| [Objectives]1.Determine whether MCMV infection can affect the autophagy status of liver tissues through establishingBALB/c mouse MCMV disseminated infection model and detecting the expression levels of autophagy-related protein LC3-Ⅱ and p62 of liver tissues;2.Investigate the role of autophagy on liver injury during MCMV infection and the host’s antiviral immune response through detecting the severity of liver pathological damage,the expressions of inflammatory cytokines and MCMV viral replication under different autophagy interventions in vivo,expecting to provide some new experimental evidence for elucidating the mechanism of HCMV immunopathogenesis.[Methods]1.Establishment of BALB/c mouse MCMV disseminated infection model:4-week-old female SPF BALB/c mice were randomly divided into normal control group(Mock)and MCMV infection group(MCMV).Normal control group mice were injected intraperitoneally with 200μl DMEM medium/every mice;MCMV infection group mice were injected intraperitoneally with 200μl MCMV Smith strain 1.0×10~4PFU/every mice.Three mice were randomly sacrificed by dislocation of cervical vertebrae after ether anesthesia of each group on days 1,3,7,14,and 21 post infection.The livers and peripheral blood were aseptically collected;2.Establishment of mouse models of autophagy intervention:4-week-old female SPF BALB/c mice were randomly divided into 3-day group and 7-day group,and then each group was further respectively divided into 6 subgroups:(1)Normal control group(Mock),(2)MCMV infection group(MCMV),(3)Normal control+starvation group(mock+starvation,Mock+S),(4)MCMV infection+starvation group(MCMV infection+starvation,MCMV+S),(5)Normal control+chloroquine group(mock+chloroquine,Mock+CQ),(6)MCMV infection+chloroquine group(MCMV infection+chloroquine,Mock+CQ).Infection methods were the same as above.3-day group and 7-day group were respectively given autophagy intervention treatment at 24hours and 120 hours after modeling:starvation 48 hours before sampling to induce autophagy and chloroquine was injected intraperitoneally to inhibit autophagy.The dose of chloroquine was 100 mg/kg and dissolved in 100μl PBS at each time,three doses were given at 48 hours,24 hours,and 4 hours before sampling.Three mice of each group were sacrificed by dislocation of cervical vertebrae after ether anesthesia in each group on days3 and 7 post infection,the livers and peripheral blood were aseptically collected;3.Pathological injury of liver tissues:Liver tissues of mice in each experimental group were collected and the pathological injury was evaluated by HE staining,and the degree of liver pathological damage was scored according to Knodell liver histological activity index(HAI);4.Serum AST and ALT levels:Microplate methods were used to detect the expression levels of serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in order to evaluate liver function;5.Transcription levels of inflammatory cytokines in liver tissues:Extracted RNA from liver tissues of each experimental group and obtained cDNA by reverse transcription.Real-Time PCR was used to detect the transcription levels of inflammatory cytokines IL-1βand IFN-αin the livers;6.MCMV viral load in liver tissues:MCMV viral protein gB mRNA transcription level of livers in each experimental group was detected by Real-Time PCR to evaluate liver MCMV viral load;7.Autophagy in liver tissues:Dynamic changes of the expression levels of autophagy-related proteins LC3-Ⅱ,p62,PI3K,Akt,p-Akt,mTOR and p-mTOR in liver tissues of each experimental group were detected by Western Blot.[Results]1.Liver pathology and autophagy in MCMV disseminated infection model mice(1)Pathological changes of liver tissues:Compared with the Mock group,inflammatory cells infiltration were observed in the livers of MCMV group on day 1 post infection,and then the pathological damage aggravated gradually until day 7 when it reached the most severe state.This condition was improved on day 14,but the liver damage continue existed until day 21 post infection;the liver HAI scores were consistent with the trend of liver pathological damage.(2)Serum AST and ALT levels:The level of AST of MCMV group was increased from day 1 post infection,and reached its peak on day 7 post infection,returned to normal on day 14 post infection;the level of ALT of MCMV group was increased slightly later than AST,but it also reached peak on day 7 post infection and returned to normal on day 21 post infection.The levels of serum AST and ALT remained normal in Mock group.(3)The transcription levels of inflammatory cytokines IL-1βand IFN-α:The mRNA transcription levels of IL-1βand IFN-αin liver tissues of MCMV group were increased from day 1 post infection,and reached the highest on day 7 post infection,and then decreased.On day 14 post infection,the transcription level of IL-1βhad fallen to that of day 1 post infection,while the transcription level of IFN-αwas still significantly higher than that of day 1 post infection(P<0.01),both of them returned to normal levels on day 21 post infection.The transcription level of IL-1βwere significantly positively correlated with the degree of pathological damage(HAI score)in liver tissues(R=0.959,P<0.01).(4)Hepatic MCMV viral load:The transcription level of MCMV virus protein gB mRNA reached to peak in MCMV group on day 3 post infection,and then decreased.The transcription level of gB mRNA was not detected on day 21 post infection.(5)Detection of liver autophagy:Compared with Mock group,decreased expression of LC3-Ⅱ(P<0.05)and increased expression of p62(P<0.01)were observed on days 3 and 7 post infection.There was no statistical difference of the expression levels of LC3-Ⅱ and p62 on days 1,14,21 post infections.The results showed that liver autophagy was inhibited on day 3 and day 7 post infections.2.Effect of autophagy induction on liver injury during MCMV infection(1)Transcription of MCMV gB mRNA was increased:Compared with MCMV group,the transcription level of MCMV gB mRNA was increased in the liver of MCMV+S group on days 3 and 7 post infection(P<0.05).(2)The pathological damage of liver tissues was aggravated:The liver pathological damage was more severe and the HAI scores were increased in MCMV+S group compared with the corresponding MCMV group on days 3 and 7 post infection(P<0.01).(3)Serum AST and ALT levels were increased:On days 3 and 7 post infection,the levels of serum AST and ALT were increased in MCMV+S group than those of corresponding MCMV group(P<0.05).(4)Increased expression of IL-1βand decreased expression of IFN-αin liver tissues:On days 3 and 7 post infection,the expression of proinflammatory cytokine IL-1βwas increased in MCMV+S group than that of corresponding MCMV group(P<0.05).However,the antiviral cytokine IFN-αwas decreassed in MCMV+S group compared to that of MCMV group(P<0.05).3.Effect of autophagy inhibition on liver injury during MCMV infection(1)Transcription of MCMV gB mRNA was decreased:Compared with MCMV group,the transcription level of MCMV gB mRNA was decreased(P<0.05)in the liver of MCMV+CQ group on days 3 and 7 post infection.(2)The pathological damage of liver tissues was alleviated:Compared with the MCMV group,the liver pathological damage was alleviated and the HAI score was decreased(P<0.05)in MCMV+CQ group on day 3 post infection.However,there was no significant difference in liver pathological damage between these two groups on day 7 post infection.(3)Serum AST and ALT levels were decreased:serum AST and ALT levels in the MCMV+CQ group were decreased than those in MCMV group on day 3 post infection(P<0.05).On day 7 post infection,the ALT level in MCMV+CQ group was also lower than that in MCMV group,while there was no statistical difference of the AST level between these two groups.(4)Decreased expression of IL-1βand increased expression of IFN-αin liver tissues:On day 3 post infection,the expression level of IL-1βwas decreased in MCMV+CQ group than that of MCMV group(P<0.01),while the expression level of IFN-αwas increased than that of MCMV group(P<0.01).On day 7 post infection,there were no statistical difference of the expression levels of IL-1βand IFN-αbetween these two groups.4.PI3K/Akt/mTOR signaling pathway Changeed in liver tissues:The expression level of PI3K,the ratios of p-Akt/Akt and p-mTOR/mTOR in liver tissues on days 3 and 7 post infection:their expression levels were increased in MCMV group than those of Mock group(P<0.05);they were decreased in Mock+S group than those of Mock group(P<0.05),while they were increased in Mock+CQ group than those of Mock group(P<0.05);they were increased in MCMV+S group than those of Mock+S group(P<0.05);there was no statistical difference of their expression levels between MCMV+CQ group and Mock+CQ group and MCMV group,however they were still higher than those of Mock group(P<0.05).[Conclusion]1.MCMV infection induced excessive activation of pro-inflammatory cytokines and MCMV virus replication both participated in the pathological damage of liver in BALB/c mice with MCMV disseminated infection.MCMV replication played the dominant role in the early stage of acute MCMV infection,and then MCMV replication and overactivation of pro-inflammatory cytokines were coexistence,in the late stage inflammatory injury predominated.2.Autophagy was inhibited in the liver of BALB/c mice on days 3 to 7 post MCMV infection.This process might be related to the activation of the PI3K/Akt/mTOR signaling pathway,and it was the outcome of the interaction between host tissue cells and the infected virus.3.Autophagy participated in liver injury during MCMV infection through up-regulating the expression of pro-inflammatory cytokine IL-1βand promoting viral replication in liver tissues;autophagy inhibition could result in increased expression of IFN-α,decreased expression of IL-1βand viral replication,and therefore alleviated the severity of liver pathological damage during MCMV infection to a certain extent. |
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