Effect of Mycoplasma bovis Infection on Mitochondrial Pathway Mediated Apoptosis in Bovine Macrophages
[Objective]The aim of this experiment was to investigate whether infection of bovine macrophages(BoMac)with Mycoplasma bovis isolate from Shihezi could affect cell proliferation activity and apoptosis through the mitochondrial apoptosis pathway.[Method]The growth curve of Mycoplasma bovis isolated from Shihezi was drawn by medium culture and color change unit(CCU)to determine the culture time.Using Mycoplasma bovis,which was at its highest level of vitality,to infect BoMac with different infection multiples(MOI)and infection time,Real-time quantitative PCR and Western blotting tests were used to detect the expression of core molecules Bax and Bcl-2 in the mitochondrial apoptosis pathway.The cell proliferation activity level was detected by CCK8 assay,and the cell apoptosis rate was measured by flow cytometry.[Result]The analysis of the growth curve showed that Mycoplasma bovis isolated from Shihezi was in the logarithmic growth phase from 12 to 54 h,and in the plateau phase from 54 to 84 h(with a titer of 109 CCU/mL).Therefore,the Mycoplasma bovis isolated from Shihezi cultured for 54 h was selected to infect the cells.The results of Real-time quantitative PCR and Western blotting were consistent,indicating that with the increase of infection time and MOI,the pro-apoptotic protein Bax gene and its protein showed an upward trend,while the expression of the anti-apoptotic protein Bcl-2 gene and its protein showed a downward trend.Among them,after 24 h of infection with Mycoplasma bovis isolated from Shihezi,the expression of the Bax gene was extremely significantly increased(P＜0.01),the expression of the Bcl-2 gene and Bcl-2/Bax were significantly decreased(P＜0.05).When the MOI was 1 000,Bax protein expression was extremely significantly increased(P＜0.01),Bcl-2 protein expression was significantly decreased(P＜0.05),and Bcl-2/Bax was extremely significantly decreased(P＜0.01).The CCK8 test results showed that with the increase of infection time and MOI,the activity of BoMac was extremely significantly decreased(P＜0.01),and the proliferation inhibition rate was 35％to 45％.The results of flow cytometry showed that the apoptosis rate of BoMac after infection was 25％to 45％.[Conclusion]The results of this experiment indicated that as the infection time and MOI increasing,the Mycoplasma bovis isolate from Shihezi could induce apoptosis of BoMac through the expression of mitochondrial apoptosis pathway markers Bax and Bcl-2,providing a theoretical basis for further elucidating the pathogenic mechanism of Mycoplasma bovis.
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