ABSTRACT
Objective:To evaluate the effect of Candida albicans ( C. albicans) hyphae on autophagic flux in murine bone marrow-derived macrophages (BMDM) . Methods:BMDM were in vitro stimulated with C. albicans hyphae for 0.5, 4 and 12 hours, and the 0-hour group treated without hyphae served as a control. Western blot analysis was performed to detect the conversion of microtubule-associated protein 1 light chain 3 (LC3) -Ⅰto LC3-Ⅱ, and determine the expression of phosphorylated mechanistic target of rapamycin (p-mTOR) at each time point. Some BMDM were divided into several groups: control group receiving no treatment, hyphae group treated with C. albicans hyphae, lysosomal inhibitor groups treated with different lysosomal inhibitors, including E-64d (a cysteine proteinase inhibitor) + pepstatin (a pepsin inhibitor) , bafilomycin-A1 (BAF-A1) , ammonium chloride and chloroquine, and hyphae combined with lysosomal inhibitor groups treated with lysosomal inhibitors immediately followed by C. albicans hyphae. After 4- or 12-hour treatment, the effect of C. albicans hyphae on basal autophagic flux in murine BMDM was evaluated. Statistical analysis was carried out by using unpaired t test, factorial design analysis of variance and least significant difference- t test. Results:After 0.5-, 4- and 12-hour in vitro treatment with C. albicans hyphae, the conversion of LC3-Ⅰ to LC3-Ⅱ significantly increased in murine BMDM (1.254±0.118, 1.629±0.391, 1.598±0.379, respectively) compared with the 0-hour group (0.983±0.030; t=3.875, 2.856, 2.804, respectively, all P< 0.05) , while there was no significant difference in the protein expression of p-mTOR among the 0-, 0.5-, 4- and 12-hour groups. After 4- and 12-hour in vitro treatment with C. albicans hyphae combined with lysosomal inhibitors E-64d and pepstatin, the accumulation level of LC3-Ⅱ significantly increased in BMDM compared with those treated with E-64d and pepstatin alone ( t=3.691, 6.648, respectively, both P< 0.05) . Compared with the corresponding lysosomal inhibitor groups, the accumulation level of LC3-Ⅱsignificantly increased in BMDM treated with C. albicans hyphae combined with BAF-A1, ammonium chloride or chloroquine for 4 and 12 hours (all P< 0.05) . Conclusion:In vitro treatment with C. albicans hyphae can increase the conversion of LC3-Ⅰto LC3-Ⅱ in the basal autophagic flux in murine BMDM.