The Role Of Histone Acetylation In The Pathogenesis Of Nasal Polyps

Part ⅠThe HAT/HDAC activity in the nasal polypsObjectives:To assess the changes of HAT/HDAC activity and HDACs genes expression in the nasal polyps.Methods:HATs and HDACs activity of nuclear protein from the nasal polyps and normal nasal mucosa obtained during2012.6～2012.11were investigated using HAT activity kit and HDAC activity kit. Meanwhile, the extent of acetylation in the nasal polyps and normal nasal mucosa was investigated using immunohistochemistry. In addition, the total RNA from the nasal polyps and normal mucosa was isolated and real-time PCR was performed to assess the HDAC genes expression.Results:The ratio of the HAT/HDAC in nasal polyps(n=20) increased to1.95±0.57, in comparation to the ratio of1.19±0.33in the normal nasal mucosa (n=6)(P<0.01). HDACs activity significantly reduced in the nasal polyps(0.16±0.07/80ug) compared to tHAT (0.35±0.08/80ug) in the nasal normal tissue (P<0.01). However, we did not find the significant difference in HATs activity between nasal polyps and normal nasal mucosa. The epithelium of the nasal polyps and normal nasal mucosa was stained positive with protein acetylation, especially the epithelium of the nasal polyps. Real-time PCR results indicated that there was no significant difference in the HDACs genes expression between nasal polyps and normal nasal mucosa (P>0.05), indicating that there was no correlated association between HDACs genes expression and reduced HDACs activity in nasal polyps.Conclusions:The reduced HDACs activity caused the increased acetylation of the nasal polyps and the reduced HDACs activity was not correlated with the HDACs genes expression in the nasal polyps. The alteration of the HAT/HDAC balance may involve in the pathogenesis of nasal polyps. Part IIThe effects of the microorganism on the acetylation and the HDACs genes expression in the nasal epithelial cells in vitroObjectives:To analyze the effect of the mircoorganism on the acetylation and the HDACs genes expression in the nasal epithelial cells.Methods:0.01%protease XIV was used to digest the nasal mucosa for the nasal epithelial cells culture. The nasal epithelial cells was stimulated with LPS or Poly (I:C) for24h. The viability of the epithelial cells in response to the stimulation was assessed using CCK-8. After the stimulation, cell supernatants and the total RNA were collected. The activity of the epithelial cells in response to the stimulation was reflected by the alteration of IL-6and IL-8expression using ELISA. The acetylation of the nasal epithelial cells induced by LPS or Poly (I:C) was assessed using immunocytochemical analysis. Meanwhile, the HDACs genes expression in the epithelial cells in response to the stimulation was investigated using real-time PCR.Results:The nasal epithelial cells were successfully cultured in vitro and displayed typical cobblestone-like morphology under inverted microscope. The primary cells can be sub-cultured about7th day after primary culture. The epithelial cells of passages2and3keep cobblestone-like morphology and proliferation. LPS(≥15ug/ml) and Poly(I:C)(≥25ug/ml) reduced the cell viability. LPS (10ug/ml) or Poly (I:C)(20ug/ml) stimulation for24h could activate the epithelial cells reflected by the IL-6or IL-8expression. Both LPS and Poly (I:C) could induce the acetylation of the epithelial cells. Interstingly, we found that the acetylation of the epithelial cells was not only in the nuclear but also in the cytoplasm. We did not find any significant difference in the HDACs genes expression in the epithelial cells in response to the stimulation.Conclusions:LPS (10ug/ml) and Poly (I:C)(20ug/ml) stimulation for24h could induce nasal epithelial cells acetylation, not the HDACs genes expression. Part ⅢThe effects of inhibition of HDACs on the expression of IL-6, IL-8and LL37in the nasal epithelial cellsObjectives:To investigate the effects of inhibition of HDACs on the expression of IL-6, IL8and LL37in the nasal epithelial cells and NCI-H292cells.Methods:The nasal epithelial cells and NCI-H292cell line were cultured. Trichostatin A (TSA) or sodium butyrate (SB) was used to inhibit the HDACs activity in the airway epithelial cells, followed by the stimulation of Poly(I:C). The experiment included six groups:TSA, SB, Poly(I:C), TSA+Poly(I:C), SB+Poly(I:C) and epithelial cells without stimulation as control.24h after the stimulation, cell supernatants, total RNA and total protein were isolated respectively. The expressions of IL-6and IL-8in different groups were measured using ELISA. LL37in human NCI-H292airway epithelial cells and the nasal epithelial cells in response to HDAC inhibitors with or without Poly (I:C) stimulation was assessed using real-time PCR and western-blot.Results:Real-time PCR results indicated that TSA (200nM) and SB (4mM) up-regulated the LL37gene expression in the airway epithelial cells independent of Poly (I:C) stimulation. HDACs inhibitors increased LL37protein expression in NCI-H292cells but not in the nasal epithelial cells. In addition, HDACs inhibitors significantly inhibited Poly (I:C)-induced IL-6and IL-8production in nasal epithelial cells.Conclusions:HDAC inhibitors directly up-regulated LL-37gene expression in human airway epithelial cells and inhibited the IL-6and IL-8expression in nasal epithelial cells in response to Poly (I:C) stimulation.