The Effects Of N-acetylcysteine On Benzoapyrene-induced Acute Lung Injury And Interstitial Pulmonary Fibrosis In Mice And Its Mechnaism

Background and ObjectiveBenzoapyrene(BaP),a representative polycyclic aromatic hydrocarbon(PAH),widely exists in automobile exhaust,incomplete combustion of fossil fuel,organic matter,wood combustion,charred meat,industrial emission,forest fires and polluted air.Additionally,BaP is a key component of atmospheric and indoor fine particulate matters.Because of its slow degradation in natural environment,human beings always suffer from BaP exposure through respiration and diet.Respiratory tract,digestive tract and skin are the three important exposed methods.In the cells,BaP is metabolized to benzoapyrene-7,8-diol-9,10-epoxide(BPDE)and other toxic metabolites through cytochrome P450.These toxic metabolites can combine with DNA to form BaP-DNA adducts.BaP-DNA adducts can result in DNA damage,genomic instability and reactive oxygen species(ROS)excessive production,oxidative damage and cellular oxidative stress of nucleic acid,lipid,protein and other macromolecules.At present,BPDE is regarded as ultimate carcinogenic of BaP in human bodies.BaP is of great concern for its mutagenesis and carcinogenesis.More and more researches have indicated that BaP is toxic to the liver,kidney,nervous system and multiple organs.In addition,the lung is the direct target of BaP.Increasing data showed that short-term or acute BaP exposure evoked other respiratory diseases except for pulmonary cancer.Acute lung injury(ALI)is one of severe and acute pulmonary inflammation.It is diffuse pulmonary parenchymal damage due to direct and indirect factors.ALI is a syndrome characterized with pulmonary neutrophil accumulation,pulmonary interstitial edema and alveolar epithelial injury.The previous research indicated that inflammatory reaction is always involved in the process of ALI.Interstitial pulmonary fibrosis(IPF)is a progressive interstitial pulmonary disease.It is unclear of cause and mechanism in IPF.IPF is an excess repair in lung tissues after different pathogenies-caused interstitial pulmonary disease in human bodies.The pathological characteristic of IPF is diffuse alveolar inflammation and alveolar structure disorder in the early stage.In addition,the pathological characteristic of IPF mainly shows fibroblast proliferation,abnormal accumulation of extracellular matrix and collagen excessive production in the late stage.Clinically,IPF patients often show dyspnea and irreversible pulmonary function injury.Finally,IPF leads to respiratory failure and death.The previous evidences reveal that epithelial-mesenchymal transition(EMT)has exerted a central role in the process of IPF.Several laboratory evidences and epidemiological studies have indicated that acute or short-term BaP exposure may induce ALI and IPF.However,the direct laboratory evidences are deficient.N-acetylcysteine(NAC)is a glutathione precursor and well-known antioxidant.NAC has the capacities of anti-inflammatory and antioxidant.The former studies have demonstrated that NAC can partially rescue ALI and IPF.However,it is obscure whether acute or short-term BaP exposure evokes ALI and IPF or not.In addition,whether NAC supplementation can alleviate BaP-induced ALI and IPF is needed to further verified.The aim of this study was to evaluate the effect of acute BaP exposure on ALI and pulmonary inflammation,and the effect of chronic BaP exposure on IPF and EMT in mice.Moreover,this study also assessed the effect and mechanism of NAC supplementation on BaP-induced ALI and IPF in mice.Methods(1)The effect of acute BaP exposure on ALI and pulmonary inflammation in miceIn order to evaluate the effect of acute BaP exposure on lung injury and pulmonary inflammation in mice,all 48 C57BL/6J male mice were divided into Control(Ctrl)and BaP groups.Mice were given an intratracheal instillation with BaP(2 mg/m L/week,45 ?L per mouse)in BaP group.Controls were dripped with the same volume of oil-in-water solvent.Mice were killed at 6,24,and 48 h after instillation(8 mice each time point).Lungs,bronchoalveolar lavage fluid(BALF)and serum were collected in mice.Pathological structure and morphological changes in mice lungs were evaluated through hematoxylin-eosin(HE)staining.Pulmonary cytokines and inflammatory signaling pathways in mice were detected via western blotting,reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry(IHC).(2)The effect of NAC pretreatment on acute BaP exposure-induced ALI and pulmonary inflammation in miceIn order to evaluate the effect of NAC pretreatment on acute BaP exposure-induced lung injury and pulmonary inflammation in mice,all 32 C57BL/6J male mice were divided into Ctrl group,NAC group,BaP group and NAC+BaP group.Mice were given an intratracheal instillation with BaP(2 mg/m L/week,45 ?L per mouse)in BaP and NAC+BaP groups.Mice were given an intratracheal instillation with equal DMSO in Ctrl and NAC+BaP groups.In NAC and NAC+BaP groups,mice were intraperitoneally injected with NAC(1 g/kg)in 8 h before and after BaP exposure.In Ctrl and BaP groups,mice were intraperitoneally injected with normal saline in the same time.Mice were killed in 8 h after BaP exposure.Lung tissues and serum were collected.Pathological structure and morphological changes in mice lungs were evaluated through HE staining.Pulmonary cytokines and inflammatory signaling pathways in mice were detected via western blotting,RT-PCR and IHC.(3)The effect of NAC pretreatment on BaP-activated NF-?B signaling in pulmonary epithelial cellsIn order to analyze the the effect of NAC pretreatment on BaP-activated NF-?B signaling in human pulmonary epithelial cells,A549 cells were randomly divided into Ctrl group,NAC group,BaP group,NAC+BaP group.A549 cells were co-cultured with BaP(0.1m M)in BaP and NAC+BaP groups.A549 cells were co-cultured with DMSO(0.1%)in Ctrl and NAC groups.A549 cells were added NAC(4 m M)in 2 h and 12 h before BaP exposure twice in NAC and NAC+BaP groups.A549 cells were harvested in 24 h after BaP exposure.NF-?B signaling was measured in A549 cells using western blotting.The m RNAs levels of pro-inflammatory cytokines and chemokines were detected in A549 cells using RT-PCR.(4)The effect of chronic BaP exposure on interstitial pulmonary fibrosis and pulmonary epithelial-mesenchymal transition in miceIn order to evaluate the effect of chronic BaP exposure on lung injury and interstitial pulmonary fibrosis in mice,all 40 C57BL/6J male mice were divided into Ctrl and BaP groups.Mice were given intratracheal instillation with BaP(25 ?g/once/week)for 6 weeks in BaP group.Ctrl mice were given instillation of normal saline into the trachea.Half of mice were killed in six weeks after BaP exposure.Mice lungs were collected.Lung weight and body weight were recorded.Pathological structure and morphological changes in mice lungs were evaluated through HE staining.Pulmonary collagen deposition was detected through Masson and sirius red staining.The proteins of EMT and collagen deposition were detected in mice lungs with western blotting.Pulmonary function was assessed in the remianing half of mice through pulmonary function instrument.(5)The effect of chronic BaP exposure on epithelial-mesenchymal transition in human pulmonary epithelial cellsIn order to evaluate chronic BaP exposure on EMT in human pulmonary epithelial cells,BEAS-2B cells were co-cultured with BaP(0.1 m M)for two weeks.EMT nuclear transcription factors were detected in BEAS-2B cells using immunofluorescence(IF).(6)The effect of NAC on chronic BaP exposure induced-IPF and EMT in mice and pulmonary epithelial cellsIn order to assess NAC supplementation on chronic BaP-evoked IPF in mice,all 60 C57BL/6J male mice were divided into Ctrl group,NAC group,BaP group and NAC+BaP group.Mice were given an intratracheal instillation with BaP(25 ?g/once/week)for 6 weeks in BaP and NAC+BaP groups.Mice were given an intratracheal instillation with equal DMSO in Ctrl and NAC+BaP groups.In NAC and NAC+BaP groups,mice were intraperitoneally injected with NAC(1 g/kg)for 6 weeks.In Ctrl and BaP groups,mice were intraperitoneally injected with normal saline in the same time.Mice were killed in 6 weeks after BaP exposure.Lung tissues and serum were collected.Collagen deposition was detected using Masson staining in mice lungs.In order to observe the the effect of NAC pretreatment on chronic BaP expsoure-induced EMT in human pulmonary epithelial cells,BEAS-2B cells were divided into Ctrl group,NAC group,BaP group,NAC+BaP group.BEAS-2B cells were co-cultured with BaP(0.1 m M)in BaP and NAC+BaP groups.BEAS-2B cells were co-cultured with DMSO(0.1%)in Ctrl and NAC groups.BEAS-2B cells were co-cultured with NAC(4 m M)for two weeks in NAC and NAC+BaP groups.EMT nuclear transcription factors and EMT markers were measured through IF.Results(1)Acute BaP exposure induced ALI and pulmonary inflammation in miceBaP exposure obviouisly elevated absolute lung weight and relative lung weight in mice.HE staining revealed that acute BaP exposure obviously induced lung injury,alveolar collapse,inflammatory cell infiltration,airway wall thickening and airway stenosis.Additionally,acute BaP exposure elevated the inflammatory cells in BALF,upregulated pulmonary m RNAs levels of pro-inflammatory cytokines and chemokine in mice.Besides,acute BaP exposure increased the phosphorylation levels of MAPK p38?MAPK JNK ? MAPK ERK1/2 in mice lungs.Moreover,acute BaP exposure promoted nuclear translation of NF-?B p65 and p50,elevated I?B? phosphorylation in mice lungs.(2)NAC pretreatment alleviated acute BaP exposure-induced ALI and pulmonary inflammation in miceThe results suggested that BaP-elevated absolute lung weight and relative lung weight in mice were significantly attenuated in NAC-pretreated mice.Moreover,NAC-pretreatment obviously alleviated BaP-induced alveolar collapse,inflammatory cell infiltration,airway wall thickening and airway stenosis in mice lungs.Furthermore,we found that NAC-pretreatment obviously alleviated BaP-induced phosphorylation of NF-?B p65 and I?B?,inhibited BaP-induced nuclear translocation of NF-?B p65 and NF-?B p50 in mice lungs.(3)NAC pretreatment attunuated BaP-activated NF-?B signaling in pulmonary epithelial cellsThe results showed that BaP exposure significantly increased the levels of phosphorylation of NF-?B p65 and I?B?.Interestingly,NAC pretreatment inhibited BaP-induced phosphorylation of NF-?B p65 and I?B?.Moreover,the m RNAs level of pro-inflammatory cytokines and chemokines were elevated in BaP-exposed A549 cells.As expected,BaP-induced upregulation of pro-inflammatory cytokines and chemokines was attenuated in NAC-supplemented A549 cells.(4)Chronic BaP exposure caused interstitial pulmonary fibrosis and pulmonary epithelial-mesenchymal transition in miceChronic BaP exposure elevated absolute lung weight and relative lung weight,induced lung injury and pulmonary inflammatory cell infiltration in mice.In addition,chronic BaP exposure elevated pulmonary collagen deposition and hydroxyproline in mice lungs.Collagen-and ?-SMA-positive cells were increased in BaP-exposed mice.Chronic BaP exposure damaged pulmonary function in mice.Besides,the marker of epithelial cells was decreaced,the marker of interstitial cells was increased in lungs of BaP-exposed mice.Moreover,we found that serum TGF-? and the phosphorylation of Smad2 and Smad3 were increased in BaP-exposed mice.(5)Chronic BaP exposure evoked epithelial-mesenchymal transition in human pulmonary epithelial cellsThe results indicated that chronic BaP exposure promoted nuclear translocation of Snail and ZEB in BEAS-2B cells.Moreover,the epithelial markers of EMT was reduced in chronic BaP-exposed BEAS-2B cells.(6)NAC supplementation inhibited chronic BaP exposure induced-IPF and EMT in pulmonary epithelial cellsIn vivo animal experiment showed that chronic BaP exposure elevated collagen deposition in lungs of mice.However,NAC supplementation obviously inhibited chronic BaP exposure-induced collagen deposition in mice lungs.In vitro cell experiment revealed that chronic BaP exposure two weeks significantly elevated ?-SMA-and Vimentin-positive cells in BEAS-2B cells.Not only that,chronic BaP exposure two weeks remarkedly promoted Twist nuclear translocation in BEAS-2B cells.Interestingly,NAC supplementation obviously alleviated chronic BaP-elevated ?-SMA-and Vimentin-positive cells,and inhibited chronic BaP-promoted Twist nuclear translocation in BEAS-2B cells.ConclusionAcute BaP exposure induces ALI and pulmonary inflammation,activates NF-?B and MAPKs signaling pathways in lungs of mice.Interestingly,NAC pretreatment alleviates BaP-induced ALI and pulmonary inflammation partially through inhibiting NF-?B signaling pathway.Moreover,chronic BaP exposure results in IPF and EMT in pulmonary epithelial cells.NAC supplementation attenuates chronic BaP exposure-induced IPF and EMT in pulmonary epithelial cells.Therefore,these results provide evidence that NAC may be used as a potential preventive and therapeutic drug for air pollutants-induced pulmonary diseases.