The Role Of HDAC6 In Inflammatory Responses Caused By Carbon Black Particles In Human Bronchial Epithelial Cells

Objective: To investigate the role of histone deacetylase 6 in the inflammatory response in bronchial epithelials cells induced by carbon black particles.Methods: The bronchial epithelial cells(16HBE)were treated with carbon black particles(Printex 90)at concentrations of 12.5、25、50、100 μg/ml for 12,24,36,48 h.Cell viability was detected by CCK-8(Cell Counting Kit-8)to determine the time of exposure.Then the 16 HBE cells were pretreated by HDAC6 si RNA,and the experiment was divided into six groups,cell blank control group,25 μg/ml carbon black particles group,50 μg/ml carbon black particles group,HDAC6 si RNA interference blank group,25 μg/ml carbon black particles group + HDAC6 si RNA interference group,50μg/ml carbon black particles group + HDAC6 si RNA Interference group,after 16 HBE cells infected with HDAC6,collect infected cells and supernatant.Using RT-PCR to detect the relative expression level of histone deacetylase 6(HDAC6)Mrna,the expression levels of HDAC6,intraflagellar transporter protein 88(IFT88)and autophagosomal protein LC3-II were assessed by Western blot after exposure to carbon black particles.ELISA was used to detect the secretions of cytokines IL-8,IL-1β,TNF-α,IL-6,TGF-β1 and IL-17 A in the cell supernatant.Results:(1)At each time point,The 16 HBE cell viability was significantly decreased in a clear dose-response relationship as the concentration of carbon black particles increased induced by carbon black particles.With the prolonged exposure time,the cell viability of 16 HBE also gradually decreased.After 16 HBE cells were exposed carbon black particles for 24 h,the relative expression of HDAC6 m RNA increased and then decreased,reached the highest at the concentration range of 12.5 μg/ml,returned to normal level when the concentration of carbon black reached 100 μg/ml.The expression of LC3-Ⅱ protein in 16 HBE cells was increased by all concentrations of carbon black particles,and the LC3-Ⅱ protein level was significantly higher than that in blank control group,when cells were treated with carbon black particles at 100 μg/ml(P<0.05).while IFT88 protein levels gradually decreased,compared with blank control group,when the carbon black particles at concentration of 50 μg/ml or higher,IFT88 protein expression levels were significantly increased(P<0.05).Compared with the blank control group,the expression levels of inflammatory cytokines IL-8,IL-1β,TGF-β1,and IL-17 A increased in cell supernatants showing a significant dose-response manner(P<0.05),while the expression levels of TNF-α and IL-6 were slightly higher than the blank control group(P>0.05).(2)Compared with normal cells,HDAC6 m RNA expression level was significantly decreased(P<0.05)in the group of carbon black particles + HDAC6 si RNA.The level of IFT88 protein in 50 μg/ml carbon black particles + HDAC6 si RNA interference group was significantly higher than that in HDAC6 si RNA interference blank group(P<0.05),but the expression level of autophagosome protein LC3-II was nomal(P>0.05).Compared with HDAC6 si RNA interference blank group,Cell in 50 μg/ml carbon black particles + HDAC6 si RNA interference group released IL-8,IL-6 and TGF-β1 levels were significantly increased.while TNF-αsignificantly decreased(P<0.05).Conclusion: Carbon black particles can lead to a significant decrease in the viability of 16 HBE cells,increase the expression of HDAC6 protein and cilia autophagy,and induce IL-8,IL-1β,TGF-β1 and IL-17 A release significantly increased.HDAC6 si RNA interference inhibited the level of autophagy induced by carbon black particles in 16 HBE cells,increased the expression of IFT88,and decreased the levels of TNF-αsecreted in cells.while IL-8,IL-6,and TGF-β1 inflammatory cytokines increased.Suggestting that HDAC6 may participate in the process of pulmonary inflammatory response inducd by carbon black particles by affecting the level of ciliary autophagy.