Fine Particulate Matter Aggravates Cigarette Smoke Extract-induced Airway Inflammation Via Wnt5a-ERK Pathway In Chronic Obstructive Pulmonary Disease

Part ?Effects of PM2.5 on pulmonary inflammation in mice induced by cigarette smoke and inflammation in 16HBE cells induced by cigarette smoke extractBackground:Exposure to environmental particulate matter ?2.5 ?m in diameter(PM2.5)and smoking are common contributors to chronic obstructive pulmonary disease(COPD),and pertinent research implicates both factors in pulmonary inflammation.Using in vivo mouse and in vitro human cellular models,we investigated the joint impact of PM2.5 pollution and cigarette smoke in mice or cigarette smoke extract(CSE)in cells on COPD inflammationMethods:A total of 32 healthy male C57BL/6 mice(18-21g,6-8 weeks old)were randomly divided into four groups:control group,PM2.5 group,smoking group and PM2.5+smoking group(n=8/group).On the other hand,16HBE cells were stimulated with different concentrations of PM2.5 and CSE for 24 hours,and 16HBE cells were also stimulated with constant concentrations of PM2.5 and CSE for different periods of time.The appropriate concentration of PM2.5 and CSE was determined by detecting the protein concentrations of inflammatory genes(IL-6 and IL-8)in the supernatant of cell culture medium by ELISA.Then,16HBE cells were divided into four groups according to the purpose of the study:control group,PM2.5 group,CSE group and PM2.5+CSE group.The pathological changes of lungs in mice were studied by light microscopy,examining hematoxylin and eosin(H&E)and immunofluorescence-stained sections.Levels of inflammatory factors induced by PM2.5/cigarette smoke in mice and PM2.5/CSE in 16 human bronchial epithelial(16HBE)cells were also monitored by quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assays(ELISAs)Results:PM2.5 at 100ug/ml and CSE at 10%concentration were selected as the optimal stimulating concentration,and 24h was selected as the optimal stimulating time.PM2.5,cigarette smoke/CSE individually upregulated inflammation in mouse and 16HBE cells In addition,the inflammatory response to combined exposures of PM2.5 and cigarette smoke/CSE in mouse and 16HBE cells surpassed responses incited separatelyConclusions:Our findings suggest that PM2.5 could aggravate cigarette smoke-induced pulmonary inflammation in vivo mouse or CSE-induced inflammation in vitro human cellular modelsPart ?The changes of Wnt5a level in airway inflammation induced by PM2.5,cigarette smoke/cigarette smoke extractBackground:The purpose of this part of the study is to investigate the expression levels of Wnt5a in COPD model induced by PM2.5 and cigarette smoke/cigarette smoke extract(CSE)Methods:Thirty-two healthy male C57BL/6 mice were randomly divided into four groups:control group,PM2.5 group,smoking group and PM2.5+ smoking group(n=8/group).16HBE cells were divided into four groups according to the purpose of the study:control group,PM2.5 group,CSE group and PM2.5+CSE group.Expressions of Wnt5a were assessed at transcriptional and protein levels using qRT-PCR and western blot in 16HBE cells.Meanwhile,the levels of Wnt5a were assessed at transcriptional and protein levels using qRT-PCR and immunofluorescence in miceResults:Although separate PM2.5 and cigarette smoke/CSE exposure upregulated the expression of Wnt5a(a member of the Wnt secreted glycoprotein family),combined PM25 and cigarette smoke/CSE exposure produced a steeper rise in Wnt5a levels Conclusions:Our findings suggest that the levels of Wnt5a upregulated in PM2.5,cigarette smoke/CSE-induced airway inflammation in the context of COPDPart ?Application of Wnt5a specific antagonist BOX5 can partially improveinflammation induced by PM2.5 and CSE in COPD modelsBackground:The purpose of this part of the study is to observe whether the application of BOX5(a specific antagonist of Wnt5a)can partially inhibit the expression of Wnt5a,and then reduce the occurrence of inflammatory reaction in 16HBE cells stimulated by PM2.5/CSE.The relationship between Wnt5a and extracellular signal-related kinase(ERK)signaling pathway was also explored in this partMethods:Thirty-two healthy male C57BL/6 mice were randomly divided into four groups:control group,PM2.5 group,smoking group and PM2.5+ smoking group(n=8/group).The protein levels of P-ERK and T-ERK in lungs of mice were detected by western blot.16HBE cells were divided into four groups according to the purpose of the study:control group,PM2.5 group,CSE group and PM2.5+CSE group.The protein levels of P-ERK and T-ERK in 16HBE cells were also detected by Western Blot.After confirmation of the activity of ERK signaling pathway in mouse and cell models,16HBE cells were pre-incubated with 200 ?M BOX5 or vehicle(PBS)for 1 h,followed by 24 h incubation with or without exposure to PM2.5/CSE.The protein concentrations of IL-6 and IL-8 in the supernatant of 16HBE cell culture medium were detected by ELISA.The mRNA levels of IL-6 and IL-8 in 16HBE cell were measured by qRT-PCR.The levels of ERK signaling pathway-related proteins P-ERK1/2,T-ERK1/2 and Wnt5a in 16HBE cells were detected by western blot,further determining their role in airway inflammation induced by PM2.5 and CSEResults:The level of P-ERK1/2 and the ratio of P-ERK1/2/T-ERK1/2 increased significantly in mice after PM2.5 and smoking exposures,respectively,becoming even more pronounced after joint exposures to PM2.5 and smoking.In 16HBE cells,PM2.5 and CSE exerted similar effects on the ERK pathway.However,the upregulated phosphorylation of ERK1/2(P-ERK1/2)induced by PM2.5/CSE in 16HBE cells was inhibited partly by BOX5.Meanwhile,the levels of IL-6,IL-8 and Wnt5a in 16HBE cells were also downregulated partly by BOX5Conclusions:Wnt5a may regulate airway inflammation induced by PM2.5,cigarette smoke/CSE in COPD models through ERK signaling pathway.