Mechanisms Of The Acute Airway Inflammation Induced By PM_2.5 And The Anti-inflammatory Effect Of Compound Essential Oils

Purpose: PM_2.5 has become one of the most critical issues in modern society,however,the mechanisms of PM_2.5-induced acute lung inflammation remains unclear.Furthermore,there is no proven therapy countering PM_2.5-induced inflammation.This study was aimed at exploring the potential mechanism of PM_2.5-induced lung inflammation and the effects of compound essential oils?CEOs?treatment on PM_2.5-induced lung inflammation.Methods: 1.Analysis of the composition of CEOs: The chemical compositions of CEOs were analyzed by gas chromatography-mass spectrometry?GC-MS?;2.PM_2.5 collection,analysis and preparation procedure: PM_2.5 was collected with a high volume air sampler in Langfang?Hebei?from January to March 2013 and the components of PM_2.5 sample were then detected.Filters containing PM_2.5 were administrated by ultrasonic sonication in sterile distilled water for 2 h.Detached PM_2.5 was then vacuum-freeze dried,weighed and suspended in certain amount of sterile saline to achieve the PM_2.5 suspension with a concentration of 10 mg/ml for mouse model;3.Grouping of experimental animals and establishment of mouse model: All 96 Balb/c mice were randomly divided into four groups and housed with food and water ad libitum.Mice in control group were treated with 50?L saline on day 0,2 of the study.Mice in PM_2.5 group were exposed with PM_2.5 suspension as the same protocol above.Mice in PM_2.5+saline group suffered PM_2.5 exposure and administrated with 200?Lsaline via static inhalation for 30 min.per day since the day before PM_2.5 exposure.Mice in PM_2.5+CEOs group exposed to PM_2.5 and treated with 200?L saline with 2 drops of CEOs via static inhalation for 30 min.per day since the day before PM_2.5 exposure;4.Measurement of inflammatory indicators in lung tissues: Animals were sacrificed on day 3,7 and 14 after PM_2.5 exposure,then lung tissues,BALF,spleen tissues and serum were obtained.Phathological alterations of lung tissues were measured under H&E staining;IL-1?,TLR4,My D88,Nf-?B,NLRP3,P2X7 m RNA levels were detected by q-PCR;TLR4,NLRP3 protein levels were assessed using immunohistochemical staining;Total and differential cell count of BALF were performed;IL-1? protein concentration of BALF was evaluated by an ELISA kit;5.Transcription factors expressions of T cells in spleen tissues and cytokines expressions in serum: m RNA expressions of T-bet,GATA3,ROR-?t and Foxp3 which respectively belong to Th1,Th2,Th17 and Treg cells were measured by q-PCR;Concentrations of IFN-?,IL-4 in BALF were detected by ELISA kits.Results: 1.The chemical compositions of CEOs: 25 compounds were identified.The main constituents of CEOs were Eucalyptol?16.7%?,?-Pinene?16.54%?,Menthol?12.86%?and Cinene?7.37%?;2.The characterization of PM_2.5: In the PM_2.5 sample,organic carbon and element carbon consisted carbolic component.NH4+,NO3-and SO42-represented the main component of water soluble ions,meanwhile Ca,Na,Al and Mg consisted main mental elements.Among the polycyclic aromatic hydrocarbons,Fluoranthene,pyrene and Benzo [b] fluoranthene represented main components of PAH;3.Pathological examination: Cells infiltration and alveolar walls changes remarkably appeared in PM_2.5 group at all three time-points compared with those in control group.Meanwhile,CEOs significantly reduced cells infiltration and alveolar walls changes time-dependently;4.Cells count in BALF: Total cells,neutrophils,lymphocytes and macrophages of BALF were remarkably increased in PM_2.5 group than those in control group.Cell count in BALF in PM_2.5+CEOs group showed a significantly decrease than those in PM_2.5+saline group;5.Expressions of lung inflammation-related indicator: IL-1?,TLR4,My D88,Nf-?B,NLRP3 and P2X7 m RNA expressions of lung tissueswere up-regulated in PM_2.5 group at all three time-points.Besides,IL-1?,TLR4 and NLRP3 protein level were increased as well.IL-1?,TLR4,My D88,Nf-?B,NLRP3 and P2X7 m RNA expressions of lung tissues were down-regulated in PM_2.5+CEOs group compared with those in PM_2.5+saline group at all three time-points.Similarly,CEOs also decreased IL-1?,TLR4 and NLRP3 protein level;6.Transcription factors of T cells in spleen tissues and cytokines contents in serum: CEOs significantly decreased m RNA expressions of T-bet,GATA3,ROR-?t and Foxp3 in spleen tissues and reduced IFN-?,IL-4 levels of BALF.Conclusion: PM_2.5 might induce acute lung inflammation by the increase of IL-1? production via the activation of TLR4/My D88 signaling pathway and NLRP3 inflammasome.In addition,CEOs might reduce PM_2.5-induced acute lung inflammation by the decrease of IL-1? production via the inhibition of TLR4/My D88 signaling pathway and NLRP3 inflammasome and suppressing the Th immune responses.Our study describes a potential mechanism of PM_2.5-induced acute lung inflammtion and might bring about novel therapies for the inflammatory diseases associated with PM_2.5 inhalation.