| BackgroundUltrafine particulate,a common industrial substance and air pollutant,has a particle size of less than 1 μm.Because of its small size,it has more drastic biological health effects.The common ultrafine particles are carbon-related ultrafine particles.CBNPs is the most traditional and widely used material due to its special physicochemical properties.BaP is the most typical representative substance in PAHs,which is found in coal tar.Bap is produced by automobile exhaust and fuel combustion.In recent years,CBNPs and BaP have been extensively studied in the field of epidemiology and toxicology.They have been found to be closely related to the respiratory,circulatory,cardiovascular,endocrine,nervous and reproductive systems.When CBNPs and BaP are inhaled,the primary target is the respiratory system.Exogenous chemicals in the environment are rarely present alone and are ingested in the form of compounds.Several studies have shown that CBNPs and BaP can cause respiratory and lung inflammation in animals when inhaled.In vitro experiments have also shown that CBNPs and BaP can induce an inflammatory response in cells.However,the disease outcome after the combined exposure of CBNPs and BaP has not been studied.The mechanisms of CBNPs and BaP combined exposure in cells or animals have not been investigated.The role and mechanisms of non-coding RNA in the combined exposure of CBNPs and BaP have not been developed.In recent years,more and more researches on non-coding RNA have been carried out.Studies from miRNA to lncRNA to circRNA have gradually enriched the scope of epigenetics.The function of non-coding RNAs are gradually improved.Non-coding RNAs can play an important role in transcriptional regulation,post-transcriptional regulation,RNA modification and even mRNA translation.The study of the function and mechanism of non-coding RNA in CBNPs and BaP combined exposure can further explain the mechanism of airway inflammation caused by CBNPs and BaP combined exposure.It provides new evidence for a more comprehensive understanding of exogenous chemicals and the regulatory mechanisms of non-coding RNAs.circRNA is a research hotspot of non-coding RNA,which is covalently closed circRNA produced by reverse splicing.It has no 3 ’and 5’ ends and no ployA tail.It plays a regulatory role at both transcriptional and post-transcriptional levels.The regulatory role of circRNA in CBNPs and BaP composite exposure has not been reported.We selected circRNA with regulatory function,tested its regulatory function in CBNPs and BaP exposure alone and in combination,and studied its mechanism of action,providing a new perspective for further understanding of the role and mechanism of airway inflammation induced by nano particles.MethodsIn the process of inflammation caused by combined exposure of CBNPs and BaP,16 HBE cells,BEAS-2B cells and BALB/c mice were mainly selected as the study subjects.CCK8 kit and LDH kit were used to detect the activity of cells.The differentially expressed inflammatory factors were found by the inflammatory factor chip.The expression of differentially expressed inflammatory factors was verified by RT-qPCR,ELISA kit.The ability of adhesion were investigated with leukocyte adhesion experiment.HE staining and immunohistochemistry were used to evaluate the pulmonary inflammation of the mice exposed to the combination of CBNPs and BaP.High-throughput sequencing was used to select differentially expressed circ RNA.Cytoplasmic or nuclear localization of circRNA was performed with FISH experiment.The anti-inflammatory function of circFAM117 was verified by interference and overexpression experiments.The interaction between circFAM117 and miR-204-5p,miR-204-5p and IL-11 were determined by double luciferase reporter assay.Overexpression both of circFAM117 and miR-204-5p further clarified the interaction between circFAM117 and miR-204-5p.At last,the proteins on the inflammatory pathway were analyzed by Western blot.ResultsTransmission electron microscopy(TEM)showed that CBNPs and BaP-CBNPs could enter 16 HBE cells and the lung tissues of mice.On the time gradient of 24-72 h,CBNPs and BaP-CBNPs inhibited cell activity on the concentration gradient of 2.5 μg/ml-80 μg/ml,and BaP inhibited cell activity on the concentration gradient of 2.5 ng/ml-80 ng/ml(p < 0.05),showing a dose-response relationship,and the inhibition effect was stronger in the group exposed to BaP alone(p < 0.05).Results of inflammatory factor microarray cluster analysis showed that IL-6,IL-11 and ICAM-1 in CBNPs,BaP and BaP-CBNPs exposure groups were equally divided in the same group(p < 0.05).The BaP exposure group alone had the ability to cause more extensive inflammatory factor changes(p < 0.05).IL-6,IL-11,and ICAM-1 showed some commonalities in the CBNPs,BaP,and BaP-CBNPs exposure groups.After 72 h of exposure to IL-6,IL-11 and ICAM-1,the time-dose response relationship in mRNA and protein levels was more obvious,presenting a gradually increased state(p < 0.05).After 72 h of exposure,more U937 cells were adhered in the BaP group than in the other two groups(p < 0.05).The mice were poisoned by calculating the actual exposure.8 ng/d and 80 ng/d were used in the CBNPs and BaP-CBNPs groups,and 0.008 ng/d and 0.08 ng/d were used in the BaP group,which nasal drops were administered for 7 d and 14 d.The mRNA levels of IL-6,IL-11 and ICAM-1 were detected from mouse lung tissues.The results showed that the mRNA levels of IL-6,IL-11 and ICAM-1 increased with time and dose gradient(p < 0.05).ELISA of mouse alveolar lavage solution showed increased levels of IL-6,IL-11 and ICAM-1(p < 0.05).We scored the pathological sections.The results showed that the score of the high-doze BaP group was higher than that of other groups(p < 0.05).Immunohistochemical results showed that the protein indexes of IL-6,IL-11 and ICAM-1 in the BaP group were higher than those in the other two groups(p < 0.05).Through high-throughput sequencing,we found the gene circFAM117 with high expression in the CBNPs,BaP and BaP-CBNPs infection group.Treatment with circFAM117 and CBNPs,BaP or BaP-CBNPs exposure,compared to the Scrambled combined exogenous chemical exposure group,the cell vitality was increased(p < 0.05).After overexpression of circFAM117 combined with exposure to exogenous chemicals,cell activity was reduced(p < 0.05).Treatment with circFAM117 with CBNPs,BaP and BaP-CBNPs exposure,compared to the Scrambled combined exogenous chemical exposure group,IL-6,IL-8 and ICAM-1 in the mRNA and protein expression level was reduced(p < 0.05).After overexpression of circFAM117 combined with exposure to exogenous chemicals,the expression of IL-6,IL-8,and ICAM-1 increased at mRNA and protein levels compared the group only treated with exposure to exogenous chemicals(p < 0.05).In Scrambled+BaP / Vec+Ba P groups the expression of IL-6,IL-11,ICAM-1 mRNA level and protein level were significantly higher than Scrambled and Vec treated with BaP or BaP-CBNPs groups after exposure(p < 0.05).The results revealed the proinflammatory function of circFAM117 after exposure to CBNPs and BaP-CBNPs.Through the double luciferase reporter gene experiment,we found that circFAM117 could directly bind to miR-204-5p,miR-204-5p could directly bind to IL-11,and there was a reverse regulation relationship between circFAM117 and miR-204-5p.When the circFAM117 was overexpressed alone,the expression of IL-11、IL-6 and ICAM-1 increased at protein and mRNA levels(p < 0.05).When the miR-204-5p was overexpressed alone,the expression of IL-11、IL-6 and ICAM-1 decreased at protein and mRNA levels(p < 0.05).When both circFAM117 and miR-204-5p were overexpressed,the expression effect at the protein and mRNA levels was between the two genes that were overexpressed alone(p < 0.05).When circFAM117 was overexpressed alone,the expression of p-STAT3 increased in each infected group(p < 0.05).When miR-204-5p was overexpressed alone,the expression of p-STAT3 decreased in each infected group(p < 0.05).When both circFAM117 and miR-204-5p were overexpressed,the expression of p-STAT3 in each infected group was between those two groups(p < 0.05).Conclusions1.CBNPs,BaP and BaP-CBNPs can promote inflammatory responses in 16 HBE cells,BEAS-2B cells,and BALB/c mice.2.Compared with CBNPs and BaP-CBNPs,the BaP induces stronger inflammatory response.3.CircFAM117 has the function of proinflammatory genes in the inflammatory process of 16 HBE cells induced by CBNPs Bap and BaP-CBNPs.4.In the inflammatory process of 16 HBE cells induced by exposure to CBNPs BaP and BaP-CBNPs,circFAM117 targeted mi R-204-5p and played an proinflammatory effect through the IL-11 /STAT3 axis. |
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