N~6-Methyladenosine-dependent MicroRNA-126 Processing Drove The Development Of Pulmonary Fibrosis Induced By Nanoscale Carbon Black Particles In Rats

Objective:Carbon black?CB?is a kind of amorphous carbon produced by incomplete combustion or thermal cracking of hydrocarbons under conditions of insufficient air.CB is widely used due to its good stability and inertness,such as black dye for the manufacture of inks,paints,and reinforcing agent for rubber.In toxicological study,CB is often used as a negative control because of its low toxicity and low solubility.However,study has shown that CB could induce inflammation and histopathological damage in lung,like pulmonary fibrosis.In our previous study,we found the thickening of the alveolar wall and collagen deposition after CB inhalation for 14days in mice.Though the fibrosis in lung could be induced by CB particles,the mechanism of fibrosis has not been known clearly.Micro RNAs?mi RNAs?,a kind of small non-coding RNAs,negatively regulates gene expression by binding to the 3'-untranslated region?3'-UTR?of their target gene m RNAs.Mi RNAs play important roles in almost all of physiology and pathophysiology process,including apoptosis,cell differentiation and proliferation as well as fibrosis.N6-methyladenine?m⁶A?is a dynamically reversible modification of RNA,and it has become a new research hotspot as the most abundant RNA modification method in eukaryotes.m⁶A plays a vital role in post-transcriptional regulation and regulate the mi RNA maturation process.Here,we established a pulmonary fibrosis model in rats after CB inhalation and explored its possible mechanism,in order to provide scientific basis for the research of particulate matter to health and the risk assessment of the health of occupational populations.Methods:1. Characterization of carbon blackThe size and morphology of the CB were measured using a Tecnai G220transmission electron microscope.The surface morphology of CB was observed by a scanning electron microscope.The specific surface area of the CB particles was calculated using the Brunauer-Emmett-Teller?BET?adsorption isotherm.2. Establishment of carbon black inhalation model in ratsThe rats were random Ly divided into control,exposure and recovery groups.Rats individually placed in nose-only exposure tanks,for 6 hours/day,respectively.The rats in 5 mg/m³ exposure group were exposed for a continuous inhalation for 28 days.And rats in 30 mg/m³ exposure group were exposed for a continuous inhalation for 14 days,28 days and 90 days,respectively.Rats in the recovery group were exposed to CB for 90 days and recovered for another 14 days.At the same time,the control rats were exposed to filtered air for 6 hours/day.3. Cell culture and treatmentThe normal human bronchial epithelial cell lines?16HBE?were cultured in Dulbecco's Modified Eagle's Medium?DMEM?supplemented with 10%bovine growth serum and 100 U/m L penicillin in 5%CO₂ at 37?.16HBE cells in logarithmic growth phase were treated with CB for 24 hours at doses of 50,100,and 200?g/m L,respectively.DMEM with 0.04%Tween 80 served as a negative control.4.Cell transfectionFor mi R-126 overexpression,16HBE cells were trans-fected with 50 n M of mi R-126 mimic or mi RNA negative control mimic?NC mimic?for 48hours,respectively.For PIK3R2 knockdown,16HBE cells were transfected with PIK3R2 si RNAfor 48 hours.5.Pulmonary function testAssessment of pulmonary function was evaluated using the Ani Res2005Lung Function System.Briefly,the rats were anesthetized with pentobarbital sodium?50 mg/kg body weight?and tracheostomized,intubated and put in a sealed whole-body plethysmograph that connected to an Ani Res2005 data collection and analysis system for monitoring respiratory mechanics.6.HistopathologyThe rat lungs were fixed with 4%paraformaldehyde at 4? and embedded in paraffin before sectioning into 5?m-thick slices.The resulting slides were stained with hematoxylin and eosin?H&E?to evaluate pathological changes.Masson Trichrome stain to assess collagen deposition.7.Effect of CB particles on expression of fibrosis indicators of pulmonary fibrosisThe expressions of?-SMA,vimentin and Collagen-I protein were detected by immunohistochemistry and Western blot.8.Hydroxyproline assayHydroxyproline?alkali hydrolysis?assay kit was used to detect the changes of hydroxyproline content in the lung from each group after exposure to CB.9.Western blotThe lungs in rats were homogenized in RIPA buffer containing 1%PMSF using a high throughput tissue grinder.PI3K,AKT,p-AKT,mTOR,p-mTOR and p70S6K were detected by Western blot.10.Quantitative reverse transcriptase polymerase chain reaction?q RT-PCR? analysisTotal RNA from lung and 16HBE cells was isolated using TRIzol reagent.And c DNA was obtained by reverse transcription with mi RNA One Script TM c DNA Synthesis Kit or Go Script^TM.mi RNA-126?pri-mi RNA-126?METTL3 and METTL14 qualification were performed with Gotaq q PCR Mix following the amplification instructions.11.RNA immunoprecipitation?RIP?The lungs were teased apart in ice-cold PBS using a Dounce homogenizer.The magnetic beads were incubated with rabbit DGCR8 antibody for 30minutes at room temperature,then thoroughly mixed with lysis buffer and incubated overnight at 4?.The protein-RNA complex was digested with proteinase K in order to purify the RNA.Then RNA was extracted with phenol:chloroform:isoamyl alcohol?125:24:1?,and subjected to reverse transcription.Finally,the pri-mi RNA-126 was analyzed by q RT-PCR.Results:1.Characterization of carbon blackTEM confirmed that the CB particles contained clusters composed of smaller particles having a size of 30 to 50 nm.The CB particles constitute aggregates of tens to hundreds of nanometers,but no intermediate structure was observed.The BET results showed that the surface area of the CB particles is about 74.85 m²/g.2.Establishment of carbon black inhalation model in ratsDuring the course of the experiment,all of the rats survived and were generally in good condition;no significant toxic effects were observed during the exposure and recovery.3.Effects of Carbon Black on Coefficients of Lung Changes in RatsAfter CB inhalation,the coefficients of lung were significantly increased compared with the control group since 14th day after CB treatment?P<0.05?.Besides,the coefficients of lung in the recovery group were still higher than control group.After exposure for 28 days,the coefficients of lung were significantly increased in a dose-dependent manner.4. Morphological changes of lung in rats after CB exposure.After exposure of 30mg/m³ for 14 days,28 days and 90 days,carbon black particles and thickened alveolar walls were found in the rat lung tissue.CB particles,macrophages and mononuclear cells were aggregated in the alveolar space.In the CB treatment groups,marked increase in collagen fibers had been found since 14th.In the recovery group,there are still many CB particles in the alveolar interstitial space and a large number of inflammatory cells infiltrations.The fibrous nodules in the lungs still existed.The pathological changes in the 5 mg/m³ group after 28 days of exposure were milder than 30 mg/m³ group.5. Pulmonary function testIn the present study,We also performed pulmonary function test and observed a declination of lung function since 28th day after CB treatment?P<0.05?,which was reflected by an decrease in Forced Vital Capacity?FVC?,forced expiratory volume at 1s?FEV1?,FEV1/FVC%and mean mid expiratory flow?MMEF?.6. Effects of carbon black on expression of fibrosis indicatorsConsistence with the results of immunohistochemical analysis,the results of western blot and IHC showed that?-SMA,Collagen-I and vimentin had statistically increased since exposure for 28 days compared with the control?P<0.05?and gradually increased in a dose-or time-dependent manner.After14 days recovery period,the expression of?-SMA,Collagen-I and vimentin still had statistical difference compared to control group?P>0.05?.In 16HBE cell line,the levels of?-SMA,vimentin and Collagen-I in CB treatment groups statistically increased in a dose dependent manner compared with the control?P<0.05?.7. The effects of mi R-126 targeting the PI3K/AKT/mTOR signaling pathway on pulmonary fibrosis formation induced by CBThe results of Western blotting showed that PI3K and mTOR in CB treatment groups statistically increased depended on the treatment time or dose compared with the control?P<0.05?,respectively.There was no significant difference in total AKT between the control group and the exposure group?P>0.05?.At the same time,the p-AKT and p-mTOR in CB treatment groups gradually increased in a time-dependent or dose-dependent manner.To further verify the function of PI3K/AKT/mTOR signaling pathway on pulmonary fibrosis formation induced by CB,the expression of PIK3R2 was silenced in the 16HBE cells?PIK3R2 si RNA?.The results of Western blotting showed that PIK3R2 silence could lead to decreased expression of p-AKT,mTOR and p-mTOR?P<0.05?,but did not affect the level of the AKT protein?P>0.05?.The expression of PIK3R2 was downregulated when the expression of mi R-126 was upregulated in 16HBE cells?P<0.05?.8. The restraint of m⁶A methylation on the maturation processing of mi RNA-126 in rats after treatment with CB miR-126 in 16HBE cells after treatment with different concentration of CB was statistically decreased whereas unprocessed pri-mi R-126 was statistically increased?P<0.05?.After RIP assay with DGCR8 antibody,we found that DGCR8 binding to pri-mi RNA-126 was significantly reduced in the CB treatment groups?P<0.05?,suggesting that CB could inhibit DGCR8 to recognize and bind pri-mi RNA-126.After RIP was performed with m⁶A antibody,we found that the content of m⁶A-modified pri-mi RNA-126 was reduced in the CB treatment group?P<0.05?,suggesting that CB could inhibit the m⁶A modification of pri-mi RNA-126.The results of q RT-PCR showed that the expression of METTL3 and METTL14 m RNA decreased gradually with the increase of CB treatment time or dose compared with the control group?P<0.05?.Conclusions:1.After inhalation of CB,most of the carbon black particles are deposited in the lung interstitial,and a small amount is present in the alveoli.In the recovery group,there were still some CB particles deposited in the bronchioles,which indicates that the carbon black cannot be cleared in the short term.2.After CB treatment,histopathology results showed that the alveolar wall and bronchial structure were disordered.CB particles,macrophages and mononuclear cells were aggregated in the alveolar space.In the CB treatment groups,impaired alveolar structure with thickening of the lung diaphragm,fibroblast formation and a marked increase in collagen fibers had been found since 14th.The content of collagen fiber gradually increased depend on the treatment time and dose.After exposure for 90 days,we found mature fibrotic nodules in lung.Mi R-126 in rats after treatment with different time of CB was statistically decreased whereas activate PI3K/AKT/mTOR signaling pathway.3.The levels of m⁶A modification of pri-mi RNA-126 in lung after treatment with CB were decreased,which weakened the recognition of pri-mi RNA-126 by DGCR8 then inhibited the maturation of mi RNA-126.4.Carbon black particles-induced pulmonary fibrosis in rat may be related to METTL3 and METTL14 regulating the methylation of pri-mi RNA-126 and further affecting its metabolism.