白念珠菌菌丝调控小鼠巨噬细胞自噬关键分子微管相关蛋白1轻链3的实验研究

Regulatory effect of Candida albicans hyphae on the key autophagy-related molecule microtubule-associated protein 1 light chain 3 in murine bone marrow-derived macrophages

目的探讨白念珠菌菌丝对小鼠骨髓来源巨噬细胞(BMDM)自噬流的影响。方法白念珠菌菌丝分别体外诱导BMDM细胞0.5、4、12 h,以不加菌丝处理的0 h组作为对照,Western印迹法检测各时间点自噬关键蛋白微管相关蛋白1轻链3(LC3)-Ⅰ向LC3-Ⅱ的转换及磷酸化雷帕霉素机制性靶蛋白(p-mTOR)的表达。白念珠菌菌丝分别联合4种溶酶体阻断剂,包括半胱氨酸蛋白酶抑制剂E-64d+胃蛋白酶抑制剂pepstatin、巴弗洛霉素-A1(BAF-A1)、氯化铵及氯喹,体外诱导小鼠BMDM细胞4、12 h,观察白念珠菌菌丝对BMDM细胞基础自噬流的影响。统计分析采用非配对t检验、析因设计的方差分析及LSD-t检验。结果白念珠菌菌丝体外处理小鼠BMDM 0.5、4和12 h后,与0 h组(0.983±0.030)相比,LC3-Ⅰ向LC3-Ⅱ转换均增加(1.254±0.118、1.629±0.391、1.598±0.379),差异有统计学意义(t值分别为3.875、2.856、2.804,均P<0.05),但各组p-mTOR的蛋白表达无明显差异。白念珠菌菌丝联合E-64d+pepstatin体外处理BMDM细胞4和12 h后,LC3-Ⅱ的蓄积水平较E-64d+pepstatin单独处理组明显增高,差异均有统计学意义(t值分别3.691、6.648,均P<0.05)。与相应溶酶体阻断剂组相比,白念珠菌菌丝联合BAF-A1、氯化铵或氯喹4和12 h后,LC3-Ⅱ的蓄积水平均显著升高(均P<0.05)。结论白念珠菌菌丝体外诱导可增加小鼠BMDM细胞基础自噬流中LC3-Ⅰ向LC3-Ⅱ的转换。

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-Ⅰto LC3-Ⅱsignificantly 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-Ⅱsignificantly 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-Ⅱin the basal autophagic flux in murine BMDM.