中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (11): 962-968.doi: 10.35541/cjd.20220007

• 论著 • 上一篇    下一篇

烟曲霉对小鼠巨噬细胞自噬流的影响初步研究

杨璐1    段志敏1    徐松1    陈旭1    李岷1，2   

  1. 1中国医学科学院、北京协和医学院皮肤病研究所真菌科  江苏省皮肤病与性病分子生物学重点实验室，南京  210042；2南京医科大学全球健康中心，南京  210029
  • 收稿日期:2022-01-06 修回日期:2022-06-07 发布日期:2022-11-03
  • 通讯作者: 陈旭; 李岷 E-mail:chenx@pumcderm.cams.cn; limin@pumcderm.cams.cn
  • 基金资助:
    国家自然科学基金;国家自然科学基金;国家自然科学基金;江苏省自然科学基金;中国医学科学院医学与健康科技创新工程项目;南京市国家级临床医学中心培育计划项目

Effect of Aspergillus fumigatus on the autophagic flux in murine macrophages： a preliminary study

Yang Lu1, Duan Zhimin1, Xu Song1, Chen Xu1, Li Min1, 2   

  1. 1Department of Medical Mycology, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China; 2Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing 210029, China
  • Received:2022-01-06 Revised:2022-06-07 Published:2022-11-03
  • Contact: Chen Xu; Li Min E-mail:chenx@pumcderm.cams.cn; limin@pumcderm.cams.cn
  • Supported by:
    National Natural Science Foundation of China;National Natural Science Foundation of China;National Natural Science Foundation of China;Natural Science Foundation of Jiangsu Province;CAMS Innovation Fund for Medical Sciences;The Nanjing Incubation Program for National Clinical Research Center

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摘要： 【摘要】 目的 探讨烟曲霉对小鼠骨髓来源巨噬细胞（BMDM）自噬流的影响。方法 灭活烟曲霉体外诱导小鼠BMDM不同时间（0、0.5、4、12 h），提取细胞蛋白，Western印迹法检测自噬关键蛋白LC3-Ⅰ型/Ⅱ型的转换及磷酸化雷帕霉素靶蛋白（p-mTOR）Ser2481的蛋白水平。用烟曲霉和溶酶体阻断剂［包括半胱氨酸蛋白酶抑制剂E-64d + 胃蛋白酶抑制剂pepstatin、巴佛洛霉素-A1（BAF-A1）、氯化铵、氯喹］单独或联合体外诱导小鼠BMDM不同时间（4、12 h）后，Western印迹法检测烟曲霉对新生的LC3-Ⅱ、细胞基础自噬流的影响，并通过共聚焦荧光显微镜观察烟曲霉与LC3、Rubicon（RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein）的共定位关系。不同处理时间数据结果采用非匹配t检验分析，烟曲霉孢子和自噬溶酶体阻断剂两因素处理数据结果采用2 × 2析因分析方法。结果 Western印迹显示，与对照组（0 h组）比较，烟曲霉体外诱导小鼠BMDM 0.5、4、12 h后细胞内LC3-Ⅱ表达逐渐增加，差异均有统计学意义（t值分别为6.58、3.28、3.02，均P < 0.05），但各组p-mTOR蛋白水平差异无统计学意义（t值分别为0.441、0.477、0.382，P值分别为0.682、0.660、0.722）。与单独氯喹处理BMDM 4 h和12 h相比，烟曲霉联合氯喹处理后 LC3-Ⅱ进一步增高，差异均有统计学意义（t = 2.13、2.78，均P < 0.05）。与单独氯化铵处理BMDM 4 h和12 h相比，烟曲霉联合氯化铵处理后 LC3-Ⅱ进一步增高，差异均有统计学意义（t = 2.92、2.92，均P < 0.05）。与单独BAF-A1处理BMDM 4 h和12 h相比，烟曲霉联合BAF-A1处理后LC3-Ⅱ进一步增高，差异有统计学意义（t = 2.13、2.13，均P < 0.05）。与单独E-64d + pepstatin处理BMDM 4 h和12 h相比，烟曲霉联合E-64d + pepstatin处理后 LC3-Ⅱ进一步增高，差异有统计学意义（t = 2.13、2.92，均P < 0.05）。用钙荧光白荧光标记的烟曲霉孢子刺激BMDM 8 h后，共聚焦荧光显微镜显示LC3、Rubicon主要包绕于烟曲霉周围，与烟曲霉均有共定位关系。结论 烟曲霉体外诱导可增加小鼠BMDM基础自噬流。

关键词: 烟曲霉菌, 巨噬细胞, 自噬, 微管相关蛋白质类, 微管相关蛋白1轻链3, LC3相关吞噬

Abstract: 【Abstract】 Objective To explore the effect of Aspergillus fumigatus （A. fumigatus）on the autophagic flux in murine bone marrow-derived macrophages （BMDM）. Methods Murine BMDM were in vitro cultured with heat-killed A. fumigatus for 0, 0.5, 4, and 12 hours. Then, cellular proteins were extracted, and Western blot analysis was performed to detect the conversion of the key autophagy protein microtubule-associated protein 1 light chain 3 （LC3）-Ⅰto LC3-Ⅱ, and to determine the protein expression of phosphorylated mammalian target of rapamycin （p-mTOR） Ser2481. Additionally, murine BMDM were in vitro cultured with A. fumigatus alone or in combination with different lysosomal inhibitors, including the cysteine cathepsin inhibitor E-64d + pepstatin, bafilomycin-A1 （BAF-A1）, ammonium chloride （NH4Cl）, and chloroquine, for 4 or 12 hours. Then, Western blot analysis was performed to investigate the effect of A. fumigatus on newly formed LC3-Ⅱ and basal autophagic flux, and confocal laser scanning fluorescence microscopy to analyze the colocalization of A. fumigatus with LC3 and Rubicon （a RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein）. Experimental results at different treatment time points were analyzed by using unpaired t test, and results of experiments evaluating the effect of two factors （A. fumigatus spores and autophagosome inhibitors） were analyzed by 2 × 2 factorial analysis. Results After in vitro co-culture with A. fumigatus for 0.5, 4, 12 hours, Western blot analysis showed that the conversion of LC3-Ⅰ to LC3-Ⅱ increased over time in murine BMDM compared with the control (0 hour) group （t = 6.58, 3.28, 3.02, respectively, all P < 0.05）, but the protein expression level of p-mTOR （Ser2481） did not significantly differ at different treatment time points（t = 0.441, 0.477, 0.382, P = 0.682, 0.660, 0.722, respectively）. After 4- and 12-hour in vitro treatment, the accumulation levels of LC3-Ⅱ in BMDM significantly increased in the A. fumigatus + chloroquine group compared with the chloroquine-alone group （t = 2.13, 2.78, respectively, both P < 0.05）, in the A. fumigatus + NH4Cl group compared with the NH4Cl-alone group （t = 2.92, 2.92, respectively, both P < 0.05）, in the A. fumigatus + BAF-A1 group compared with the BAF-A1-alone group （t = 2.13, 2.13, respectively, both P < 0.05）, and in the A. fumigatus + E-64d + pepstatin group compared with the E-64d + pepstatin group （t = 2.13, 2.92, respectively, both P < 0.05）. After 8-hour treatment with calcofluor white-labeled A. fumigatus spores, confocal laser scanning fluorescence microscopy showed that LC3 and Rubicon mainly surrounded A. fumigatus, suggesting their colocalization with A. fumigatus. Conclusion A. fumigatus can in vitro increase the basal autophagic flux in murine BMDM.

Key words: Aspergillus fumigatus, Macrophages, Autophagy, Microtubule-associated proteins, Microtubule-associated protein 1 light chain 3, LC3-associated phagocytosis