Effects Of Mycobacterium Tuberculosis ESTA-6 And CFP-10 On NLRP3 Inflammasome Formation In Macrophages And Related Mechanisms

Tuberculosis(TB)is one of the bacterial infectious diseases that endanger humans.Its pathogen is Mycobacterium tuberculosis(MTB).MTB is phagocytized by alveolar cells after it enters the lungs from the respiratory tract.The first line of defense,as recognized by macrophages,can inhibit fusion of phagolysosomes,interfere with cell signal transduction,inhibit ROS(Reactive Oxygen Species)and RNS(Reactive Nitrogen Species)toxicity,inhibit autophagy,etc.Resist host cell immune killing.At present,the use of BCG to prevent tuberculosis has gradually shown many drawbacks.Therefore,this study used RAW264.7 cells as the research object,and studied the TB protein ESAT-6 and CFP-10 to cause corresponding infection when infected macrophages.Inflammatory response,qRT-PCR method was used to study the expression levels of related inflammatory factors and apoptosis-related factors,Western blot was used to detect the expression of inflammatory components in NLRP3,and flow cytometry was used to determine the apoptosis rate.Apoptosis-related enzyme activity,a preliminary exploration of the NLRP3 inflammasome activation mechanism,the results show that: successfully transfected pEGFP-ESAT-6,pEGFP-CFP-10 vector,when the ratio of the carrier and transfection reagent is 1:4,After transfection,the medium was replaced with complete medium at 6 h,and the transfection efficiency was the best.After infecting macrophages with ESAT-6 and CFP-10,the inflammation-related factors caspase-1,IL-1?,IL-18,TNF-? The expression levels of IFN-? were up-regulated in different degrees;the expression levels of NLRP3,ASC,IL-1?,and caspase-1 were up-regulated at the protein level,demonstrating the production of NLRP3 inflammasome;flow cytometry,ESAT-6 and CFP-10 infected macrophages Compared with the control group,the apoptosis rate increased.At the same time,the expression of apoptosis-related factors caspase-3,caspase-7,caspase-8,and caspase-9 was up-regulated;after macrophages were infected by ESAT-6 and CFP-10,the expression of burn-related factors GSDMD and caspase-11 was increased.,The cells are in a state of persecution.Therefore,we preliminary confirmed that ESAT-6 and CFP-10 can induce macrophage inflammatory reactions,produce NLRP3 inflammasome,and promote macrophages apoptosis and apoptosis.