Preliminary Study On The Effect Of Mycobacterium Fortuitum On THP-1 Cell Apoptosis

Nontuberculous mycobacteria(NTM)are free-living saprophytic organisms that are not members of the Mycobacterium tuberculosis complex(MTC),also known as environmental mycobacteria.NTM is an opportunistic pathogen and has a variety of variable pathogenic potentials which is widely distributed in the natural environment.To date,more than 170 NTM species have been recognized,approximately one-third have been associated with diseases in humans.After infection with Mycobacterium tuberculosis(MTB),macrophages as a major immune cell,through a variety of ways to kill MTB,one of the important ways for macrophage is the apoptosis.MTB can also avoid the remove of the immune system through the intervention and control of the macrophage apoptosis.Recent studies have shown that the clinical manifestations and pathogenesis of NTM are very similar to MTB.In view of the relatively few reports on NTM research at home and abroad,this paper mainly refers to some MTB related researches.M.fortuitum is a rapid growth type of NTM,which is commonly isolated from the soil and water,and often causes trauma and soft tissue infection.It can lead to disease when the immunity is low.To investigate the effect of M.fortuitum on macrophage apoptosis,the following experimental studies were carried out:Firstly,we had recultivated and identified the M.fortuitum which was isolated from the mesenteric lymph nodes of livestock,and then established the model of THP-1cells infected with the bacterium.The apoptosis of THP-1 cells was preliminarily observed by Hoechst 33258 staining and transmission electron microscopy.The results showed that the apoptosis of THP-1 cells could be induced by the M.fortuitum,especially after 6 h to 24 hours after infection.The mRNA and protein expression levels of caspase-3,caspase-8 and bcl-2 were detected by fluorescence quantitative PCR and Western-blot.The results showed that the expression level of caspase-3 and caspase-8 was increased at the level of mRNA transcription and protein level with the prolongation of the time,and it was time-dependent.The difference was caspase-3 mRNA transcription level was the highest at 12 h after infection,and its protein level reached the highest at 24 h after infection.This result may be because the expression of protein level has a certain time delay compared with mRNA transcription level;the expression level of Bcl-2 was different from that of caspase-3 and caspase-8.The mRNA transcription level of Bcl-2 was higher at the initial stage of infection,and began to decrease gradually from 4 h,reached the lowest at 12 h,especially the decrease of the isolated strain,and the difference was significant.The expression of Bcl-2 protein was lower in the THP-1 cells than that in the control group.These data suggested M.fortuitum infection can cause THP-1 cell apoptosis with the increased expression of caspase-3 and caspase-8 and inhibition of the expression of anti-apoptotic gene,Bcl-2.The expression of TNF-?,IL-6 and IL-10 in THP-1 cells after infection with Mycobacterium infection was detected by ELISA.The results showed that the expression of TNF-? increased with the prolongation of the time,and reached the highest at the 12 h,showing a certain time-dependent;the IL-6 was highly expressed at 0h-4h and then decreased;the expression level of IL-10 was higher at the initial stage of infection,and there was a significant decrease from 6 h.This result indicates that M.fortuitum infection can induce the secretion of pro-apoptotic cytokine TNF-? and contribute to the process of intracellular apoptosis,while the decrease of IL-6 and IL-10 expression may be that mycobacterium could not resist macrophage clearance.The above results were used to investigate the effect of M.fortuitum infection on THP-1 cell apoptosis.The results showed that through up-regulating the expression of caspase-3 and caspase-8 in THP-1 cells,inhibiting the expression of anti-apoptotic gene Bcl-2,and regulate the secretion of apoptosis-related cytokines: TNF-?,IL-6 and IL-10,the M.fortuitum infection can ultimately cause the apoptosis of macrophages.These results will provide scientific data for revealing the effect of NTM on macrophage apoptosis and provide a theoretical basis for further study of the immune mechanism of NTM disease.