| 【Objectives】To study the effect of p202 on the expression of AIM2 inflammasome signaling pathway related molecules and downstream signaling molecules in MCMV-infected J774 A.1 cells to explore its effect on MCMV replication and whether it participates in the molecular mechanism of virus evading host immune response.【Methods】1.p202 transcription and protein expression after MCMV infection: MCMV infects J774 A.1 cells and RAW264.7 cells respectively,the multiplicity of infection(MOI)is 1,and a mock infection control group(Mock group)is set up at the same time.Establishment of disseminated MCMV infection model in BALB/c mice: 40 4-week-old SPF female BALB/c mice were randomly divided into MCMV infection group(MCMV group)and normal control group(Mock group);Mock group mice were intraperitoneal injected with DMEM medium/every mouce(200μl);MCMV infection group mice were intraperitoneally injected with the MCMV Smith strain,with an injection volume of 1.0×104 PFU/every mouse(200μl).After ether anesthesia,three mice were sacrificed randomly by cervical dislocation on days 1,3,7,and 14 post infection respectively,and bilateral salivary gland tissues were collected;2.si RNA transfection of J774 A.1 cells: J774 A.1 cells were transfected with p202 specific si RNA for 24 hours to obtain p202-silenced J774 A.1 macrophages(S+),and at the same time transfected with irrelevant si RNA(Stealth negative control LO GC)to obtain simulated silent control cells(S-);S+ and S-cells were respectively infected with MCMV Simth strain(MOI=1),namely S+/V+ group and S-/V+ Group,and also set up a simulated infection control group,namely S+/V-group and S-/V-group;3.Transcription levels and protein expression of AIM2 inflammasomes after MCMV infection of p202 pre-silenced J774 A.1 cells: At 6h,12 h and 24 h post infection/simulated infection,four sets of cell samples were collected,total RNA was extracted,and the m RNA expression levels of p202,AIM2,caspase-1 and IL-1β were detected by RT-q PCR method;the total protein of four groups of cells was collected after infection/simulated infection for 6h,12 h and 24 h,and Western Blot detected the protein expression levels of p202,AIM2,pro-caspase-1,p20(activated caspase-1 fragment),pro-IL-1β and IL-1β;4.Immunofluorescence method was used to detect the distribution of p202 and AIM2 protein expression in J774 A.1 cells after MCMV infection in p202 silent/simulated silent state;5.The effect of p202 pre-silencing on EA protein expression in infected cells: In the state of silence/simulated silence of p202 gene,the expression of MCMV EA in each group of cells was detected at 6h,12 h and 24 h post infection by immunofluorescence method.【Results】1.The transcription level and protein expression of p202 increased after MCMV infection(1)After MCMV infects J774 A.1 cells and RAW264.7 cells,the expression levels of p202 m RNA and protein in the cells were significantly increased compared with the mock infection group.(2)After MCMV infection of BALB/c mice,compared with the mock infection group,the p202 m RNA and protein expression levels in the salivary gland tissues of the infected group were significantly increased,and they increased at 1 day after infection,and then gradually increased to 14 days reaching the highest.2.The effect of p202 gene pre-silencing on the m RNA transcription level of AIM2 inflammasome and downstream molecules induced by MCMV(1)p202 m RNA expression: In S-V+ cells,the p202 m RNA expression level increased significantly at 6h post MCMV infection,and at 12 h and 24 h post infection,though its expression level was lower than that at 6h,it was still higher than that of mock-infected S-/V-group;while the p202 m RNA expression level of the p202-specific si RNA silencing group(S+V+)was significantly lower than the corresponding simulated silencing group(S-V+)at all time points;(2)AIM2 m RNA expression: At 6h,12 h and 24 h post infection,the AIM2 m RNA expression level of the p202-specific si RNA silencing group(S+V+)was higher than the corresponding simulated silencing group(S-V+);(3)m RNA expression of caspase-1 and IL-1β: At 6h,12 h and 24 h post infection,the expression of caspase-1 and IL-1β m RNA in S+V+ group cells was higher than the corresponding S-V+ group.3.The effect of p202 pre-silencing on AIM2 inflammasome pathway and downstream protein expression in MCMV-infected cells(1)The expression level of p202 protein: Regardless of whether p202 is silenced or not,the expression of p202 is almost undetectable in J774 A.1 cells when they are not infected with MCMV.The expression of p202 protein increased significantly after virus infection,but the p202 protein content of the p202-specific si RNA silencing group(S+V+)was significantly lower than that of the simulated silencing group(S-V+)(P<0.05);(2)AIM2 protein expression level: AIM2 protein is almost not expressed in uninfected J774 A.1 cells.Regardless of whether p202 was silenced or not,the expression of AIM2 protein increased after infection,and it increased most significantly at 6h post infection,and then gradually decreased.In the case of p202 silence,the AIM2 protein content of the MCMV-infected S+V+ group was higher than the corresponding simulated silence S-V+ group at 6h,12 h and 24 h post infection(P<0.05).Under simulated infection conditions,the expression of AIM2 protein when p202 was silenced was slightly higher than that of the simulated silence group,but there was no statistical difference;(3)Distribution of p202 and AIM2 protein expression: In the cytoplasm of J774 A.1 cells not infected with MCMV,the expression of AIM2 protein in the p202 silenced group(S+V-)was slightly higher than that of the simulated silence group(S-V-).After MCMV infection,the expression of p202 and AIM2 protein increased significantly,and both of them were mainly located in the cytoplasm.Compared with the S-V+ group,the AIM2 protein expression in the cells of the S+V+ group was significantly increased at 6h post infection(P<0.05),and the AIM2 protein distributed both in the cytoplasm and nucleus,while the fluorescence intensity of the cytoplasm was higher than that in the nucleus.At 12 h and 24 h post infection,the protein expression trend of p202 and AIM2 protein was consistent with that at 6h(12h:P<0.05,24h:P<0.05);(4)The expression of pro-caspase-1 and activated caspase-1: In the unsilenced state of p202,the expression of pro-caspase-1 after MCMV infection was slightly higher than that of the mock infection group,but there was no statistical difference(P>0.05).In the S-V+ group of cells,caspase-1 was significantly activated at 6h post infection.The activation at 12 h and 24 h was less than that of 6h post infection,but it was still higher than that in the mock infection group.The activation of caspase-1 in the S+V+ group was higher than that in the corresponding S-V+ group at 6h,12 h and 24 h post infection(6h:P<0.01,12h:P<0.05,24h:P<0.05);(5)The expression of pro-IL-1β and mature IL-1β: At 6h,12 h and 24 h post infection,pro-IL-1β in the S+V+ group was higher than the corresponding S-V+ group(all P<0.05).Consistent with the changes in the expression of pro-IL-1β,mature IL-1β increased at 6h post MCMV infection and continued to 24 h,and mature IL-1β in S+V+ group was higher than the corresponding S-V+ group at 6h,12 h and 24 h post infection(6h:P<0.05,12h:P<0.01,24h:P<0.05).4.The effect of p202 pre-silencing on EA protein expression in infected cellsMCMV EA has been expressed in J774 A.1 cells at 6h post infection,and its expression increased significantly at 12 h and 24 h.In addition,at 6h and 12 h post infection,the expression of MCMV EA in the p202-specific si RNA silencing group(S+V+)J774A.1 cells was significantly lower than the corresponding simulated silencing(S-V+)(P<0.01).There was no significant difference in the fluorescence intensity of MCMV EA in J774 A.1 cells between the two groups at 24 h post infection(P>0.05).【Conclusion】1.After infection with MCMV,it can significantly promote the transcription level and protein expression of p202;2.p202 can inhibit the AIM2 inflammasome pathway to a certain extent;3.p202 promotes MCMV virus replication by inhibiting the AIM2 inflammasome pathway,and may be involved in the immune escape mechanism of the virus. |
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