Study On Mechanism Of Apoptosis Regulation Of Megalophage Infected With BCG By The Effect Of VDAC1

Tuberculosis is a chronic,wasting zoonotic infectious disease caused by mycobacterium tuberculosis and mycobacterium bovis.It is a great threat to public health and animal husbandry.Macrophages are the main target cells during mycobacterium tuberculosis infection.After infection,mcobacterium tuberculosis will face a variety of innate and adaptive immune cells,among which activating apoptosis through various ways is one of the active defense mechanisms of the host against mycobacterium tuberculosis infection.However,mycobacterium tuberculosis can inhibit cell apoptosis through its own virulence factors,inducing anti-apoptotic factors and regulating host gene expression,so as to make itself survive better.Recent studies have shown that VDAC 1 can regulate apoptosis through oligomerization in other disease models.VDAC1 induces apoptosis by mediating the release of Cyt C,AIF,Smac/DIABLO and endonuclease G,which plays an important role in pathogen infection and treatment.However,in the process of mycobacterium tuberculosis infection,whether the host cells regulate apoptosis through the participation of VDAC1 and the mechanism of action remain unclear.Therefore,it is of great significance to explore the regulatory role of VDAC1 in macrophage apoptosis during Mtb infection.In this study,the mouse macrophage RAW264.7 was infected by BCG strain,and the expression of VDAC1 was inhibited by designing small interfering RNA(Si-VDAC1),and then realtime-PCR,western blot,immunofluorescence and flow cytometry were used.To investigate whether VDAC1 is involved in the regulation of apoptosis in mouse macrophage RAW264.7 infected by BCG,and to study the mechanism of VDAC1 involved in the regulation of apoptosis in mouse macrophage RAW264.7 infected by BCG by blocking the oligomerization of VDAC1 inhibitor VBIT-4.The results are as follows:(1)By nucleic acid and protein level detection,it was found that the mRNA expression of VDAC1 was different at different times of BCG infection,and the time of BCG infection was most significant after 12 h(p<0.001),and the intracellular protein expression level of VDAC1 was the highest,which was significantly up-regulated compared with the control group(p<0.001).(2)The design and synthesis of VDAC1 small interfering RNA successfully reduced the expression of VDAC1,combined with BCG infection,can still significantly down-regulate the expression of VDAC1 in BCG-infected macrophages(p<0.001).(3)In the process of BCG infection,VDAC1 knockdown significantly down-regulated the expression of macrophage apoptosis-related factors Caspase3,Caspase9,PARP1 and intracellular Cyt C(p<0.001),and significantly reduced the apoptosis rate of macrophages and alleviated the survival rate of macrophages(p<0.001);Further studies showed that the mitochondrial membrane potential of VDAC1 was significantly reduced by knockdown(p<0.001).(4)During macrophage infection,BCG can up-regulate ROS accumulation in macrophages(p<0.001),but this phenomenon was alleviated by knocking out VDAC1(p<0.001).(5)After treatment with VDAC1 oligomer inhibitor VBIT-4,compared with BCG treatment group,Vbit-4 significantly inhibited the formation of VDAC1 oligomers and Cleaved Caspase 3,Cleaved PARP1,Cleaved Caspase 9 cleavages(p<0.001).As a conclusion,we found that BCG infection can induce apoptosis of macrophages and lead to up-regulation of VDAC1 expression.Knockdown of VDAC1 can reduce the expression of apoptosis-related genes and proteins,and reduce the apoptosis rate and accumulation of intracellular ROS content,and VBIT-4 can reduce vDAC1-mediated apoptosis by inhibiting the formation of VDAC1 oligomer.These results suggest that VDAC1 participates in the regulation of apoptosis of RAW264.7 macrophages infected with BCG through oligomerization.