形态。提取肝组织线粒体,利用细胞能量代谢仪依次检测加入复合物Ⅴ底物二磷酸腺苷(ADP)、复合物Ⅴ抑制剂寡霉素、解偶联剂FCCP、复合物Ⅱ和Ⅲ的抑制剂抗霉素A后5 min线粒体的耗氧率。 结果 HE和Masson染色结果显示,感染组小鼠肝脏形成肉芽肿,肉芽肿区域发生明显纤维化。Western blotting分析结果显示,感染组肝组织中VDAC1表达水平未发生改变;Beclin1和p-Beclin1的相对表达水平分别为0.65 ± 0.05、0.78 ± 0.03,均较健康对照组下降(P < 0.05)。荧光定量PCR结果显示,感染组小鼠肝组织中Col 1a1、Col 3a1 mRNA的相对转录水平分别为4.05 ± 0.23、2.71 ± 0.14,均较健康对照组上升(P < 0.01);柠檬酸合酶、异柠檬酸脱氢酶、α酮戊二酸脱氢酶及苹果酸脱氢酶mRNA的相对转录水平分别为0.61 ± 0.03、0.65 ± 0.01、0.41 ± 0.03、0.55 ± 0.01,均较健康对照组下降(P < 0.01)。电镜观察可见,感染组肝组织线粒体结构受损,嵴断裂。线粒体功能检测显示,加入复合物Ⅴ底物ADP后,健康对照组和感染组耗氧率分别为(335 ± 29)、(78 ± 23)pmol/min,差异有统计学意义(P < 0.01);加入复合物Ⅴ抑制剂寡霉素后,健康对照组和感染组耗氧率分别为(80 ± 2)、(31 ± 6)pmol/min,两组耗氧率差异减小但仍有统计学意义(P < 0.05);加入解偶联剂FCCP后,健康对照组和感染组耗氧率分别为(159 ± 4)、(42 ± 5)pmol/min,差异有统计学意义(P < 0.01);加入复合物Ⅱ和Ⅲ的抑制剂抗霉素A后,两组耗氧率均降为0。 结论 血吸虫病肝纤维化过程中,Beclin1表达量下降,柠檬酸循环受到抑制,线粒体耗氧率进一步下降。线粒体复合物Ⅴ是血吸虫病肝纤维化过程中线粒体活性调节的关键复合物。

关键词: 日本血吸虫, 肝纤维化, 线粒体, 柠檬酸循环, Beclin1

Abstract: Objective To explore the relationship between Beclin1 and mitochondrial function in hepatic fibrosis in schistosomiasis. Methods Forty female C57BL/6 mice were randomly divided into infected group and healthy control group (20 mice in each group). Mice in the infected group were infected with 20 Schistosoma japonicum cercariae by the abdominal patch method. Ten weeks after infection, fresh liver tissues were collected from the two groups. Liver pathology was assessed by HE and Masson staining of liver tissue sections. The relative protein levels of Beclin1, p-Beclin1 and voltage-dependent anion channel protein 1 (VDAC1) in liver were assessed by Western blotting. Fluorescence quantitative PCR was performed to analyze the relative mRNA expression of collagen Col 1a1 and Col 3a1, as well as enzymes involved in the mitochondrial citric acid cycle reaction, including citrate synthase, isocitrate dehydrogenase, alpha ketoglutarate dehydrogenase and malate dehydrogenase. Another part of liver tissues was sectioned for visualizing mitochondrion morphology by electron microscopy. To examine the mitochondria oxygen consumption rate (OCR) using cell energy metabolizer, the mitochondria extracted from liver tissues were added to the culture plates, to which complex V substrate ADP, complex V inhibitor oligomycin, uncoupler FCCP, and antimycin A (an inhibitor for complex Ⅱ plus Ⅲ) were sequentially added, allowing culture for 5 min, then the OCR was detected. Results HE and Masson staining showed that granulomas were formed in mouse liver in the infected group, and obvious fibrosis occurred around the granulomas. Western blotting showed that the protein level of VDAC1 remained unchanged in the infected liver, while Beclin1 (0.65 ± 0.05) and p-Beclin1 (0.78 ± 0.03) were decreased, compared with the control (P < 0.05). The fluorescence quantitative PCR results indicated that the relative transcription levels of Col 1a1 and Col 3a1 mRNA in livers of the infected group were 4.05 ± 0.23 and 2.71 ± 0.14, respectively, which were higher than those of the healthy control group (P < 0.01). The relative transcription levels of citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase mRNA were 0.61 ± 0.03, 0.65 ± 0.01, 0.41 ± 0.03 and 0.55 ± 0.01, respectively, which were lower than those of the healthy control group (P < 0.01). Electron microscopy revealed that in the infected group, the mitochondrial structure was damaged and the ridge was broken. Mitochondrial function test showed that after addition of complex Ⅴ substrate ADP, the OCR of the healthy control group [(335 ± 29) pmol/min] was higher than that of the infected group [(78 ± 23) pmol/min] (P < 0.01); addition of complex Ⅴ inhibitor oligomycin reduced the difference [(80 ± 2) pmol/min in the control group and (31 ± 6) pmol/min in the infected group], although significance of difference still existed (P < 0.05); after addition of uncoupler FCCP, the OCRs of the healthy control and the infected groups increased to (159 ± 4) pmol/min and (42 ± 5) pmol/min(P < 0.01), respectively. After addition of complex Ⅱ plus Ⅲ inhibitor, the OCRs of both groups decreased to zero. Conclusion In the development of liver fibrosis in schistosomiasis, Beclin1 is reduced, the citric acid cycle is restrained, and the mitochondrial OCR further declines. The complex Ⅴ plays an important role in the regulation of mitochondrial activity during liver fibrosis in schistosomiasis.

Key words: Schistosoma japonicum, Liver fibrosis, Mitochondria, Citric acid cycle, Beclin1

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