The Role Of IPO13 In The Insensitive Of Glucocorticoid Treatment After RSV Infection That NS1 Protein Of Respiratory Syncytial Virus Blocks Glucocorticoid Receptor Nuclear Translocation

PART ?ANALYSIS OF THE EFFECT OF GLUCOCORTICOID ON RSV INFECTED MICE MODELObjective: respiratory syncytial virus(RSV)is the most common and important virus causing acute lower respiratory tract infection in infants worldwide.About 39% of infants with severe RSV bronchiolitis still experience recurrent wheezing at age 18.As a non-specific anti-inflammatory drug,glucocorticoid is widely used in the treatment of acute RSV infection.However,meta-analysis found that GC treatment of bronchiolitis caused by RSV is no clinical benefit,neither can shorten hospitalization time,nor can it reduce the occurrence of recurrent wheezing in the late stage of RSV infection,and its mechanism is not clear.Methods: The mice,aged at 6-8 weeks,were randomly divided into three groups and infected intranasally(i.n.)with 100 ?l of a stock suspension of 1.5×10 PFU/ml RSV(RSV group),UV-inactivated virus(UV-RSV group)and HEp-2 suspension without virus(Control group)under sodium pentobarbital anesthesia.After 2 hours of RSV infection,dexamethasone was injected intraperitoneally.Bronchoalveolar lavage fluid and lung tissue were collected at different time points after RSV infection.IL-6 and IFN-? levels in BALF were detected by ELISA.Inflammatory response was evaluated by pathological H&E staining and BALF cell counts and classification counts.Airway hyperresponsiveness was assessed by airway resistance.Results: The airway inflammation and airway hyperresponsiveness of RSV infected mice were more obvious than those of the control group.The pathological damage was more serious than that of the control group.The cytokines IL-6 and IFN-? were significantly increased.After high dose dexamethasone treatment,lung pathological damage was more serious than RSV,airway inflammation and airway AHR did not change significantly;cytokine IFN-? did not change significantly,but IL-6 level was higher than RSV group,and was dose-dependent.Different doses of dexamethasone also had no significant changes in airway inflammation,airway hyperresponsiveness and cytokines.Conclusion: Dexamethasone could not inhibit airway inflammation and airway hyperresponsiveness caused by RSV infection.PART II EFFECT OF RSV INFECTION ON GLUCOCORTICOID RECEPTORObjective: Only when glucocorticoid bind to glucocorticoid receptor in cytoplasm and enter the nucleus can they further exert anti-inflammatory effects through transcriptional activation or transcriptional inhibition.In vivo and in vitro studies in hormone-insensitive asthma,COPD and psoriasis showed reduced GR nuclear translocation.In hormone-resistant neutrophil airway inflammatory diseases(including neutrophil asthma,COPD,etc.),GR can not enter the nucleus and retain the cytoplasm.It has been found that RSV and Poly(I:C)can inhibit GR-mediated gene transcription in epithelial cells.The impact of RSV infection on GR has not been systematically reported.Methods: After RSV infected infants nasopharyngeal aspirate(NPA)collection,cell counts,cytokines,GR expression were detected by immunofluorescence and GR? and GR? expression were detected by PCR.The mice,aged at 6-8 weeks,were randomly divided into three groups and infected intranasally with RSV.Lung tissues were collected at different time points after RSV infection.Total GR protein and cytoplasmic nucleus levels were detected by Western blot.The expression of GR? and GR? genes and GR-mediated anti-inflammatory genes FKBP51,GILZ and MKP-1 were detected by polymerase chain reaction.The GR entry into nuclear was detected by immunofluorescence in the A549 cell after RSV infection.The expression of anti-inflammatory genes FKBP51,GILZ and MKP-1 mediated by GR was detected by immunofluorescence and PCR in A549 cell line.Results: The expression of GR? in NPA of children with RSV i nfection decreased,GR? did not decrease significantly,and GR nucleation decreased significantly.The expression of GR?,GR? and GR p rotein in lung tissue of mice infected with RSV decreased significantl y,and the expression of GR protein in nucleus decreased significantl y.Meanwhile,the expression of anti-inflammatory genes FKBP51,GI LZ and MKP-1 mediated by GR decreased significantly.Immunofluor escence showed that GR entry into nucleus decreased significantly.In A549 cells,GR entry into nucleus was also significantly reduced,ac companied by GR-mediated anti-inflammatory genesGILZ and MKP-1 expression decreased,while UV-RSV infection had no significant eff ect on GR expression and nuclear entry.Conclusion: RSV infection down-regulate GR expression and inhibit GR nuclear tranlocation.PART ? STUDY ON THE MECHANISM OF GR ENTRY BARRIER INDUCED BY RSV NS1 PROTEIN THROUGH COMPETITIVE BINDING TO IPO13Objective: At present,the mechanism of GR entry barrier caused by RSV infection is not clear.Studies have confirmed that the IPOs family is a key molecule mediating GR nucleation.Studies in airway or lung epithelial cells have found that IPO7 and IPO13 play a key role in GR nucleation.Some studies have shown that RSV NS1 protein can directly act on IPO7 and IPO8.Therefore,it is necessary to find out whether NS1 can interfere with the combination of IPO and GR by directly acting on the IPO family,thus leading to GR entry barrier.Methods: The expression of IPO13 and IPO7 in children with RSV infection and control group were detected by PCR after NPA collection.The lung tissues of BALB/c mice were collected at different time points after RSV infection.The total protein level of IPO13 was detected by Western blot and the gene expression of IPO13 was detected by PCR.Establishment of A549 epithelial cells infected with RSV.Immunofluorescence was used to detect GR nucleation after silencing NS1 with siRNA,and the expression of GR-mediated anti-inflammatory genes GILZ and MKP-1 was detected by PCR.The interaction of IPO13,GR and NS1 was detected by co-immunoprecipitation.GST pull down was used to determine whether IPO13 and GR,NS1 and IPO13 interact directly in vitro.Results: The expression of IPO13 gene in NPA of children with RSV infection decreased.The expression of IPO13 gene and protein in lung tissue of RSV infected mice decreased.After siRNA silencing NS1,GR nucleus entry was significantly increased in A549 cells,and the expressionof GR-mediated anti-inflammatory genes GILZ and MKP-1 was increased.The protein interaction showed that GR and IPO13,NS1 and IPO13 had direct interaction,and the binding of GR and IPO13 increased after siRNA silenced NS1.Conclusion: RSV NS1 may lead to GR nuclear entry barrier and insensitivity to hormone therapy through competitive inhibition of the combination of IPO13 and GR.