| Part One:PM2.5 Induced Inflammatory Changes in Mice Lung Tissue and MH-S CellsObjective:Mice lungs and MH-S cells were stimulated with different concentrations of particles matter with diameter less than or equal to 2.5 microns（PM2.5）,and acute local inflammation models were established in vitro and in vivo to investigate the inflammatory changes of mice lungs and MH-S cells under the stimulation of PM2.5 gradient concentration.Methods:1.Twenty-four male C57BL/6J mice were randomly separated into 4groups（n=6 per group）:control group,low（4mg/kg）,medium（8mg/kg）,and high（16mg/kg）PM2.5 concentration intervention group.After 100ul PM2.5suspension of different concentrations was injected into the trachea,mice were rotated 360°to make PM2.5 injected into the lungs of mice and distributed evenly.Mice in the control group were given the equal volume normal saline in the same operation.24h later,the mice were anesthetized and euthanized after receiving samples.The serum levels of IL-1β,IL-18 and TNF-αwere detected by ELISA.Mouse lung tissue samples were collected for HE staining to observe the pathological changes of lung tissue.The changes of NLRP3protein were detected by western blot after the protein was extracted from lung tissue by grinding homogenate.2.MH-S cells were divided into four groups:control group（given the same amount of normal saline as the experimental group）,low-concentration PM2.5 intervention group（working solution 500ug/ml,100ul,final concentration 50ug/ml）,medium concentration PM2.5 intervention group（working solution 1000ug/ml,100ul,final concentration 100ug/ml）,high concentration PM2.5 intervention group（2000ug/ml working fluid,100ul,final concentration of 200ug/ml）.MH-S cells were incubated in a 5%CO2cell incubator at 37℃for 8h.The levels of IL-1β,IL-18 and TNF-αwere detected by ELISA.The NLRP3 protein changes were detected by western blot.In96-well plates,MH-S cells were stimulated with different concentrations of PM2.5（the final concentration was 0,50,100 and 200ug/ml）for 8h.CCK-8was used to detect changes in MH-S cell activity after PM2.5 intervention.Results:1.PM2.5 Exposure Caused Lung Injury and Systemic Inflammatory Changes in Mice（1）Compared with the control group,thickening and inflammatory cell infiltration,alveolar fusion and interstitial edema were observed in the alveolar wall and pulmonary septum respectively in the low,medium and high concentration PM2.5 exposure groups,and the inflammatory cell aggregation was more obvious,more extensive and more serious with the increase of concentration.Moreover,the pathological scores of lung tissue in the medium and high concentration PM2.5 exposure groups were significantly higher than those in the control group（p<0.05）.（2）The levels of IL-1β,IL-18 and TNF-αwere up-regulated by PM2.5 in mouse peripheral blood serum in a concentration-dependent manner.Compared with the control group,the expression level of inflammatory cytokines in the medium and high concentration PM2.5 group was significantly increased（p<0.05）.（3）Compared with the control group,there was no significant difference in NLRP3 protein level in the low-concentration PM2.5 exposure group（p>0.05）,but the NLRP3 protein level in the medium-high concentration PM2.5 group was significantly increased（p<0.05）.2.PM2.5 Intervention Caused Inflammatory Changes in MH-S Cells（1）In the supernatant of MH-S cells,the levels of IL-1β,IL-18 and TNF-αincreased after PM2.5 treatment.Compared with the control group,there was no significant change in the level of proinflammatory cytokines in low-concentration PM2.5 treatment（p>0.05）.The levels of IL-1βand TNF-αwere significantly increased in the treatment of medium and high concentration PM2.5（p<0.05）,and IL-18 was significantly increased in the treatment of medium concentration PM2.5（p<0.05）.（2）Compared with the control group,the NLRP3 protein level in the low-concentration PM2.5intervention group was not significantly up-regulated（p>0.05）,while the NLRP3 protein level in the medium-high concentration PM2.5 intervention group was significantly increased compared with the control group（p<0.05）.（3）Compared with the group without PM2.5,the survival rate of MH-S cells was decreased by PM2.5 stimulation,and the survival rate of MH-S cells was decreased with the increase of PM2.5 concentration（p<0.05）.Summary:1.PM2.5 exposure caused infiltration and aggregation of inflammatory cells,alveolar fusion and interstitial edema in lung tissue of mice.2.PM2.5 exposure induced the up-regulation of NLRP3 protein expression in mouse lung tissue and MH-S cells,and the increase of pro-inflammatory cytokines IL-1β,IL-18 and TNF-α.Part Two:Irisin Attenuates PM2.5 Induced Acute Lung Inflammation by Regulating Nod2/NF-κB Signaling PathwayObjective:The role of irisin in lung inflammation after exposure to PM2.5 is unclear.We will observe the effect of irisin on PM2.5-induced inflammatory lung injury model in mice and MH-S cell models in vitro,so as to demonstrate the hypothesis that irisin is an important defense factor in the body’s anti-inflammation,and explore the molecular mechanism of its lung anti-inflammation.Methods:1.Twenty-four male C57BL/6J mice were randomly separated into 4groups（n=6 per group）:（1）control group:given the same amount of normal saline as the experimental group.（2）PM2.5 group:the final concentration of PM2.5 in mice was 8 mg/kg after tracheal perfusion of PM2.5 suspension.（3）PM2.5+irisin group:irisin（250ug/kg）was injected intraperitoneally 30minutes before PM2.5 was administered.（4）PM2.5+dexamethasone group:dexamethasone（1mg/kg）was injected intraperitoneally 30 minutes before PM2.5 was administered.The mice were exposed to PM2.5 for 24h,euthanized after anesthesia,and peripheral blood and lung tissues were collected.The expression of FNDC5/irisin in lung tissues of mice after exposure to PM2.5 was detected by immunofluorescence staining.Lung tissue samples of mice in each group were collected for hematoxylin-eosin staining to observe the pathological changes of lung tissue.The levels of IL-1β,IL-18and TNF-αwere detected by ELISA in peripheral blood of mice.Nod2,NF-κB p65 and NLRP3 levels were detected by q RT-PCR,and Nod2,NF-κB,IκB,p-IκB levels were detected by western blot.2.The cells were separated into 4 groups:（1）control group:given the same amount of normal saline as the experimental group.（2）PM2.5 group:100ul PM2.5 suspension was given,and the final concentration of PM2.5 was100ug/ml.（3）PM2.5+irisin group:irisin（200ng/ml）was administered for 2h,and PM2.5 suspension（100ul）was added.（4）PM2.5+dexamethasone group:dexamethasone（10ug/ml）was administered for 2h,and PM2.5 suspension（100ul）was added.After the intervention of PM2.5,the cells were incubated in a 5%CO2cell incubator at 37℃for 8h.The viability of MH-S cells treated with irisin and dexamethasone was determined using a CCK-8 assay.The levels of IL-1β,IL-18 and TNF-αin the supernatant were detected by ELISA.Subcellular localization of NLRP3 by cellular immunofluorescence.m RNA and protein were extracted and Nod2,NF-κB p65 and NLRP3 levels were detected by q RT-PCR.The levels of Nod2,NF-κB,I-κB and p-I-κB were detected by western blot.Results:1.Compared with the control group,the fluorescence expression of FNDC5/irisin in the lung tissue of mice was significantly enhanced after exposure to PM2.5（p<0.05）.2.In vivo studies showed that compared with PM2.5 group,PM2.5+irisin group could significantly reduce the area of pulmonary interstitial hemorrhage,edema and inflammatory cell infiltration,lung injury score and IL-1β,IL-18and TNF-αconcentrations（p<0.05）.Nod2,NF-κB p65 and NLRP3 m RNA levels were significantly decreased（p<0.05）,Nod2,NF-κB and p-IκB protein levels were significantly decreased（p<0.05）,but there was no significant difference in the level of IκB protein（p>0.05）.3.Compared with control group,irisin did not affect MH-S cell viability（p>0.05）.4.In vitro studies showed that compared with PM2.5 alone,the concentrations of IL-1βand TNF-αwere significantly decreased in PM2.5+irisin group（p<0.05）,and there was no statistical difference in IL-18levels（p>0.05）.Compared with PM2.5 group,the NLRP3 fluorescence intensity of PM2.5+irisin group had no statistical significance after 4h（p>0.05）,however after 8,12&24 hours,the PM2.5+irisin group had significantly lower NLRP3 specific fluorescence than the PM2.5 alone treatment group（p<0.05）.Nod2,NF-κB p65 and NLRP3 m RNA levels in PM2.5+irisin group were significantly decreased（p<0.05）,Nod2,NF-κB and p-IκB protein levels were also significantly decreased after the administration of irisin cells（p<0.05）,but there was no significant difference in the level of IκB protein（p>0.05）.Summary:1.Acute exposure to PM2.5 induced FNDC5/irisin aggregation in lung tissue of mice.2.This study found that pre-administration of irisin in vivo or in vitro can reduce the production and release of inflammatory mediators induced by PM2.5.This process may be related to irisin inhibiting Nod2/NF-κB signaling to prevent fine particles from causing acute lung injury.Conclusions:This study found that pre-administration of irisin in vivo or in vitro can reduce the production and release of inflammatory mediators induced by PM2.5.This process may be related to irisin inhibiting Nod2/NF-κB signaling to prevent fine particles from causing ALI.It was also observed that irisin inhibited the activation of the NLRP3 inflammasome.These findings provide preliminary basic experimental data for the use of irisin in the treatment of pulmonary inflammatory injuries associated with PM2.5 air pollution. |
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