Role Of Histone H3K27me3 Methylation Mediated By NF-κB Signaling Pathway In Azithromycin Inhibition Of Lung Inflammation

Part Ⅰ Role of EZH2 mediated histone H3K27me3 modification imbalance in post infection wheezingObjective:To investigate the methylation regulation mechanism of histone H3K27me3 modification imbalance mediated by EZH2 in post infection wheezing,and to provide a new idea for clinical prevention and treatment of wheezing diseases.Methods:The children who underwent fiberoptic bronchoscopy for pulmonary infection in our hospital were selected as the research objects.According to whether there is wheezing,the subjects were divided Non-wheeze group [diagnosed as asthmatic bronchitis,asthmatic pneumonia and bronchiolitis without wheezing and not treated with azithromycin(AZM)] and Wheeze group(diagnosed as asthmatic bronchitis,asthmatic pneumonia and bronchiolitis with wheezing and not treated with AZM).At the same time,Control group(diagnosed as bronchial foreign body in acute stage,congenital laryngeal cartilage dysplasia,airway malformation.None of them was associated with infection)was set up.In addition,the wheezing cases were divided into two groups: Wheeze group(Intravenous infusion of 5% glucose injection without AZM,and the rest were treated routinely);Wheeze + AZM group(5% glucose injection containing AZM was given intravenously at a dose of 10 mg/kg·d,and the rest of the treatment was the same as the Wheeze group).According to whether it was infected with mycoplasma,the Wheeze + AZM group was subdivided into AZM +MP positive group and AZM + MP negative group.The bronchoalveolar lavage fluid(BALF)of children in each group was collected,centrifuged at 1500 rpm and 4℃ for10 minutes,and the supernatant and precipitation were separated.The following tests were carried out:(1)Cytometric Bead Array(CBA)to detect the level of the cytokines IFN-γ,IL-6,IL-2 and IL-10 in BALF;(2)The methylation of histone H3K27me3 and the expression of histone methyltransferase EZH2 in BALF were observed by immunofluorescence staining.(3)The change of macrophage related inflammatory factors in post infection wheezing cases were tested by ELISA.Results:(1)The CBA results showed that the expression of IL-2 and IL-6 in BALF in the Wheeze group were significantly higher than those in the Non-wheeze group and the Control group(both P < 0.05),and the expression level of IL-10 in the Wheeze group was higher lower that in the Non-wheeze group(P < 0.05).There was no significant difference between the Control group and Non-Wheeze group(both P > 0.05).While the expression level of IFN-γ had no change in the same samples(P > 0.05).(2)The results of immunofluorescence staining showed that the expression levels of histone H3K27me3 and EZH2 in the Wheeze group were significantly increased compared with the Control group(both P < 0.05).(3)ELISA results showed that the expression of IL-6 was significantly decreased in the Wheeze + AZM group compared with the Wheeze group(P ﹤0.05),IL-2 and IL-10 expression was significantly increased in the same samples(both P ﹤0.05).Conclusions:(1)Histone H3K27me3 mediated by histone methyltransferase EZH2 may be involved in the occurrence of post-infection wheezing in children.(2)AZM may ameliorate wheezing by standardizing abnormal secretion of macrophage-related inflammatory factors.Part Ⅱ Role of histone H3K27me3 methylation imbalance mediated by NF-κB signaling pathway in azithromycin inhibition of lung inflammationObjective:To investigate the regulation of histone methylation imbalance mediated by NF-κB signaling pathway in azithromycin(AZM)non-specific anti-inflammatory,and to provide a new intervention target for clinical prevention and treatment of post-inflammatory wheeze.Methods:Rat alveolar macrophages(NR8383)were cultured.The experimental subjects were randomly divided into eight groups,as following: Control group;Vehicle group(DMSO);LPS group(LPS 2 μg/m L);AZM group(LPS 2 μg/m L + AZM 8 μg/m L);GSK126 group(LPS 2 μg/m L + GSK126 4 μg/m L);SN50 group(LPS 2μg/m L+SN50 8 μg/m L);AZM + GSK126 group(LPS 2 μg/m L + AZM 8 μg/m L +GSK126 4 μg/m L);AZM + SN50 group(LPS 2 μg/m L + AZM 8 μg/m L + SN50 8μg/m L);NR8383 cells were collected after intervention 24 hours for analysis.(1)The protein levels expression of TLR4,p65,p-p65,EZH2,p-EZH2,histone H3K27me3 and IL-10 were determined by western blot.(2)The nucleation of p65 was detected by immunofluorescence staining.(3)The interaction between histone H3K27me3 and EZH2,p65 protein was verified by Co-immunoprecipitation(Co-IP).Results:(1)The results of western blot showed that the expression levels of TLR4,p65,p-p65,EZH2,p-EZH2 and histone H3K27me3 in the LPS group were significantly increased compared with the Control group(all P < 0.05);while IL-10 was significantly decreased(P < 0.05).Compared with the LPS group,the expression levels of TLR4,p65,p-p65,EZH2,p-EZH2 and histone H3K27me3 in the AZM group,GSK126 group,SN50 group,AZM + GSK126 group and AZM + SN50 group were significantly decreased(all P < 0.05);while IL-10 was significantly increased(P< 0.05).(2)The results of Co-IP showed that histone H3K27me3 directly interacts with EZH2,and p65 in NR8383 cells.(3)The immunofluorescence staining results showed that AZM(8 μg/m L)or SN50(4 μg/m L)significantly inhibited LPS-induced nuclear NF-κB P65 translocation.Conclusions:EZH2-mediated histone H3K27me3 methylation involving NF-κB signaling pathway may be one of the key pathways to non-specific anti-inflammatory effects of AZM.