Regulatory Of Glutamine On BCG-induced Apoptosis Of Macrophage RAW264.7 Cells

Background:Tuberculosis(TB)is a highly lethal zoonotic disease caused by Mycobacterium tuberculosis(Mtb)infection.Macrophages,as the main target cells of Mtb,can kill invading pathogens through immune processes such as apoptosis,thereby preventing Mtb infection and spread.In this process,in order to effectively exert their own immune function,macrophages will reprogram their own metabolic pathways and change the level of specific metabolites in the cells.Metabolism and immunity interact to maintain cellular homeostasis.Glutamine is an important metabolic substrate of cells.On the one hand,it can be degraded to α-ketoglutaric acid through the glutamine decomposition pathway,participating in the tricarboxylic acid cycle to provide energy for cells.On the other hand,glutamine and its metabolites are important signaling molecules that can affect the activation of multiple signaling pathways such as PI3K/AKT/mTPR,NF-κB,and endoplasmic reticulum stress.However,the regulatory effect of glutamine metabolism on cell apoptosis during MTB-infected macrophages and the underlying molecular mechanisms have not been elucidated.Methods:In this study,mouse macrophage RAW264.7 was used as the research model.First,the cells were infected with Mycobacterium tuberculosis vaccine strain BCG,and the proteins were extracted at different stages of infection,and the expression of apoptosis-related factors was detected by Western blotting to determine the optimal time and multiplicity of infection of BCG.Then the intracellular levels of glutamine and its metabolites,glutamine transporters and glutamine metabolizing enzymes were detected by ELISA and Western blotting to determine the effect of BCG on glutamine metabolism in RAW264.7 cells.Then,the cells were cultured in different concentrations of glutamine and combined with BCG infection.ELISA,Western blotting,immunofluorescence and flow cytometry were used to clarify the regulatory effect of glutamine on BCG-induced apoptosis of macrophages,and to explore its mechanism.Results:1.BCG infection significantly promoted the expression of macrophage apoptosis-related factors Caspase 3 and PARP(p<0.001),significantly increased the apoptosis rate of RAW264.7 cells(p<0.001),the optimal infection time was 12 hours,and the optimal multiplicity of infection was 10.BCG infection promoted the catabolism of glutamine in RAW264.7 cells,and the contents of intracellular glutamine and α-ketoglutaric acid were significantly decreased(p<0.01),while the contents of glutamate and glutathione were significantly increased(p<0.05),and glutamine transporter(SLC1A5)and glutaminase(GLS)were significantly up-regulated(p<0.001),glutamine dehydrogenase(GLUD)was significantly down-regulated(p<0.01);3.Glutamine deprivation decreased the intracellular levels of glutamine,glutamate,and glutathione during BCG infection of macrophages.Furthermore,glutamine deprivation promoted the expression of apoptosis-related factors Caspase 3 and PARP(p<0.001),and significantly increased the apoptosis rate of RAW264.7 cells(p<0.05)and promoted the accumulation of intracellular ROS(p<0.001).In addition,glutamine deprivation inhibited the survival of BCG in RAW264.7 cells.4.Glutamine deprivation promoted the expression of endogenous apoptotic proteins Bax,Caspase 9 and Cytc during BCG-infected macrophages(p<0.001)and significantly down-regulated the expression of anti-apoptotic protein Bcl-2(p<0.001).Glutamine deprivation inhibited the opening of mitochondrial permeability transition pore,significantly changed mitochondrial membrane potential(p<0.01),and promoted the expression of MAPK pathway proteins ASK1 and JNK(p<0.001).Conclusion:BCG infection of macrophages promotes glutamine catabolism in macrophages.After glutamine deprivation,the intracellular glutathione content is reduced,which in turn promotes the accumulation of ROS.ROS,as a second messenger,activates the downstream ASK1/JNK signaling pathway,and then JNK promotes the endogenous apoptosis of macrophages by regulating the balance between the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax.