Effects Of Mtb H37Ra Infection On Mitochondrial Function And Metabolism Of Macrophages

Tuberculosis(TB)is a chronic immune disease caused by Mycobacterium tuberculosis(Mtb)infection.So far,it is still the single infectious disease with the highest mortality rate worldwide.Macrophages are the natural host cells of Mtb H37 Ra.Mitochondria are not only the energy power plant of cells,but also provide multiple signal transduction platforms for the functioning of cells.Maintaining normal mitochondrial function is crucial for combating Mtb H37 Ra infection.As a result,mitochondria have become targets for various pathogenic attacks,including Mtb H37 Ra.Changes in mitochondrial metabolites can alter mitochondrial signal transduction under pathogenic stress conditions,thereby affecting the anti Mtb H37 Ra immune response.However,there are currently no studies on the regulation of mitochondrial metabolites by Mtb to affect mitochondrial function.Therefore,this study uses LC-MS to analyze mitochondrial metabolites,identify key metabolic molecules regulated by Mtb H37 Ra,and predict,analyze,and preliminarily validate relevant signal pathways.In order to deeply understand the pathogenesis of TB and provide ideas and targets for the development of anti TB drugs.1.Mtb H37 Ra infection alters mitochondrial morphology of macrophagesTo study the effect of Mtb H37 Ra infection on the mitochondria of macrophages,we used macrophages differentiated from THP-1 monocytes induced by PMA to infect Mtb H37 Ra,and observed the morphology of mitochondria of macrophages under transmission electron microscopy.The results showed that infection with Mtb H37 Ra caused the mitochondria of macrophages to change from linear to punctuate.2.Infection with Mtb H37 Ra alters mitochondrial membrane potential of macrophagesMitochondrial membrane potential(MMP)is an important indicator for mitochondria to perform normal physiological functions.Fluorescence microscope observation showed that infection with Mtb H37 Ra resulted in a decrease in mitochondrial membrane potential of macrophages.3.Mtb H37 Ra infection promotes mitochondrial ROS production in macrophagesTo investigate the effect of Mtb H37 Ra infection on the production of reactive oxygen species(ROS)in macrophages’ mitochondria,we used a reactive oxygen species(ROS)specific probe DCFH-DA to detect intracellular ROS.The results showed that infection with Mtb H37 Ra increased ROS production.4.Mtb H37 Ra infection increases mitochondrial ATP production in macrophagesATP production is one of the important functions of mitochondria.To investigate whether infection with Mtb H37 Ra affects the production of ATP by macrophages’ mitochondria,we used ATP probe dual luciferase assay to detect intracellular ATP.The results showed that infection with Mtb H37 Ra can increase ATP production.5.Mtb H37 Ra infection increases mitochondrial MPTP opening in macrophagesTo investigate the effect of Mtb H37 Ra infection on the mitochondrial membrane permeability transition pore(MPTP)of macrophages,we used the MPTP specific probe,Calcein AM,to detect the opening and closing of MPTP on the mitochondrial membrane of macrophages.The results showed that infection with Mtb H37 Ra could increase the degree of MPTP opening.6.Mtb H37 Ra infection alters mitochondrial energy metabolism in macrophagesTo study the effect of Mtb H37 Ra infection on mitochondrial energy metabolism of macrophages,we used a cell energy metabolism analysis experiment to detect changes in mitochondrial pressure(i.e.oxygen consumption)and energy metabolism.The results showed that Mtb H37 Ra infection increased the rate of oxygen consumption by macrophages.7.Mtb H37 Ra infection regulates changes in multiple metabolites of macrophages’ mitochondriaIn order to study the effect of Mtb H37 Ra infection on mitochondrial metabolites of macrophages,we isolated mitochondria from macrophages and used LC-MS to conduct metabonomic analysis of mitochondria.We found differential metabolites between the Control group and the Mtb H37 Ra infected group.We screened and identified a total of 1008 differential metabolites from macrophages,excluding 156 exogenous metabolites,and identified 100 specific endogenous differential metabolites,Among them,53 differential metabolites were upregulated and 47 differential metabolites were downregulated.Compared with the control group,the first 18 different metabolites in the treatment group increased in content,including:cholic acid,L-glutamine,stearic acid,norcholecalciferol,valine,arachidonic tyrosine,Lyso PC(16:0),PC(18:1(9Z)/18:1(9Z)),choline phosphate,PE(20:1(11Z)/15:0),and other compounds;Compared with the Control group,the relative content of the treatment group decreased by 14 different metabolites,including compounds such as PC(15:0/16:1(9Z)),N-adenosine tyrosine,L-glutamine,L-tyrosine,Lyso PC(18:3(9Z,12 Z,15Z)),1-palmitoylglycerolcholine,indole,dihydroneopterin phosphate,N-acetyl-L-histidine,Leu Val Trp Met,and so on.8.Prediction and validation of metabolic pathways regulated by Mtb H37 Ra infectionThe prediction of metabolic pathways for mitochondrial differential metabolites in macrophages showed that a total of 27 differential metabolic pathways were enriched,among which the most important type of pathway was metabolic pathway,with a total of 21 pathways.In addition,there were 1 pathway related to organic systems,3 pathways related to human diseases,and 2 pathways related to cellular processes.Metabolic metabolic pathways account for the highest proportion of all affected metabolic pathways,indicating that Mtb H37 Ra infected macrophages regulate mitochondrial metabolism.Analyze the mitochondrial metabolic pathway of macrophages,select a metabolic pathway of interest,and validate it using biochemical and molecular biology techniques.Finally,select the metabolic pathway ko04750(TRP inflammatory mediators regulate metabolic pathway)for validation.To investigate whether Mtb H37 Ra infection regulates the mechanism of TRP metabolic pathway expression,q PCR experiments first demonstrated that Mtb H37 Ra infection causes changes in the expression of TRP genes.It was found that there are differences in TRP expression.Some genes are down-regulated at the initial stage of stimulation,while some genes are up-regulated.At 24 hours,the expression of TRP channel genes is mostly up-regulated,indicating that TRP ion channels are opened 24 hours after infection,To further observe the effect of Mtb H37 Ra infection on the regulation of intracellular calcium concentration,we used a specific fluorescence probe for intracellular calcium ions,Fluor-4 AM,to detect the concentration of calcium ions.Fluorescence microscopy observation showed that the intracellular calcium ionomer concentration increased 24 hours after infection,indicating that the calcium ion channels on the cell membrane were opened after infection.