A Study On The Mechanisms Of PPARγDNA Methylation Regulating HMGB1/NLRP3 Axis In PM_2.5-induced Pulmonary Fibrosis

Objective:Based on PPARγDNA methylation,we explored the mechanism of its role in regulating the HMGB1/NLRP3 axis in PM_2.5-induced lung fibrosis.And the results will provide scientific basis to find early biomarkers and novel measures of preventing and treating the hazards caused by PM_2.5.Methods:Animal model establishment:Thirty-two SD male rats were randomly divided into 4 groups（n=8 per group）as follows:the negative control group（NC）,the low-dose exposure group（L）,the medium-dose exposure group（M）,and the high-dose exposure group（H）.The animals in all four groups received nonexposed intratracheal instillation of PM_2.5once a week for 16 weeks.The pulmonary fibrosis in each group was detected by Masson staining,the PPARγprotein in lung tissues were detected by IHC,and the pathological alterations of lung tissues were detected by HE staining.Western blot was used to detect the protein expression levels of PPARγ,HMGB1,NLRP3,Caspase-1,α-SMA,and Collagen I in lung tissues.The BSP method was used to detect the DNA methylation rate of PPARγ,and q RT-PCR was used to detect the m RNA expression level of PPARγ.The levels of IL-1βand IL-18in BALF were detected by Elisa.Cell model establishment:According to the results of the CCK-8 assay,human bronchial epithelial cells（HBE）were divided into ten groups.They were the control group,PM_2.5exposure group（25μg/m L,50μg/m L,100μg/m L,200μg/m L）,PM_2.5+5-Aza group,PM_2.5+RSG group,PM_2.5+GW9662group,PM_2.5+GL group,PM_2.5+DMSO control group.The morphological changes of cells in each group were observed,and the protein expression levels of PPARγ,HMGB1,NLRP3,Caspase-1,α-SMA,and Collagen I,the m RNA expression level and methylation rate of PPARγwere measured.The levels of IL-1βand IL-18 in the culture supernatant of each group were detected.Results:1.Pathological changes of lung tissues and pulmonary fibrosis in rats after PM_2.5exposureThe structure of the lung tissues in the negative control group was normal,and no abnormal phenomena such as thickening of alveolar wall,edema of the alveolar septum,hemorrhage,and fibrosis were observed.The low,medium and high dose PM_2.5exposure groups showed different degrees of inflammatory cell aggregation,alveolar wall thickening,and alveolar structure destruction,respectively.With the increase of exposure dose,the lung injury is aggravated,and the lung septum is widened and fractured,which eventually leads to alveolar fusion and"honeycomb lung".The lung tissues of rats showed fibrosis changes,and with the increase of PM_2.5concentration,the degree of pulmonary fibrosis and the injury became more serious with a dose-effect relationship.These results indicate that chronic PM_2.5exposure causes pathological damage and fibrotic changes in the lung tissues of rats.2.PM_2.5exposure induced changes in the expression of related proteins and cytokines in lung tissue and HBE cells of ratsAfter PM_2.5exposure,the protein expression of PPARγin rat lung tissue and HBE cells decreased,the protein expression of HMGB1,NLRP3,Caspase-1,α-SMA,and Collagen I increased,and the levels of IL-18 and IL-1βin BALF and cell culture supernatant increased.The results indicate that PM_2.5exposure can cause an inflammatory response and pulmonary fibrosis-related expression changes in rat lung tissue and HBE.3.RSG,GW9662,and GL were used to regulate PPARγ/HMGB1/NLRP3signaling pathway in HBE to verify the mechanism of PM_2.5induced pulmonary fibrosisUp-regulation of PPARγby RSG could lead to the expression inhibition of HMGB1/NLRP3 signaling pathway and pulmonary fibrosis-related proteinsα-SMA and Collagen I,accompanied by the decrease of IL-1βand IL-18.The down-regulation of PPARγby GW9662 significantly increased the above indicators.Down-regulation of HMGB1 using GL showed that NLRP3 and Caspase-1 protein levels were inhibited and IL-1βand IL-18 levels were reduced.Those results suggested that PPARγ/HMGB1/NLRP3 signaling axis is involved in PM_2.5-induced lung injury and pulmonary fibrosis through an inflammatory response of HBE.4.PM_2.5exposure induced an increase in PPARγmethylation rate and a decrease in PPARγm RNA in lung tissues of rats and HBECompared with the negative control group,the methylation rate of PPARγwas increased with the decreased m RNA expression of PPARγin lung tissues and HBE cells in each PM_2.5exposure group,which could be reversed by 5-Aza,indicating that PM_2.5exposure caused DNA hypermethylation of PPARγpromoter.Moreover,5-Aza could induce the demethylation of PPARγ.Conclusion:1.Chronic PM_2.5exposure can lead to lung injury and pulmonary fibrosis in rats.2.PM_2.5promotes HBE cells to secrete IL-1βand IL-18 through PPARγ/HMGB1/NLRP3 axis,induces an inflammatory response,and eventually involved to pulmonary fibrosis.3.PM_2.5exposure can regulate the expression of PPARγthrough DNA promoter methylation to participate in the process of pulmonary fibrosis.