Protective Effect Of Compound Essential Oils Against Pyroptosis And Cell Cycle Arrest In G2 Induced By PM_2.5 Exposure Via Down-regulating ROS

Objective:In recent years,PM_2.5 exposure is the main cause of lung disease and aggravate lung diseases.However,the research on PM_2.5 is still limited to the basic inflammatory mechanism and there are few studies on pyroptosis caused by PM_2.5exposure.In this study,PM_2.5 exposure caused inflammation and oxidative stress in lung tissue and alveolar macrophages,which in turn stimulated pyroptosis,apoptosis and accompanied by cell cycle changes.In this process,the production of reactive oxygen species（ROS）is regulated by PM_2.5.At the same time,this article also discussed the effect of compound essential oils（CEOs）on the damage caused by PM_2.5 exposure.At the same time,the effect of CEOs on PM_2.5 exposure was discussed.It provides a new idea for the treatment and repair of lung injury after PM_2.5 exposure.Methods:PM_2.5 sample collection,preparation:In this study,the samples were collected by PM_2.5sampler.PM_2.5was collected from October 2015 to March 2016 in Langfang,Hebei Province.The collected PM_2.5 quartz filter were put into the double distilled water and sonicated 3-5 times for 20 minutes each time to separate and suspend PM_2.5 particles from the filter and suspended in the liquid.The weight of the filter membrane is weighed and the PM_2.5 concentration is calculated.When preparing in vivo model samples,a certain amount of PM_2.5 solution were weighed out and PM_2.5 suspension was prepared with physiological saline.The concentration of PM_2.5 is 10 mg/m L（300μg/m³）.Mixed by ultrasonic shaking before use.（1）Construction and grouping of experimental models:This study was divided into two parts:in vivo experiments and in vitro experiments.（2）In vivo experiments:24 male Balb/c mice of 6-8 weeks were randomly divided into control groups;the PM_2.5 group,mice were dynamically exposed to PM_2.5 at a concentration of 300μg/m³;the CEOs group,mice were statically inhaled with 1%CEOs before exposure.During the model construction,the mice were in an appropriate temperature,freely eating and drinking water and a regular diurnal environment.The expression of NLRP3,NF-κB,Bax,Bcl-2,Caspase-1,Caspase-3,GSDMD and IL-1βwere detected by real-time quantitative PCR（qPCR）and Western blotting.（3）In vitro experiments:The RAW264.7 cell was used to construct PM_2.5 exposure models in vitro.The RAW264.7 cell were exposed to PM_2.5 for 0 h,12 h and 24 h.The CEOs was used to cure the RAW264.7 cell which had been stimulated by PM_2.5 for 24 hours.The expression of NLRP3,NF-κB,Bax,Bcl-2,Caspase-1,Caspase-3,GSDMD and IL-1βin RAW264.7were detected by qPCR and Western blotting.The fluorescence intensity of NLRP3 and GSDMD were measured by immunofluorescence.Annexin FITC/PI was used to detect the activity and apoptosis of RAW264.7 cells.The expression of MDA and SOD in cells was detected to reflect the level of oxidative stress.The change of cell cycle and ROS were detected by flow cytometry.Results:（1）In vivo experiment:the expression of NLRP3,NF-κB,Bax,Caspase-1,Caspase-3,GSDMD and IL-1βwere significantly increased in PM_2.5 group.The level of Bcl-2decreased significantly.Compared with PM_2.5 group,the expression of NLRP3,NF-κB,Bax,Caspase-1,Caspase-3,GSDMD and IL-1βin CEOs group decreased significantly,while the expression levels of Bcl-2 increased slightly.（2）In vitro experiment:the results of qPCR were consistent with that of in vivo experiment.In PM_2.5 group,the expression of NLRP3,NF-κB,Bax,Caspase-1,Caspase-3,GSDMD and IL-1βincreased in 12 h and 24 h,while the expression of Bcl-2 decreased significantly.Compared with PM_2.5 group,the expression of NLRP3,NF-κB,Bax,Caspase-1,Caspase-3,GSDMD and IL-1βin CEOs group decreased significantly,while the level of Bcl-2 increased but no statistical significance.However,Bax/Bcl-2 ratio decreased,which had statistically significant.The results of immunofluorescence showed that the trend of fluorescence intensity was consistent with the above experiments.The results of Bax/Bcl-2 of qPCR and Western blotting in vitro were confirmed by Annexin FITC/PI.The detection of oxidative stress by DCFH-DA showed that RAW264.7 had oxidative stress after exposed to PM_2.5.The detection results of MDA and SOD confirmed the above conclusion,while the CEOs repaired the damage caused by PM_2.5.The results of cell cycle showed that the proportion of RAW264.7 cells in G2 phase increased when exposed to PM_2.5,while the CEOs alleviated that.After adding the inhibitor NAC of ROS,Western blotting showed that the inflammation,apoptosis,pyroptosis and cell cycle arrest in G2 caused by PM_2.5 through down-regulating ROS production.Conclusion:This study revealed that PM_2.5 exposure is based on inflammation and oxidative stress in lung tissues and alveolar macrophages,which subsequently triggered pyroptosis,apoptosis and cell cycle arrest in G2.In this process,ROS production regulated by PM_2.5 exposure.At the same time,CEOs alleviated the damage caused by PM_2.5 exposure.The protective effect of CEOs against pyroptosis and cell cycle arrest in G2 induced by PM_2.5 exposure via down-regulating ROS.