Mechanistic Study Of The Role Of PI3K/Akt Signaling Pathway In AMDV-induced Apoptosis Of CrFK Cells

Aleμtian mink disease virus(AMDV)is the caμsative agent of Aleutian mink disease(AMD),to which mink of all ages are susceptible.And it is one of the most pressing diseases in the countries worldwide specializing in mink farming.Currently,there is no vaccine can effectively prevent the dissemination of the AMDV.Therefore,studying the pathogenic mechanism of AMDV to explore effective prevention and control methods is an urgent issue in this study.In order to investigate whether the virulent strain of AMDV can induce apoptosis in Cr FK cells in vitro.This study used the virulent strain of AMDV-DL125,which are capable of proliferating in vitro and acting on Cr FK cells.Application of fluorescence quantitative PCR method to plot viral growth curves;CCK-8 assay was applied to detect Cr FK cell viability after infection with AMDV-DL125 strains;Hochest 33258 staining was applied to detect morphological changes at different time points after viral infection in Cr FK cells;flow cytometry was applied to detect apoptosis rate after viral infection.The results showed that AMDV-DL125 strain could proliferate in Cr FK cells;AMDV-DL125 strain infection had a significant inhibitory effect on the activity of Cr FK cells;after AMDV-DL125 strain infection,the Cr FK cells showed significant apoptotic features such as chromatin fixation,nuclear crinkling and fragmentation;the results of flow cytometry detection showed that the apoptosis rate of Cr FK cells showed a positive correlation with the time of virus infection,which indicates that AMDV-DL125 strain could induce apoptosis of Cr FK cells.To investigate which key genes could regulate the apoptosis of Cr FK cells induced by AMDV-DL125 strain,the m RNA expression profile of Cr FK cells was obtained by transcriptome sequencing technology at 12 h after AMDV-DL125 strain infection in Cr FK cells,and the results were normalized using DESeq2 software to screen for differential genes with significant changes in expression.Conducting functional annotations of differential genes through Gene Ontology(GO)analysis and pathway enrichment assessments via Pathway-Analysis.Subsequently,validating the differentially expressed genes employing quantitative reverse transcription PCR(RT-q PCR).The results of the analysis showed that 2412 differentially expressed genes were screened,of which 483 genes were up-regμlated and 1929 genes were down-regulated.The GO annotation of differentially expressed genes focused on biological processes,cellular composition and molecular functions and involved metabolic processes,macromolecular metabolic processes,macromolecular complexes and structμral molecular activities,etc.The results of KEGG pathway enrichment analysis showed that differentially expressed genes were mainly involved in Ras signaling pathway,PI3K/Akt signaling pathway,apoptosis pathway,etc.Among them,there were obvious differential expression in PI3K/Akt signaling pathway related to apoptosis.Five genes related to apoptosis were identified and their RT-q PCR detection results matched with sequencing results.In roder to study the role of PI3K/Akt pathway in Cr FK apoptosis induced by AMDV-DL125 strain.Indirect immunofluorescence was applied to detect the phosphorylation of Akt in Cr FK cells activated by the AMDV-DL125 strain.RT-q PCR was applied to detect the variations in the transcription levels of PI3 K and Akt at different time periods;MTT method was applied to detect the effect of different concentrations of LY294002 on the activity of Cr FK cells.Western Blot was applied to detect the inhibition effect of PI3K/Akt pathway on Cr FK cells after LY294002inhibition;RT-q PCR was applied to detect the variations of mitochondrial apoptosis-related factors expression level after PI3K/Akt pathway inhibition;finally,flow cytometry was applied to detect the alterations in Cr FK cell apoptosis induction following the inhibition of the PI3K/Akt pathway in AMDV-DL125 strains.Indirect immunofluorescence results showed that infection of Cr FK cells by AMDV-DL125 strain resulted in phosphorylation of Akt,and RT-q PCR results showed that infection of Cr FK cells by AMDV-DL125 strain significantly upregulated PI3 K and Akt m RNA expression levels in the early stage and then became decreasing.Western Blot results showed that 30 μmol of LY294002 could effectively inhibit the phosphorylation of Akt in Cr FK cells caused by AMDV infection.RT-q PCR results showed that the expression levels of Caspase-3,Caspase-9,and Bax were significantly increased.In contrast,the expression level of Bcl-2 was decreased after using the inhibitor compared with AMDV commonly infected Cr FK cells.Flow cytometric assay results showed that the apoptosis rate of Cr FK cells induced by AMDV-DL125 strain was significantly increased after blocking the PI3K/Akt signaling pathway,indicating that the PI3K/Akt signaling pathway plays a vital role in inhibiting the apoptosis of Cr FK cells induced by AMDV infection.The above findings suggest that AMDV-DL125 infection in Cr FK cells induces Akt phosphorylation in a PI3K-dependent manner to activate the PI3K/Akt signaling pathway and inhibit apoptosis.The results of this study are helpful for understanding the infection and pathogenesis of AMDV and have important implications for the prevention and treatment of this disease.