谷氨酰胺酶1对卡介苗诱导的巨噬细胞自噬的调控作用

Regulatory effect of glutaminase 1 on BCG-induced autophagy of macrophages

目的:探讨谷氨酰胺酶1(glutaminase 1,GLS1)在卡介苗(Bacille Calmette-Guérin,BCG)感染的巨噬细胞中对自噬的调控作用。方法:培养小鼠单核巨噬细胞RAW264.7,利用小干扰RNA敲减巨噬细胞内GLS1的表达并结合BCG感染,采用免疫荧光染色、流式细胞术、ELISA和Western blot等技术检测自噬相关指标,阐明GLS1对BCG诱导的巨噬细胞自噬的调控作用,并初步探讨其机制。结果:(1)BCG感染巨噬细胞显著增加了LC3B和GLS1的蛋白表达量(P<0.01),促进了谷氨酰胺分解;(2)敲减GLS1显著增加了巨噬细胞内谷氨酰胺的含量(P<0.05),同时显著降低了巨噬细胞的自噬率以及自噬相关因子LC3B(P<0.01)、beclin-1(P<0.01)和ATG12(P<0.05)的蛋白表达量;(3)剥夺培养液中的谷氨酰胺处理细胞后,敲减GLS1对BCG感染的巨噬细胞中自噬相关因子LC3B、ATG5和ATG12的表达以及自噬流的发生无显著影响。结论:在BCG感染巨噬细胞RAW264.7过程中,敲减GLS1后可通过增加巨噬细胞内的谷氨酰胺含量抑制细胞自噬。

AIM:To investigate the regulatory effect of glutaminase 1(GLS1)on Bacillus Calmette-Guérin(BCG)infection-induced macrophage autophagy.METHODS:Small interfering RNA was used to knock down GLS1 in mouse macrophage RAW264.7 cells,and then the cells were infected with BCG.Immunofluorescence staining,flow cytometry,ELISA and Western blot were used to investigate autophagy-related indicators in RAW264.7 cells.RESULTS:Infection of BCG significantly increased the expression of LC3B and GLS1(P<0.01),accompanied with enhanced glutaminolysis in RAW264.7 cells.Furthermore,knockdown of GLS1 increased glutamine level(P<0.05),reduced the autophagy rate,and decreased the expression of autophagy-related factors LC3B(P<0.01),beclin-1(P<0.01)and ATG12(P<0.05)in RAW264.7 cells.In addition,after glutamine deprivation,knockdown of GLS1 excert no significant effect on the expression of autophagy-related factors LC3B,ATG5 and ATG12 in BCG-infected macrophages.CONCLUSION:GLS1 increases BCG-induced autophagy by promoting glutaminolysis in macrophages.