Regulatory Effect Of IL-1? On BCG-induced Apoptosis Of Mouse Macrophage RAW264.7

Background and objectives:Tuberculosis(TB)is caused by Mycobacterium tuberculosis(MTB).A chronic zoonotic infectious disease poses a great threat to the health and safety of human society.As the main host cell and target cell of MTB,macrophage can kill free pathogens in cytoplasm through apoptosis,effectively inhibit the proliferation and proliferation of MTB in host cells;and MTB can escape macrophages through immune evasion mechanism.The killing,while proliferating in the cell.However,apoptosis is an extremely complex biological process,and its regulatory mechanisms are involved in numerous regulatory factors and cytokines.As an important pro-inflammatory cytokine,IL-1? plays a key role in different immune responses.In recent years,it has been found to be involved in the regulation of apoptosis in a variety of diseases,including neurodegenerative and infectious diseases.Whether IL-1?is involved in the regulation of M.tuberculosis infection-induced apoptosis of macrophages is not clear.Therefore,this study was to investigate the role of IL-1? in the regulation of macrophage apoptosis induced by M.tuberculosis infection.Methods:In this study,mouse macrophage RAW264.7 was infected by BCG strain BCG,and IL-1? small interfering RNA(si-IL-1?)was designed to inhibit IL-1? expression,and MAPK signaling pathway was blocked by MAPK signaling pathway inhibitor SB203580.To study the relationship between the immunoregulatory function of IL-1? and the MAPK signaling pathway.Using RealTime-PCR,enzyme-linked immunosorbent assay,Western blot and TUNEL staining,transmission electron microscopy and flow cytometry,IL-1? was studied from nucleic acid level,cytokine level,protein level and cell morphology.Regulatory effect of M.tuberculosis infection on apoptosis of mouse macrophages.Results:(1)Detection of nucleic acid and protein levels revealed that high levels of IL-1? were induced at different times and different infections of BCG infection,and the expression of intracellular IL-1? was observed when BCG infection time was 24 h and multiplicity of infection was 10.The highest level was significantly higher than the control group(p<0.01).Subsequently,IL-1? interfering RNA interfered with intracellular IL-1(3,and the results showed that IL-1? small interfering RNA alone or combined with BCG infection group can significantly reduce IL-1? expression(p<0.01),successfully constructed.IL-1?interferes with the vector.(2)Infecting RAW264.7 cells with BCG and blocking the expression of IL-1?,the cell survival rate was up-regulated and the apoptosis rate was also up-regulated.Morphological examination by TUNEL staining and transmission electron microscopy showed that IL-1? knockdown significantly increased BCG-induced apoptosis.It also inhibited the levels of ROS and NO,and finally changed the mitochondrial membrane potential,and down-regulated the expression of Bcl-2 family-promoting proteins Bax and Bad,and affected the efflux of cytochrome C to regulate apoptosis of mitochondrial pathway.The expression of Caspase3 and PARP in the expression of apoptosis was detected at the level of nucleic acid and protein.The results showed that the knockdown of IL-1? down-regulated its expression.This result indicates that IL-1? may be involved in the regulation of BCG-induced apoptosis.(3)After BCG infection of RAW264.7 cells,activation of the Toll-like signaling pathway releases active IL-1?.Down-regulation of IL-1? inhibits the activation of MAPK signaling pathway,down-regulates the expression of p38,p44 and JNK proteins.The MAPK signaling pathway is blocked by MAPK specific inhibitors,and the expression of Bax and Bad is down-regulated and Bcl-2 protein is down-regulated.Level up.Conclusion:BCG infection significantly increased the expression of IL-1? in a time-and concentration-dependent manner,with the most significant up-regulation at 24 h BCG infection and 10 at the multiplicity of infection.Subsequently,IL-1? was used to study the constructed IL-1? knockdown combined with BCG infection model,up-regulating the apoptotic rate,up-regulating the expression of ROS,NO and mitochondrial membrane potential,and affecting the expression of apoptosis-related proteins;1? expression can inhibit the apoptosis of mitochondrial pathway and inhibit the occurrence of apoptosis by affecting the activation of TLR-MAPK signaling pathway.