The Mechanism Of Histone Acetylation Inducing IL-8 Release In COPD And The Regulative Effect Of MAPK Pathway

Background:Chronic obstructive pulmonary disease(COPD) is a chronic inflammatory disease of airway,characterized by excessive inflammatory gene expression.Cigarette smoke is the main etiological factor in the pathogenesis of COPD.It can cause oxidative stress,activate transcription factors nuclear factor and enhance proinflammatory gene transcription.Cigarette smoke-triggered inflammation is considered to play a central role in the development of COPD.Histone acetylation and deacetylation is a key regulator of the specificity and duration of gene transcription.Disruption in the nuclear histone acetylation/deacetylation balance may result in excessive transcription of specific proinflammatory genes in the lungs,finally,chronic inflammatory cycle will develop,and it may have great influence on COPD.It was indicated that oxidative stress has direct relationship with the disruption of histone acetylation/ deacetylation balance and it can enhance the acetylation of histone protein.Oxidative stress has been suggested to influence histone acetylation depending on some signal transduction pathway.It has been reported that oxidative stress can activate MAPK(Mitogen-activated protein kinases) pathway,which play an important role in the inflammation of airway.Furthermore,MAPK pathway can influence the structure of histone,it maybe induce histone phosphorylation or acetylation in some condition.SO,MAPK pathway may participate in the regulation of histone acetylation in COPD.Based on these results,we explored the histone acetylation regulating IL-8 release mediated by cigarette smoke and the regulative effect of MAPK pathway in COPD rat and bronchial epithelail cell. Chapter One Expression of acetylated histone H4 and HDAC2 in the lung tissues of COPD rat and the anti-inflammatory effect of AminophyllineObjective:To study the expression of acetylated histone H4 and histone deacetylase(HDAC2) in the lung tissues of COPD rat model and its influence on IL-8 expression.At the same time,to explore the anti-inflammatory effect of Aminophylline.Methods:36 Wister rats were divided into three groups at random:control group,COPD group and Aminophylline group.Rat COPD model was established by intratracheal instillation of lipopolysaccharide(LPS) twice and exposure to cigarette smoke daily. Aminophylline group received Aminophylline(5 mg·kg^-1·d ) daily via the abdominal cavity injection.The spirometry was conducted and the pathological changes were observed after the model was established.HDAC activity was examined by colorimetric method.The expression of protein of acetylated H4 and HDAC2 in the lung tissues was examined by using immunohistochemistry and western blot.The concentrations of IL-8 in bronchoalveolar lavage fluid(BALF) was measured by enzyme-linked inmunosorbent assay(ELISA).And the mRNA of IL-8 and HDAC2 in the lung tissues was detected by reverse transcription-polymerase chain reaction(RT-PCR) respectively.Results:Significant decrease of PEF,FEV0.3,FEV_0.3/FVC were found in the COPD rat model group compared with the control group. Aminophylline could improve lung function.The activity of HDAC was lower in the COPD group than in the control group,and Aminophylline could restore HDAC activity partly.In the COPD group,the expression of acetylated H4 protein was increased markedly compared with the control group,and Aminophylline could decrease acetylated H4 protein.The expression of HDAC2 protein was decreased in the COPD group and Aminophylline could enhance the expression of HDAC2 protein.The expression of IL-8 mRNA and the concentrations of IL-8 in BALF were higher in the COPD group than in the control group.Aminophylline could decrease IL-8 levels,and this was associated with increased HDAC activity and HDAC2 protein levels.But there was no difference in HDAC2 mRNA levels between these three groups.Conclusion:In COPD,histones were highly acetylated in the lung tissue.The acetylation of histones can induce increased expression of IL-8.While,Aminophylline can activate HDAC and decrease acetylation of histones as well as the expression of IL-8,So it has the effect of anti-inflammation.Chapter Two Cigarette smoke induces histone acetylation and up-regulates IL-8 expression in NHBE cells via MAPK pathwayObjective:To investigate the mechanism of histone acetylation mediated by cigarette smoke inducing bronchial epithelial IL-8 release and the regulative effect of MAPK pathway in this process.Methods:We treated normal human bronchial epithelial(NHBE)cells with 5%and 10%cigarette smoke extract (CSE)respectivly,then the expression of p-p38,p-ERK1/2,p-JNK(activated MAPK pathway protein)and HDAC2 in NHBE cells were detected by immunocytochemistry and western blot.After that,we treated NHBE cells with 10%CSE,TSA(HDAC inhibitor),Aminophylline+CSE,SB203580(p38 inhibitor)+CSE,PD98059 (ERKinhibitor)+CSE and SP600125(JNK inhibitor)+CSE respectively. HDAC activity was examined by colorimetric method.The expression of protein of acetylated H4 in NHBE cells was examined by using immunocytochemistry and western blot.The concentrations of IL-8 in NHBE cells supernatant was measured by enzyme-linked inmunosorbent assay(ELISA).And the level of IL-8mRNA in NHBE cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) respectively.Results:CSE stimulation could activate MAPK pathway.In the presence of CSE,the expression of p-p38,p-ERK1/2 and p-JNK significantly increased.While,the the expression of HDAC2 decreased in the CSE group.In the CSE and TSA group,the activity of HDAC was lower than in the control group,but the expression of acetylated H4 protein was increased markedly compared with the control group,and the concentrations of IL-8 in supernatant and the expression of IL-8 mRNA were higher than in the control group.Aminophylline could restore HDAC activity partly,as a result,decreasing the expression of acetylated H4 and IL-8.The expression of acetylated H4 and IL-8 which were induced by CSE was inhibited by SB203580 and PD98059,but not by SP600125.SB203580,PD98059 and SP600125 had no effect on HDAC activity.Conclusion:In NHBE cells,CSE stimulation could induce histone acetylation associated with IL-8 expression subsequently.Aminophylline may fulfill the effect of anti-inflammation through activating HDAC and decreasing acetylation of histone.p38 and ERK1/2 MAPK pathway could down-regulate histone acetylation and inhibit the expression of IL-8 subsequently which were induced by CSE.