| Backgrounds: 1-Nitropyrene(1-NP)is a key component of fine particulate matter(PM2.5).Exist widely in the atmosphere,soil and water sources.Previous reports have shown that acute 1-NP exposure can cause inflammation of the respiratory tract.However,there are still no relevant studies and reports on the consequences of longterm exposure to 1-NP.This study aims to investigate whether chronic 1-NP exposure induces pulmonary fibrosis and its possible mechanisms.Purpose: This study aims to investigate whether chronic 1-NP exposure can induce pulmonary fibrosis in mice and explore the mechanism of chronic 1-NP exposure inducing pulmonary fibrosis.Methods: Animal experiment: According to the random method,80 male C57BL6/J mice(6 weeks,20-22g)were divided into two groups: 1-NP group and control groupMice in the 1-NP group were instilled with 1-NP(20 μg/mouse/week)into the trachea.The control group was treated with blank preparation.The exposure was continued for six weeks.After six weeks,some of the mice in the two groups were tested for lung function,and the remaining mice were sacrificed by cervicadislocation.After sacrificed,the mice were opened thorax to preserve the lung tissueRandomly select a part of the left lung in each group and place it in 4%paraformaldehyde.The right lung was stored in a refrigerator at-80°C.HE staining was used to observe the degree of lung injury and inflammation in mice,and immunohistochemistry(IHC)was used to detect the distribution and expression of smooth muscle actin(a-SMA),a marker of mouse epithelial-mesenchymal transition(EMT).Masson staining measures the deposition of collagen in the lungs.Cell experiment: Human non-small cell lung cancer cells(A549)that have not received any treatment are divided into two groups: 1-NP group and control group.Cells in the 1-NP group were exposed to 1-NP(5 μM/L)for a long period of time,and the control group was exposed to medium containing 1/1000 of DMSO.After6 weeks of culture,observe the changes of cell morphology,and do cell scratch,cell migration and invasion experiments.Cell protein was extracted and Western blotting was used to detect E-cadherin,Vimentin,N-cadherin,α-SMA,pSmad2/3,Smad2 / 3 in A549 cells expression situation.Retain the cell culture supernatant and use the human TGF-β ELISA kit to detect the TGF-β secretion of A549 cells.In addition,a part of A549 cells without any treatment were divided into six groups: 6h group,12 h group,24 h group and 6h control group,12 h control group,24 h control group.Use a reactive oxygen detection kit to detect the production of reactive oxygen species in cells.The cell protein was extracted and Western blotting was used to detect the expression of p-IRE1α,p-PERK,p-e IF2α,GRP78,HO-1 and NOX-4 proteins in A549 cells.Results: Diffuse interstitial inflammation,a-SMA-positive cells,a marker of epithelial-mesenchymal transition(EMT),and an extensive collagen deposition,measured by Masson staining,were observed in 1-NP-exposed mouse lungs.Pulmonary function showed that lung dynamic compliance(Cydn-min)was reduced in 1-NP-exposed mice.Conversely,inspiratory resistance(Ri)and expiratory resistance(Re)were elevated in 1-NP-exposed mice.Mechanistically,cell migration and invasion were accelerated in 1-NP-exposed pulmonary epithelial cells.In addition,E-cadherin,an epithelial marker,was downregulated,and vimentin,a-SMA and N-cadherin,three mesenchymal markers,were upregulated in 1-NP-exposed pulmonary epithelial cells.Although TGF-β wasn’t altered,phosphorylated Smad2/3were enhanced in 1-NP-exposed pulmonary epithelial cells.Moreover,reactive oxygen species were increased and endoplasmic reticulum stress were activated in 1-NP-exposed human non-small cell lung cancer cells.Conclusion: Long-term exposure to 1-NP can cause lung interstitial inflammation,epithelial-mesenchymal transition and pulmonary fibrosis.The endoplasmic reticulum stress induced by reactive oxygen species at least partially led to 1-NPinduced epithelial-mesenchymal transition and pulmonary interstitial fibrosis. |
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