Study On The Molecular Mechanism Of Brucella 16M By The ROS Pathways Mediated AIR Inhibition Of Cell Autophagy Regulation Inflammatory Corpuscle

Brucellosis is a zoonotic disease caused by the genus Brucella. It is widely popular in the world, especially in developing countries. Recently, the rapid increasing cases in human and animals have caused widespread concern around the world. Autophagy is closely related to the persistant infection of Brucella. It has been reported that the domain AIR of the membrane fusion proteins TECPR1 played an important role to the start of autophagy, However,the role of AIR played in the autophagy induced by Brucella was not clear. Reactive oxygen species(ROS) which was closely related to the processes of autophagy, inflammation and apoptosis. The relationship between ROS and autophagy, inflammation, and apoptosis induced by Brucella is not yet reported. Our study explored that whether inflammation, autophagy and apoptosis was related to AIR through the ROS pathway by Brucella infection, laying the foundation for the development of new drugs.Objective: To seperately construct the AIR domain in silence(I-A), over expressing(O-A), interference recovering(OA-IA). After the 16 M infection we tested:(1)Whether the process of inflammation, autophagy and apoptosis is induced through the ROS pathway;(2) The role of AIR domain to the inflammation, autophagy and apoptosis in macrophages;(3) The relationship between the AIR and ROS;(4)To further reveal the mechanism of Brucella to persistant infection which could provide the basis for treating brucellosis.Methods: To construct the AIR domain silence(I-A), over expressing(O-A), interference recovering(OA-IA). Selected cell lines of stable expression, and then infected the cell lines by 16M(with untreated group and NAC pretreatment group). After different time periods, it was detected the expression level of proteins related to inflammation, autophagy and apoptosis. The expression level of ROS and the distribution of mitochondrial in cells were detected by Laser Scanning Confocal Microscope(CLSM) and Transmission Electron Microscopy(TEM), Multifunctional Enzyme Standard Instrument, Real Time quantitative PCR, ELISA and Western blot.Results: ⑴ The I-A, O-A and OA-IA had been successfully constructed, and then the related cell lines had been built; ⑵ By time-dependant way,16 M could induce RAW264.7 cells to generate ROS and stimulate RAW264.7 cells to produce ROS; AIR interference produced more ROS and the generation of ROS was inhibited by the overexpression and reversion of AIR domain; I-A group and O-A group showed more abnormal accumulation of mitochondrial; The results of qRT-PCR suggested that ASC, Caspase-1 and NLRP3 were differently changed in mRNA level; Compared with the control group, the release of inflammatory cytokines IL-18 of I-A group significantly raised at 12 h(P<0.05), and the release of inflammatory cytokines IL-1β and IL-18 of O-A group significantly raised at 12 h(P<0.05). NAC pretreatment cells could significantly reduce ROS by 16 M and enhanced the expression of NLRP3/AIM2 inflammation complex; ⑶The results of confocal laser scanning microscopy showed that compared with control group, the number of autophagic bodies containing LC3 in RAW264.7 infected by 16 M was significantly accumulated. Abnormal accumulation of mitochondrial was showed by qRT-PCR in I-A, O-A, OA-IA groups. We found that the expression of LC3B/A reached the highest level at 6 h which is significantly different in AIR gene deleted group. Compared with the control group, I-A and O-A group inhibited the p62 expression at 6 h. Then we observed the inhibitory effects of NAC, compared with control group, the expression of p62 reached the highest and difference significantly in AIR gene deleted group(P<0.05). By western blot, the results suggested that the expression of p62 protein were significantly lower than the control group. After NAC pretreatment, we tested that the expression of p62 protein were significantly higher than the control group; ⑷ After the 16 M infection, the number of cells with FITC green and PI red dye were increased. After NAC pretreatment, 16M-infected cells with FITC green dye and PI dyed red were less. Under the white light, there were visible changes in cell morphology. 16 M could induce the mRNA expressions of Bax, Bcl-2 in RAW264.7 cells. Moreover, flow cytometry testing showed that 16M-infected cell apoptosis rate were significantly increased(P < 0.01), along with the extension of time, the apoptosis rate was proportional to the growth. After NAC pretreatment, the level of 16M-induced caspase-3 protein induced by 16 M in RAW264.7 cells significantly decreased.Conclusion: ⑴ Those results indicated that after AIR gene deletion and over expression, the release amount of ROS changed, mitochondrial clustered abnormally, and AIR was closely related to the activation of inflammasomes and induction of inflammatory reactions. By ROS pathway, 16 M activated NLRP3/AIM2 inflammation complex, and induced the secretion of IL-1β and IL-18; ⑵ AIR silence can cause abnormal mitochondria gathering and the change of autophagy related genes and increase the activity of autophagy; after NAC pretreatment, at 6 h, I-A group inhibits cell autophagy; ⑶ It could be mediated apoptosis via ROS signaling pathway by 16 M infected RAW264.7 cells; ⑷ 16M-induced ROS can cause autophagy, inflammation, and apoptosis in cells. AIR cans inhibite the mature of autophagosome. Autophagy inhibits the inflammatory reactions.