The Alleviative Effect Of Compound Essential Oils Regulating Autophagy In Lung Injury Induced By PM_2.5 Exposure

Purpose:To investigate the mechanism of chronic PM_2.5 exposure to lung injury in Balb/c mice and the changes of macrophage autophagy induced by PM_2.5 exposure,and to explore the relationship between autophagy activity and PM_2.5-induced lung injury.To explain its possible regulatory mechanisms and the protective effects of compound essential oils.These findings provide new potential therapeutic strategies for PM_2.5-induced body damage.Methods:1.PM_2.5 Sample Collection,Treatment and component analysis:PM_2.5 was collected by a special PM_2.5 sampler in Langfang District,Hebei Province,China,from October 2015 to March 2016.Immersing the silica filter membrane with PM_2.5 in 70%ethanol solution,and ultrasonic for 6-8 times,each time 20-30 min,when the PM_2.5 particles were separated from the filter membrane and suspended in the solution.The PM_2.5 suspension was filtered through 8 layers of gauze,then removed by rotary evaporation and lyophilization,and placed in a refrigerator at-20°C for later use.Prepared a 10 mg/mL suspension with normal saline and PM_2.5 particles,and ultrasonically shaken before use.The component of PM_2.5 was analyzed using carbon-sulfur analyzer,ion chromatograph,spectrometer,and gas chromatography-mass spectrometer for elemental carbon,water-soluble ions,metal elements,organic carbon,and polycyclic aromatic hydrocarbon compounds,respectively.2.In vivo experiments:Balb/c mice were exposed to PM_2.5?300 mg/m³?and inhaled with 1%compound essential oils?normal saline configuration?.After the experiment,the weight of 16weeks mice and the weight of lung tissue after euthanasia were recorded,the lung coefficient was calculated,and the effects of PM_2.5 on the weight and lung of mice were analyzed.The pathological changes of lung tissue were observed by hematoxylin-eosin?HE?staining;the number of white blood cells in BALF were counted;immunohistochemistry were performance for the activition of macrophages.Transmission electron microscopy was used to detect the changes of autophagosomes in mice lung tissue after PM_2.5 exposure;The expression of beclin1,TLR4,P53,AMPK and mTOR in lung tissue was detected by real-time fluorescence quantitative q-PCR.The proteins of beclin1,LC3B,TLR4,P53,AMPK,P-AMPK,mTOR and P-mTORin lung tissue was detected by Western blotting.3.In vitro experiments:CCK-8 method was used to detet the effect of PM_2.5 on the proliferation rate of RAW264.7 cells;MDC and immunofluorescence method were used to detect the up-regulation of autophagy level of RAW264.7 cells after PM_2.5 exposure;and 3-MA was used to determine the role of autophagy that PM_2.5-induced the damage of RAW264.7 cells.TLR4 inhibitor TAK-242 was used to determine the relationship between PM_2.5-induced autophagy and TLR4 activation.After compound essential oil pretreatment,investigate compound essential oil on PM_2.5 induced inrury protective effect of the molecular mechanism.To investigate the role of TLR4-mediated P53/AMPK/mTOR pathway in PM_2.5-induced autophagy up-regulation,we used real-time qPCR to detect the expression of the autophagy gene beclin1,the pathway genes TLR4,P53,AMPK,and mTOR.,and the changes in beclin1,LC3B,and expression of TLR4,P53,AMPK,P-AMPK,mTOR,and P-mTOR were detected by Western blotting.Results:1.Component analysis of PM_2.5:The main components of PM_2.5samples were as follows:organic carbon,elemental carbon,water-soluble ions(mainly NO^3-,SO₄^2-and Na⁺),metal elements?mainly Na,Ca,Al?,heavy metals?mainlyFe,Zn and Cu?,however,polycyclic aromatic hydrocarbon compounds were not detected.2.In vivo experiments:After 16 weeks,the body weight of three groups of mice showed an upward trend,but after treated with PM_2.5 and essential oil,the decrease of partial mice weight was observed,the weight of lung tissue and lung coefficient of PM_2.5 group mice increased compared with the control group,with statistical significance,indicating that PM_2.5 may deposit in the lung,causing pulmonary edema,fibrosis and other pathological changes.HE staining and white blood cell count results showed that the hemorrhage and increased inflammatory cells in mice lung by PM_2.5,while after essential oil intervention,the lung hemorrhage and inflammatory cells has decreased.Immunohistochemical results showed that macrophage were activited by PM_2.5exposure.In PM_2.5 exposed group,transmission electron microscopy showed that autophagosomes and autophagic lysosomes appeared.Immunohistochemical results showed that LC3B expression increased.Real-time fluorescent quantitative qPCR showed that autophagy-related gene Beclin1 increased by PM_2.5 exposed.Western blot results showed that autophagy-related protein LC3B and beclin1 expression increased due to PM_2.5 exposed.These results indicated that chronic exposure of PM_2.5 resulted in the increase of autophagy level in lung tissue of mice.These results indicated that the autophagy level of mouse lung tissue incresed after PM_2.5 exposure.real-time qPCR and Western blot showed that TLR4 may mediate P53/AMPK/mTOR pathway involved in autophagy induced by PM_2.5 exposure.3.In vitro experiments:CCK-8 results showed that the survival rate of RAW264.7 cells decreased gradually with the increase of PM_2.5exposure dose;MDC,LC3B immunofluorescence staining,real-time fluorescence quantitative qPCR,Western blot results illustrate that PM_2.5 exposure induced autophagy level increased,and the autophagy level decreased significantly after treatment with autophagy inhibition 3-MA.Real-time fluorescence quantitative qPCR and Western blot showed that the expression of TLR4,P53 and AMPK genes increased and the level of mTOR gene protein decreased.After treatment with TLR4 inhibitor TAK-242,PM_2.5-induced autophagy was inhibited,the expression of TLR4,P53 and AMPK-related genes decreased significantly and the expression of mTOR gene protein increased.After compound oil treatment,the expression of autophagy-related proteins,TLR4,P53 and AMPK proteins in RAW264.7 cells were inhibited,and the expression of mTOR was increased.Consistent with in vivo experiments,the upregulation of autophagy was inhibited by PM_2.5 exposure,and the lung injury was alleviated with compound oil treatment.Conclusion:PM_2.5 exposure causes pathological changes such as hemorrhage and inflammatory cell aggregation in lung tissue,which reduces the survival rate of macrophages;and it causes up-regulation of autophagy in lung tissue and macrophages,Autophagy by PM_2.5.5 induced may be caused by activation of the TLR4receptor-mediated P53/AMPK/mTOR pathway.Compound essential oil can alleviate lung tissue and macrophage damage caused by PM_2.5 exposure;compound essential oil may improve lung and macrophage damage caused by excessive autophagy by inhibiting TLR4.