LncRNA Mediate Macrophage Activation And The Repairing Effect Of Compound Essential Oils On Its Inflammation Under PM_2.5 Exposure

Objective:Atmospheric PM_2.5 exposure as the main cause of lung diseases has attracted much attention in recent years.Long non-coding RNA?lnc RNA?is widely involved in the occurrence and development of diseases.However,for PM_2.5 pollution,research was mainly limited to apparent damage,and there were a few studies on the mechanism of lnc RNA caused by PM_2.5 exposure.In this study,microarray was used to screen differentially expressed lnc RNAs in lung tissues of mice exposed to PM_2.5,and to explore the potential mechanism of lnc RNAs involved in the regulation of macrophage activity in lung tissues of mice exposed to PM_2.5.Furthermore,it was explored whether the compound essential oils?CEOs?can intervene in lung inflammation caused by macrophages to achieve the purpose of repair and treatment.Methods:?1?PM_2.5 sample collection,preparation and analysis:The samples in this study were collected by PM_2.5 sampler in the winter of 2015 and the spring of 2016 in Langfang city,Hebei province.The collected PM_2.5 quartz filter were put into the ethanol solution 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,and then the solution was evaporated,freezed dry.When preparing in vivo model samples,a certain amount of PM_2.5 particles were weighed out,and PM_2.5suspension was prepared using physiological saline,and mixed by ultrasonic shaking before use.?2?Construction and grouping of experimental models:This study was divided into two parts:in vivo experiments and in vitro experiments.In vitro experiments:The RAW264.7 and THP-1 cells were used to construct PM_2.5 exposure models in vitro.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 300?g/m³;the CEOs group,mice were statically inhaled with 1%compound essential oil before exposure.During the model construction,the mice were in an appropriate temperature,freely eating and drinking water,and a regular diurnal environment.?3?Expression level of lnc RNA in lung tissue of mice exposed to PM_2.5:a)Microarray was used to detect differentially expressed lnc RNA in mouse lung tissue exposed to PM_2.5;b)Quantitative Real-time PCR?q RT-PCR?was further used to detect and determine the expression of lnc Gm16410 in mouse alveolar macrophages/RAW264.7 exposed to PM_2.5.?4?The mechanism of lnc Gm16410 in PM_2.5-induced macrophage activation:a)Overexpression of lnc Gm16410 on RAW264.7 cells by plasmid transfection;b)q RT-PCR,Cell Counting Kit?CCK8?,Annexin-FITC/PI were used to detect the transfection efficiency of lnc Gm16410,the changes of RAW264.7 cells'viability and apoptosis level by lnc Gm16410;c)Western Blot,q RT-PCR and immunofluorescence to detect the effect of lnc Gm16410 on the active change of RAW264.7.?5?SRC participated in the mechanism of lnc Gm16410-induced macrophage activation:a)SRC protein expression was inhibited by the inhibitor Dasatinib;b)WB detection of the expression of SRC by lnc Gm16410 regulated;c)WB,immunofluorescence detection of the effect of SRC on the active change of RAW264.7.?6?SRC activates PI3K/AKT pathway:WB was used to detect the effect of before and after inhibition SRC on PI3K/AKT pathway and downstream protein in RAW264.7 and THP-1 cells.?7?CEOs was involved in repairing lung inflammation caused by macrophages exposed to PM_2.5:a)Hematoxylin-eosin staining?HE?for detecting pathological changes in mouse lung tissue;b)Detection of macrophage activation level in mouse lung tissue by tissue immunofluorescence;c)q RT-PCR and WB were used to detect the difference of inflammatory expression in mouse lung tissue after PM_2.5 exposure and CEOs repair;d)Immunohistochemistry and WB were used to detect the effects of PI3K/AKT pathway and downstream proteins in mouse lung tissue.Results:?1?The composition of PM_2.5 was found that it mainly contains carbon components such as organic carbon and inorganic carbon,and a small amount of water-soluble ions,metal elements and heavy metal elements.?2?307 lnc RNAs were differentially expressed in the PM_2.5-treated group compared with the control group.201 of these lnc RNAs were upregulated,and 106 were downregulated in the lung of mouse PM_2.5 model.The expressions of lnc Gm16410 were decreased in mouse lung tissue,and similarly,it was significantly lower in PM_2.5-treated than control group in the RAW264.7.?3?Through CCK8 cell proliferation assay,Annexin-FITC/PI,it was found that the proliferation of cells in vitro showed an upward trend after lnc Gm16410 overexpression.Experimental results such as WB and cell immunofluorescence showed that overexpression of lnc Gm16410 was involved in the regulation of RAW264.7 activation induced by PM_2.5.?4?PM_2.5 regulated the expression of SRC in RAW264.7,SRC may be involved in regulating the process of PM_2.5-induced RAW264.7 activation.SRC acted as an upstream molecule of the PI3K/AKT pathway,When the specific expression of SRC was downregulated,it was found that the expression of key proteins in the PI3K/AKT pathway was inhibited and the activation of the pathway was inhibited.When lnc Gm16410 was overexpressed,it was found that the expression of SRC protein in RAW264.7 was significantly decreased,thereby participating in the regulation of RAW264.7 activation.?5?While PM_2.5 regulated the activation of RAW264.7,it also released inflammatory factors such as TNF-?,IL-1?,and IL-6,which disrupted the lung tissue structure of mice and increase inflammatory cell exudation.lnc Gm16410 and CEOs can attenuate PM_2.5-induced macrophage activation and inflammatory factor secretion.At the same time,after CEOs treatment,lung tissue inflammatory cell exudation was reduced,lung tissue returns to normal,and lung inflammation was significantly improved.Conclusion:This study revealed that PM_2.5 exposure induces macrophage activation,which caused the release of inflammatory factors,which in turn led to the process of lung inflammation in mice;lnc Gm16410 inhibited the PI3K/AKT signaling pathway by regulating SRC expression in this process.It exerted the effect of controlling PM_2.5exposure on macrophage injury;and CEOs can also extenuate lung inflammation caused by PM_2.5 exposure by reducing the release of inflammatory factors.