Regulatory Mechanism Of MiR-140-5p/TLR4 In PM_2.5-induced Inflammatory Response Of Bronchial Epithelial Cells

Objective:In the process of economic development,factors such as burning of fossil fuels,emissions of industrial waste gas and reduction of vegetation coverage have jointly caused the increase of PM_2.5concentrations in atmosphere.Human inhales PM_2.5 mainly through respiratory movement.The bronchus have immune defense functions and secrete mucus,which contains immunoglobulin,to clear antigens.The ciliary movement on the bronchial surface and the cough reflex contribute to discharge of foreign bodies.In addition,the bronchus can also humidify and warm the inhaled gas.Studies in vitro using bronchial epithelial cells as model cells have shown that the main pathophysiological mechanisms currently known for PM_2.5are oxidative stress,DNA damage,activation of inflammatory signaling pathways and epigenetic changes.Toll like receptor 4（TLR4）is a key signal protein in the innate immune system,and micro RNA is one of the most important contents of epigenetics.Therefore,in this study,human bronchial epithelial cells（16HBE）were selected as the model of PM_2.5 exposure,in order to clarify the mechanism of TLR4signaling pathway in PM_2.5-induced bronchial inflammation response.Micro RNA molecules were used as the research object to verify whether there will be a differential expression after PM_2.5 intervention,and to explore its specific regulatory mechanism on expression of TLR4 gene.Methods:（1）The PM_2.5 sampling site was in Shijiazhuang,Hebei province,China;laser particle size analyzer was used to detect the particle size distribution of PM_2.5 sample;the PM_2.5 phase was identified by X-ray diffraction spectrometry;the concentration of Al,As and Fe in PM_2.5 were determined using inductively coupled plasma spectrometer;gel limulus reagent was used for qualitative detection of endotoxin in PM_2.5supernatant.（2）16HBE cells were exposed to different doses of PM_2.5suspension for 12,24 and 48 h,the relative viability of 16HBE cells was detected by CCK-8 cell counting kit;After treating the cells with a PM_2.5suspension at a dose of 100μg/m L for 24 h,calcein-AM/propidium iodide double staining kit combined with laser confocal microscope was used to observe the 16HBE cells viability ratio,Hoechst 33342 staining solution together with inverted fluorescence microscope was used to observe the apoptotic bodies,Wright’s-Giemsa staining and field emission scanning electron microscope was used to observe the morphology of 16HBE cells.（3）TAK 242（1μg/m L）,a specific inhibitor of TLR4,was used to pretreat the cells for 1 h to inhibit the activation of TLR4 signaling pathway.Real-time fluorescence quantitative PCR was used to detect interleukin-6（IL-6）and CXC motif chemokine 8（CXCL8）m RNA in the cell lysate,and enzyme-linked immunosorbent assay kit was used to detect the protein content of IL-6 and CXCL 8 in the supernatant of cells.（4）After the cells were treated with PM_2.5 suspensions of 0,25,50,75 and 100μg/m L for 24hours,the protein concentrations of TLR4,My D88,IKK and p-p65 were detected by Western blot.（5）Target Scan,mi RWalk and Star Base were used to predict the targeted regulatory micro RNA of TLR4 gene,real-time fluorescent quantitative PCR was used to detect the expression of mi R-140-5p in cells lysate treated with PM_2.5 suspension（75μg/m L,24 h）;After transfection of mi R-140-5p mimic and inhibitor,Western blot was used to detect the expression of TLR4 protein.（6）The dual-luciferase reporter assay was used to verify the interaction site between mi R-140-5p and TLR4.Results:（1）The cumulative percentage of particles in PM_2.5 sample smaller than _2.5 microns was 94.47%;and the main phase of it was quartz,and contained some calcite;the main elements were Al,As,Fe and Zn;the endotoxin titer in the PM_2.5 supernatant was higher than 2.5 EU/m L.（2）The relative viability of 16HBE cells decreased with the prolongation of PM_2.5 exposure time and the increase of dose;after the cells was treated with 100μg/m L PM_2.5 suspension for 24 h,Wright’s-Giemsa staining showed that boundary of 16HBE cells was blurred,and cytoplasm was dissolved.Scanning electron microscopy depicted that the cells were collapsed and particles were attached to the surface of the cell membrane.The Hoechst 33342 staining showed that the nucleus was broken into fragments,chromatin was concentrated,and there were obvious apoptotic bodies appeared.After AM-PI probes labeled intracellular markers,the dead cells in the poisoned group showed bright red fluorescence.（3）PM_2.5stimulated high expression of inflammatory factors（IL-6 and CXCL 8）in16HBE cells from m RNA and protein levels;after pretreatment with TAK242 for 1 hour,the protein expression of IL-6 and CXCL 8 in the supernatant of 16HBE cells treated with PM_2.5 suspension at a dose of 50,75 and 100μg/m L decreased,the difference was statistically significant.（4）PM_2.5 promoted the expression of TLR4,My D88,IKK and p-p65 protein in a dose-dependent manner.When the concentration of PM_2.5 suspension was higher than or equal to 75μg/m L,the expression level of TLR4,My D88,IKK and p-p65 protein decreased in TAK 242 pretreatment group.（5）Target Scan,mi RWalk and Star Base predicted that the upstream targeted regulatory micro RNA molecule of TLR4 gene probably was mi R-140-5p.After exposure to PM_2.5（75μg/m L）for 24 hours,the expression of mi R-140-5p nucleic acid was significantly decrease;compared with the NC group,the expression of TLR4 protein in the mimic transfection group decreased significantly,and the expression of TLR4 in the inhibitor transfection group increased（P<0.01）.（6）The overexpression of mi R-140-5p significantly reduced the luciferase activity of the TLR4 gene wild-type reporter vector（P<0.01）,while the luciferase activity of the TLR4gene mutant reporter vector was almost unchanged,these indicated that mi R-140-5p negatively regulated the expression of TLR4（binding positions:720-726）by directly binding to the 3?Untranslated Regions（3?UTR）sequence of TLR4.Conclusion:（1）PM_2.5 can induce inflammatory response in human bronchial epithelial cells through TLR4/NF-κB signaling pathway,and lipopolysaccharide（LPS）may be an important factor triggering inflammatory response.（2）Exposure to PM_2.5 can reduce the expression of mi R-140-5p,activate the TLR4 signaling pathway and aggravate the inflammatory response of cells.（3）mi R-140-5p can interact with TLR43?UTR（5?AACCACT3?）to inhibit the expression of TLR4 protein.