| Objective: The purpose of this study was to investigate the effect of progesterone on type I IFN signaling pathway in A549 cells infected with HEV,and to provide a theoretical basis for the clinical treatment and preventive of pregnant women with hepatitis E in the future.Methods: After HEV infected A549 cells,the level of HEV was detected by fluorescence quantitative PCR.After successful infection,the A549 cells infected with HEV were treated with different doses of PG,the same concentration of MF and IFN-?at the same time or separately.The A549 cells infected with HEV without other treatment factors and the A549 cells not infected with HEV were used as control group.Western-blotting was used to detect the phosphorylation levels of Jak1,Tyk2,Stat1,Stat2 and the expression levels of MX1,PKR and OAS1 in JAK-STAT signaling pathway.Results: 1.After HEV infected A549 cells,RNA was extracted and quantified by fluorescence quantitative PCR.The results showed that there were no primer dimers and non-specific products at the dissolution temperature Tm=80?.HEV directly infected A549 cells and entered the cell body.2.The expression of phosphorylated Jak1 protein was up-regulated in model group(vs blank control group,P<0.001);the expression of phosphorylated Jak1 protein was down-regulated in IFN-? intervention group(vs blank control group,P<0.001);the expression of phosphorylated Jak1 protein was down-regulated in 200 ng/ml PG intervention group(vs blank control group,P<0.001);different concentrations of PG(50ng/ml,100ng/ml,200ng/ml,400ng/ml)and IFN-? treatment.The highest expression level of phosphorylated Jak1 protein was found in 200 ng/ml PG group and 100 ng/mlPG group(vs blank control group,P<0.001;vs blank control group,P<0.001).Compared with the progesterone intervention group,the expression level of phosphorylated Jak1 protein in the mifepristone and progesterone co-intervention group had no significant difference.3.Compared with the blank control group,the expression of phosphorylated Tyk2 protein was up-regulated in the model group(P<0.001);the expression of phosphorylated Tyk2 protein was up-regulated in HEV-A549 cells by IFN-?intervention(P<0.001);The expression level of phosphorylated Tyk2 in HEV-A549 cells treated with 200 ng/ml PG alone was lower than that in model group(P<0.001),but higher than that in blank control group(P<0.001).HEV-A549 cells were treated with different concentrations of PG(50ng/ml,100ng/ml,200ng/ml,400ng/ml)and IFN-?.The expression level in experimental group 3 was higher than that in other groups(vs experimental group 2,P<0.001;vs experimental group 4,P<0.001;vs experimental group 5,P<0.001).Progesterone may affect the expression of phosphorylated Tyk2 protein by binding or acting on receptors other than progesterone receptor.4.There was no significant difference in the expression of phosphorylated Stat1 protein before and after the establishment of cell infection model(compared with blank control group,P=0.501).Intervention of HEV-A549 cells with IFN-? resulted in up-regulation of phosphorylated Stat1 protein expression(compared with blank control group,P<0.001).The expression level of phosphorylated Stat1 protein in 200ng/ml PG group was higher than that in model group(P=0.021)and blank control group(P=0.005).When HEV-A549 cells were treated with different concentrations of PG(50ng/ml,100ng/ml,200ng/ml,400ng/ml)and the same concentration of IFN-?,the expression level of phosphorylated Stat1 protein in 200ng/ml PG group was higher than that in other PG concentration groups(vs experimental group 2,P<0.001;vs experimental group 3,P<0.001;vs experimental group 5,P<0.001).Progesterone may affect the expression of phosphorylated Stat1 protein by binding or acting on receptorsother than progesterone receptors.5.The expression of phosphorylated Stat2 protein could not be induced in A549 cells infected with HEV.Intervention of HEV-A549 cells with IFN-? resulted in the expression of phosphorylated Stat2 protein.In experimental group 6,the expression of phosphorylated Stat2 protein was also induced by the intervention of PG.The expression of phosphorylated Stat2 protein in 200 ng/ml PG group was higher than that in other PG concentration groups(vs experimental group 2,P<0.001;vs experimental group 3,P<0.001;vs experimental group 5,P<0.001).Progesterone may affect the expression of phosphorylated Stat2 protein by binding or acting on receptors other than progesterone receptors.6.The expression of MX1 protein was similar to that of phosphorylated Stat2 protein.HEV infected A549 cells could not induce the expression of MX1 protein.Intervention with IFN-? could increase the expression of MX1 protein(compared with blank control group,P<0.001).When HEV-A549 cells were treated with different concentrations of PG and IFN-? at the same concentration,the expression level of MX1 protein in experimental group 3 was higher than that in group 1(P=0.001)and higher than that in other concentration groups(vs experimental group 2,P<0.001;vs experimental group 4,P=0.260;vs experimental group 5,P < 0.001).Progesterone may bind or interact with receptors other than progesterone receptor PR,and progesterone affects the expression of phosphorylated MX1 protein.7.The expression of PKR protein in A549 cells infected with HEV was up-regulated(compared with blank control group,P=0.013).The expression of PKR protein was up-regulated after IFN-? intervention(P<0.001).After HEV infection,the expression of PKR protein was up-regulated with different concentrations of PG(50ng/ml,100ng/ml,200ng/ml,400ng/ml)and the IFN-?,there was no significant difference in the expression of PKR between 100ng/ml and 200ng/ml PG groups(P=0.596),but they were higher than other PG concentration groups(vs group 2,P<0.001;vs group 5,P<0.001).After progesterone antagonist mifepristone fully bindsto progesterone receptor,the expression of phosphorylated PKR protein is up-regulated by adding progesterone.8.HEV had no effect on the expression of OAS1 in A549 cells(compared with blank control group,P=1.000).After HEV infected A549 cells,the expression of OAS1 protein was up-regulated by IFN-? intervention(compared with blank control group,P<0.001);100ng/ml PG was higher than other PG concentration groups(vs experimental group 2,P=0.005;vs experimental group 4,P < 0.001;vs experimental group 5,P<0.001);Progesterone may up-regulate the expression of phosphorylated OAS1 protein by binding or acting on receptors other than progesterone receptors.Conclusion: 1.HEV infection has an effect on type I IFN signaling pathway in A549 cells.It can up-regulate the expression of PKR,phosphorylated Tyk2 and phosphorylated Jak1 protein,but has no effect on the expression of phosphorylated Stat1 and OAS1 protein.Single HEV infection can not induce the expression of phosphorylated Stat2 and MX1 protein.2.PG,IFN and HEV may interact with each other.The levels of signaling pathway proteins in 100ng/ml and 200ng/ml progesterone groups were higher than those in 50ng/ml and 400ng/ml progesterone groups.Progesterone may bind or interact with other receptors besides progesterone receptors,and down-regulate the expression levels of phosphorylated Tyk2,phosphorylated Stat2,MX1,PKR and OAS1 proteins. |
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