Grape Seed Proanthocyanidin Extract Influences Susceptibility To Steroid Resistance Via Regulating Nrf2-iNOS-HDAC2 Axis In Murine Models Of Asthma

Objective:To explore the mechanisms of Nrf2-iNOS-HDAC2 pathway in the regulation of glucocorticoid sensitivity in asthma and whether proanthocyanidins could restore glucocorticoid responsiveness via Nrf2-iNOS-HDAC2 axis in a murine model of asthma.Methods:BALB/c mice(n=48;aged 8 weeks)were randomly and averagely divided into 6 groups:control group,asthma group,dexamethasone group and GSPE group(low dose,medium dose and high dose).The mice were sensitized and challenged with ovalbumin(OVA)and lipopolysaccharide(LPS)to produce a murine model of asthma.The mice Pulmonary expiratory resistance maximum was measured by connecting the animal lung function instrument.Alteration of the airway inflanmmtion was observed by haematoxylin—easin(HE)and Periodic Acid-Schiff(PAS)staining.The expression of inducible nitric oxide synthase(iNOS)in the lung were evaluated by immunohistochemistry.The levels of interleukin(IL)-8 and monocyte chemotactic protein(MCP)-1 in the serum of mice were measured by enzyme linked immunosorbent assay(ELISA).The protein and mRNA expressions of nuclear factor erythroid 2-related factor 2(Nrf2),iNOS,histone deacetylase 2(HDAC2),Glutamatecysteine ligase catalytic subunit(GCLC)and Glutamatecysteine ligase modifier subunit(GCLM)were detected by Western blot and qRT-PCR respectively.Results:According to the results of lung function,the dexamethasone can not reduce airway resistance,while the GSPE group can reduce airway resistance regardless of the low medium or high dose.There were inflammatory cell infiltration,airway stenosis and bronchial smooth muscle hypertrophy in the asthmatic group;but these changes were significantly ameliorated in the GSPE treatment group.The area of the iNOS-stained smooth muscle layer in the GSPE group and dexamethasone group was significantly lower than that in the asthmatic group(P<0.05).Compared with dexamethasone group,the area of the iNOS-stained smooth muscle was lower in the middle or high dose GSPE group(P<0.05).As compared with the asthmatic group,the GSPE group showed significant decrease of IL-8 and MCP-1 in the serum of mice lung tissues.The results of Western blot and qRT-PCR analysis showed that the expression levels of Nrf2,HDAC2,GCLM and GCLC in the asthmatic group were lower than that in the control group,while the expression level of iNOS was significantly higher than that in the control group.The expression levels of Nrf2,HDAC2,GCLM and GCLC in the GSPE group were higher than that in the dexamethasone group,while the expression level of iNOS was lower than that in the dexamethasone group(P<0.05).Conclusion:GSPE relieves the steroid resistance via regulating Nrf2-iNOS-HDAC2 axis in mice,which is promising to be a potential drug in the treatment of asthma.