Effect Of Drug-Resistant Mycobacterium Tuberculosis On Four Cytokines And Preliminary Study On Inhibition Of NF-κB Pathway

Tuberculosis(TB)is a chronic,consumptive and zoonotic infectious disease caused by Mycobacterium tuberculosis.Every year,about 10 million cases of tuberculosis are infected worldwide,and about 3 million people die of tuberculosis.Nowadays,the emergence of tuberculosis,especially the emergence of tuberculosis resistant bacteria,not only poses a great threat to human health,but also seriously threatens the development of aquaculture.Anti-tuberculosis chemical drugs have been used for clinical treatment of tuberculosis for several decades,and the unreasonable use of drugs and the prevalence of AIDS have led to the emergence of drug-resistant tuberculosis,making tuberculosis more difficult to treat.Therefore,this study used isonicotinic acid hydrazide(INH)to induce the standard strain H37 Rv with different gradients,and preliminarily studied the effects of drug-resistant strains on major cytokines related to infection and immune escape after interaction with macrophages.The minimum inhibitory concentration(MIC)value of the isoniazid drug was determined for the standard strain H37 Rv and the wild strain.The MIC value of standard strain was set as the standard,then the induction of H37 Rv was carried out with 1/8MIC,1/2MIC,1/4MIC,MIC,2MIC and 4MIC respectively.The MIC value of the induced strain was determined and was compared with the standard strain H37 Rv.The MIC value of the strain after induction was larger than that of H37 Rv,and the larger the drug concentration of the induced strain,the larger the MIC.RAW264.7 macrophages were infected with standard strain H37 Rv,wild strain and strains induced by different gradients.Cell proliferation was detected by CCK-8 method.The expression and transcription levels of cytokines IL-6,IL-10,IL-1β and TNF-α were detected by ELISA and quantitative fluorescence PCR.The results showed that cell proliferation increased in the first 8h and reached the maximum at 8h,and decreased cell proliferation after 8h.The secretion and transcription levels of cytokines increased after infection by different virulence strains,and the expression levels and transcription levels of MIC,2MIC and 4MIC induced strains were higher than other induced strains.The infection of the strain can cause the activation or inhibition of MAPK and other pathways.The content of p38,JNK,ERK and phosphorylated protein in the MAPK pathway was detected by western blot.The results showed that the protein expression level of the standard strain H37 Rv was higher than that of the induced strain and the wild strain,and the protein expression of the low-concentration drug induced strain was higher than that of the high-concentration drug induced strain.After treatment of macrophages with nuclear factor kappa-B(NF-κB)activation inhibitor,the secretion of NO and the change of NF-κB protein content of macrophages at different time points were detected by Griess method.The results showed that NO secretion and NF-κB protein content increased as a whole,and then decreased with time.The expression level was the highest at 8h and 4h without inhibitor,and the time at which the highest expression level was delayed after the addition of inhibitor.The NO secretion and NF-κB protein content of the induced strains were lower than those of H37 Rv strains,and the higher the induction gradient,the lower the expression level.The expression level of wild strain and 4MIC-induced strains were similar.