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�
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-Ⅱand 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-Ⅰto LC3-Ⅱincreased 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-Ⅱin 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+NH4
作者
杨璐
段志敏
徐松
陈旭
李岷
Yang Lu;Duan Zhimin;Xu Song;Chen Xu;Li Min(Department 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;Center for Global Health,School of Public Health,Nanjing Medical University,Nanjing 210029,China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2022年第11期962-968,共7页
Chinese Journal of Dermatology
基金
国家自然科学基金(81773338、82103749、82173432)
江苏省自然科学基金(BK20190144)
中国医学科学院医学与健康科技创新工程项目(2017-I2M-1-017)
南京市国家级临床医学中心培育计划项目(2019060001)。
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
Aspergillus fumigatus
Macrophages
Autophagy
Microtubule-associated proteins
Microtubule-associated protein 1 light chain 3
LC3-associated phagocytosis
