Protective Effects Of VGX-1027 In PM_2.5-induced Airway Inflammation And Airway Hyperresponsiveness

Background In recent years,Air pollution has become increasingly severe in China with the rapid development of industrialization and urbanization.Fine particulate matters,known as PM2.5,is easily inhaled into lungs,deposits on the distal small airways and alveoli,and induces airway hyperresponsiveness(AHR)and pulmonary inflammation by stimulating alveolar epithelial cells and pulmonary microcirculation.Studies have shown that the activation of Toll-like receptor 4(TLR4)signaling may be involved in airway inflammation and airway hyperresponsiveness induced by PM2.5 through regulating lung tissue immune damage and the release of inflammatory factors.Therefore,we investigated the preventive protective effect of VGX-1027,a TLR4 blocker,on PM2.5-induced airway inflammation and AHR in a mouse model in vivo and on inflammatory mechanisms in vitro in human airway epithelial cells(Beas-2b cells).Objective To investigate the protective effects of VGX-1027,a TLR4 blocker,on PM2.5-induced AHR and pulmonary inflammation and its underlying protective mechanisms.Methods1.In vivo,detect the protective effects of VGX-1027 on PM2.5-induced lung inflammation and airway hyperresponsiveness(AHR)in mice Firstly,a mouse model of lung inflammation induced by PM2.5 was established.Secondly,Mice were injected intraperitoneally with PBS or corresponding doses of VGX-1027 one hour before intranasal instillation of PBS or PM2.5(7.8 mg/kg)for two consecutive days.24 hours after last intranasal instillation,mouse AHR and bronchoalveolar lavage fluid(BALF)cell numbers were measured.Lung inflammation scores were evaluated by HE staining and the levels of inflammatory cytokines in BALF were detected by ELISA,TLR4 protein expression levels of lung tissues was determined by immunohistochemistry and Western blot analysis.And,the expressed levels of mitochondrial fusion/fission proteins and NLRP3 and caspase-1 proteins,as well as the phosphorylation levels of NF-?B protein,were determined using Western blot analysis.2.In vitro,detect the protective mechanism of VGX-1027 on PM2.5-induced human bronchial epithelial cell injury with activated inflammatory protein pathways and increased proinflammatory cytokines.Human bronchial epithelial cells were treated with vehicle(PBS)or VGX-1027(50 mM)and incubated for 60 min.Then,cells were stimulated with the PBS or PM2.5 suspension(150 ng/ml)for 24 hours.Proinflammatory cytokine levels in cells were assayed by Reverse transcription PCR(RT-PCR).The expressed levels of TLR4,NLRP3,caspase-1,and mitochondrial fusion/fission proteins,as well as the phosphorylation levels of NF-?B protein,were determined by Western blot analysis.Results1.VGX-1027 inhibited PM2.5-induced mouse AHR,lung inflammation and mitochondrial damage PM2.5(7.8 mg/kg)instillation induced significant AHR and lung inflammatory infiltration;VGX-1027(25 mg/kg)prevented PM2.5-induced BHR and lung inflammation,down-regulated levels of proinflammatory cytokines(TNF-a,CXCL1(KC),IL-1b,IL-6 and IL-18)in BALF;inhibited the activation of TLR4/NF-?B pathway and NLRP3/Caspase-1 pathway,as well as the expressed levels of mitochondrial fusion/fission proteins2.In vitro,VGX-1027 inhibited the activation of inflammatory signaling pathways and the expression of proinflammatory cytokines induced by PM2.5In Beas-2b cells,PM2.5 stimulated increased proinflammatory cytokine levels,activated inflammatory signaling pathways Compared with the PBS control group.Pretreatment with VGX-1027 reduced expressed levels of proinflammatory cytokines(TNF-a,IL-1b,IL-6 and IL-18);inhibited the activation of TLR4/NF-?B pathway and NLRP3/Caspase-1 pathway,as well as the expressed levels of mitochondrial fusion/fission proteins in cells.Conclusions1.PM2.5 induced significant AHR,lung inflammation and the activation of inflammatory signaling pathways.2.VGX-1027 decreased PM2.5-induced AHR and lung inflammation.3.The protective effects of VGX-1027 may be involved in mediating TLR4-NF-?B/p38 MAPK pathway and NLRP3/Caspase-1 pathway,as well as the expression of mitochondrial fusion/fission proteins.