铜绿假单胞菌的信号分子3-oxo-C12-HSL促进小鼠MH-S肺泡巨噬细胞自噬

摘要

目的 探讨3-oxo-C12-HSL对MH-S小鼠肺泡巨噬细胞自噬的影响.方法 用LasI基因(3-oxo-C12-HSL合成基因)缺失型和野生型的铜绿假单胞菌(PA)菌株的培养物上清液以及化学合成的3-oxo-C12-HSL信号分子处理MH-S细胞,激光共聚焦荧光显微镜观察LC3-GFP荧光斑点以及Western blot检测LC3Ⅱ/LC3Ⅰ比值来监测自噬的形成,同时检测p62的降解以及氯喹对LC3Ⅱ/LC3Ⅰ比值的影响来监测自噬流(autophagic flux)的变化.结果 野生型PA菌株的培养物上清液使MH-S细胞LC3-GFP荧光斑点增多(P<0.05),LC3Ⅱ/LC3Ⅰ比值增加(P<0.01),LasⅠ基因缺失型PA菌株不能引起上述自噬相关指标的改变.化学合成的3-oxo-C12-HSL信号分子能够明显增加自噬体数量,上调LC3Ⅱ的表达(P<0.01);自噬底物p62随着3-oxo-C12-HSL作用时间的延长而进行性降解,溶酶体抑制剂氯喹可增强3-oxo-C12-HSL引起的LC3Ⅱ累积(P<0.05,P<0.01).结论3-oxo-C12-HSL可增加MH-S细胞的自噬水平.%Objective To investigate the effect of 3-oxo-C12-HSL on autophagy in mouse alveolar macrophages MH-S cells. Methods MH-S cells were treated with culture supernatants of the mutant and wild type Pseudomonas aeruginosa(PA) strains of LasI gene(3-oxo-C12-HSL synthetic gene) and chemically synthesized 3-oxo-C12-HSL signaling molecules. GFP puncta was observed by laser confocal fluorescence microscopy and the ratio of LC3Ⅱ/LC3Ⅰ was detected by Western blot to detect the formation of autophagic.Autophagic flux was also detected by mo-nitoring the degradation of p62 and the change of chloroquine to LC3Ⅱ/LC3Ⅰratio. Results The supernatant of the culture medium of the wild type PA strain increased the GFP puncta of the MH-S cells(P<0.05) and the ra-tio of LC3Ⅱ/LC3Ⅰ(P<0.01),The mutant PA strain of LasI gene could not cause the above changes related to autophagy. The chemically synthesized 3-oxo-C12-HSL signal molecules could increase the number of autophagic bodies and the expression of LC3Ⅱ (P<0.01). Autophagic substrate p62 was degraded by 3-oxo-C12-HSL. Chloroquine, a lysosomal inhibitor, enhanced LC3Ⅱaccumulation caused by 3-oxo-C12-HSL (P<0.05,P<0.01).Conclusions 3-oxo-C12-HSL increases the level of autophagy in MH-S cells.