The Mechanism Of MFN2 Regulating Apoptosis Mediated By Endoplasmic Reticulum Stress In Brucella Infection

Brucellosis is a kind of zoonosis caused by Brucella infection.A detailed understanding of its pathogenic mechanism is the premise of prevention and treatment of the disease.The apoptosis mediated by Brucella is closely related to its intracellular activity.In recent years,the pathway of apoptosis induced by endoplasmic reticulum stress has increasingly come into the view of scholars.Mitofusin 2(MFN2)is located in the outer membrane of mitochondria,which is closely related to apoptosis and endoplasmic reticulum stress.It is also potentially associated with virus and pathogen infection.However,what role it plays in Brucella infection is still unknown.Objective:(1)Objective to explore the effect of Brucella on endoplasmic reticulum stressinduced macrophage apoptosis;(2)To establish MFN2 gene interference and overexpression transient transfection cell model;(3)To explore whether MFN2 is involved in the regulation of endoplasmic reticulum stress-induced macrophage apoptosis during Brucella infection.Methods:(1)In this study,RAW264.7 cells were infected with Brucella A19 in vitro.The activation of endoplasmic reticulum stress markers(GRP78,CHOP)and apoptosis related genes(BAX,BCL-2)were detected by q RT-PCR,Western blot and flow cytometry,and the expression of MFN2 was detected.(2)The interference sequence and recombinant plasmid were transfected into RAW264.7 cells with the help of transfection medium,and the interference and overexpression efficiency of MFN2 were detected by q RT-PCR and Western blot.(3)In the MFN2interference/overexpression state,Brucella was used to infect RAW264.7 cells,The expression of endoplasmic reticulum stress marker molecules and apoptosis related genes were detected by blot and flow cytometry,and the apoptosis rate was detected.Results:(1)Brucella could increase the expression levels of GRP78,CHOP and BAX,but decrease the expression of BCL-2 and increase the apoptosis rate;A19 could inhibit the expression of MFN2.(2)si MFN2-1661 was selected from the three pre selected si RNA sequences was the best interference sequence,and the efficiency was 68%.The overexpression efficiency of p CDNA3.1-EGFP-MFN2 was about 8 times.(3)MFN2 interference increased the expression of GRP78 and BAX induced by Brucella,increased the apoptosis rate and decreased the number of viable cells;MFN2overexpression inhibited the expression of GRP78 and BAX induced by Brucella,decreased the apoptosis rate and increased the number of viable cells.Conclusion:(1)This study confirmed that Brucella could induce endoplasmic reticulum stress and induce macrophage apoptosis.(2)MFN2 gene was successfully interfered and overexpressed by RNA interference and gene recombination.(3)It was found that MFN2 deletion could promote the ability of Brucella to induce endoplasmic reticulum stress and apoptosis,and inhibit the intracellular survival of Brucella to a certain extent;while MFN2 overexpression could inhibit the ability of Brucella to induce endoplasmic reticulum stress and apoptosis,and promote the intracellular survival of Brucella to a certain extent.