The Role And Regulation Of Histone Deacetylase 2 In Tracheal Stenosis After Injury

There are many causes of benign airway stenosis,including traumatic,infectious,inflammatory,benign tumors and idiopathic.In tracheal stenosis after injury,stenosis caused by trauma and iatrogenic factors is the most common.Airway stenosis after tracheotomy and tracheal intubation is the most common in iatrogenic airway injury.Trauma and anastomosis of tracheobronchial ends after surgery are also common causes.In addition,physical and chemical damage is also one of the important reasons,including chemical(toxic agents,acid and alkali)inhalation injury,radiation injury,bronchoscopy intervention airway thermal ablation(laser,argon plasma coagulation,electrocoagulation).Compared with malignant airway stenosis,benign airway stenosis is more difficult to treat and more likely to cause long-term complications.At the same time,due to the long survival period of patients,patients and their families have higher expectations,and the serious short-term and long-term complications caused by surgery are difficult to accept.Therefore,the treatment of benign airway stenosis is a difficult point in the field of respiratory diseases.At present,transbronchoscopy intervention therapy has gradually become one of the main methods for the treatment of benign airway stenosis.However,due to the inevitable secondary injury of the airway in the process of the treatment of airway stenosis,restenosis after endoscopic intervention therapy has always been a problem for scholars in the field of interventional respiratory disease.The pathogenesis of benign tracheal stenosis is very complex.In the multi-gene genetic background,it involves various factors such as immunity,inflammation,cell proliferation,differentiation,apoptosis and oxidative stress.The inflammatory response is an important pathogenesis.Airway epithelial integrity and function are impaired after tracheal injury.Repeated or sustained inflammatory reactions promote the production of a large number of inflammatory factors and profibrotic cytokines involved in repair activities,fibroblast cell activation and proliferation,inhibition of apoptosis,extracellular matrix deposition,imbalance of collagen synthesis and degradation,and eventually the excessive proliferation of granulation tissue leads to tracheal stenosis.How to reduce or block airway inflammation will be the key to prevent and treat benign airway stenosis.The activation and release of inflammatory cells and inflammatory mediators are achieved through the transcription and regulation of inflammatory genes.Histone acetylation and deacetylation are the "switches" that regulate the transcription of inflammatory genes.Histone deacetylases(HDACs)remove acetyl groups from the tail of histones and inhibit the transcriptional expression of inflammatory genes.HDAC2,a subtype of type I HDAC,is located in the nucleus and is primarily involved in inflammation inhibition.In chronic airway inflammatory diseases such as asthma and COPD,HDAC2 activity is significantly reduced and is closely related to disease progression,and can be restored by inhaled corticosteroid therapy.Studies have shown that low concentrations of theophylline and erythromycin can up-regulate the protein expression of HDAC2,enhance the activity of HDAC2,and improve the prognosis of COPD.Erythromycin may also increase the activity of HDAC2 by inhibiting the PI3K/Akt pathway and reduce the release of inflammatory factors such as IL-8,thereby increasing the anti-inflammatory activity of budesonide.Vorinostat is an HDAC inhibitor and has a strong inhibitory effect on HDAC2.Does the regulation of HDAC2 improve or aggravate tracheal granulation tissue proliferation? Is erythromycin better than other anti-inflammatory drugs in treating tracheal stenosis after injury? Does it work synergistically with other anti-inflammatory drugs? Does blocking HDAC2 target reduce the therapeutic effect of erythromycin? What is the potential therapeutic mechanism of erythromycin for tracheal stenosis after injury? In order to explore the above problems,we will conduct research in two parts,and the two parts are expressed as a progressive relationship.The main content of the paper is as follows:Part 1: Expression of histone deacetylase 2 in tracheal stenosis models and its relationship with tracheal granulation tissue proliferationObjective:To observe the expression of histone deacetylase 2 in different models of tracheal stenosis,and to explore its relationship with tracheal granulation tissue proliferation and its potential as a new target for prevention and treatment of benign tracheal stenosis.Methods:Animal models of tracheal stenosis were constructed.HE staining was used to judge whether the models were successful.Twenty-four New Zealand white rabbits were randomly divided into control group,erythromycin(ERY)group,budesonide group and vorinostat group,with 6 rats in each group.Stenotic tracheal tissues were collected on day 11 after continuous administration for 10 days as described above.The tracheal stenosis of each group was calculated and the pathological changes of each group were observed by HE staining.The m RNA expression of HDAC2,IL-8,TGF-?1 and VEGF in each group was detected by q PCR.The expression of HDAC2 protein was detected by immunofluorescence,and the expression of type I collagen and type III collagen was detected by immunohistochemistry.Results:The models were successfully constructed.HE staining showed that there were different degrees of tracheal stenosis,tissue hyperplasia and mucosal epithelial hyperplasia in each group.Compared with the control group,the tracheal epithelial hyperplasia was significantly improved in the erythromycin group,the degree of hyperplasia was the lightest,and the tracheal stenosis was significantly reduced.In the vorinostat group,the tracheal epithelial tissue hyperplasia was aggravated,and the stenosis was increased;the difference in tracheal stenosis was statistically significant(P < 0.05),no significant changes in the budesonide group.Compared with the model control group,the m RNA and protein expression of HDAC2 in the erythromycin group was significantly increased,and the vorinostat group was significantly decreased(P<0.05),no significant changes in the budesonide group.The m RNA expression of IL-8 in the erythromycin group was significantly decreased,and that in the vorinostat group was significantly increased(P<0.05),no significant changes in the budesonide group.The expression of TGF-?1,VEGF,type I and type III collagen in the erythromycin group decreased significantly,and the expression of TGF-?1,VEGF and type III collagen in the vorinostat group increased significantly(P< 0.05),no significant changes in the budesonide group.Conclusions:Erythromycin up-regulates HDAC2 expression,inhibits inflammatory response,reduces tracheal granulation tissue proliferation,and improves tracheal stenosis.Vorinostat down-regulates HDAC2 expression,promotes inflammatory response,increases tracheal granulation tissue proliferation,and increases tracheal stenosis.It suggests that the regulation of histone deacetylase 2 expression may be a new strategy for the prevention and treatment of benign tracheal stenosis.Part 2: Study on the effect of regulating histone deacetylase 2 on tracheal stenosis after injuryObjective:There is currently no effective treatment and prevention measures for tracheal stenosis after injury.We observed the effects of different combinations of different anti-inflammatory drugs such as erythromycin and histone deacetylase 2 inhibitor on animal models of tracheal stenosis after injury,and explored the protective role of erythromycin in tracheal stenosis by regulating histone deacetylase 2 and its underlying mechanism.Methods:The rabbit model of tracheal stenosis was established and HE staining method was used to determine whether the model was successful.Forty-eight New Zealand white rabbits were randomly divided into control group,normal saline(NS)group,erythromycin(ERY)group,budesonide group,ERY+budesonide group,vorinostat group,ERY+vorinostat group,ERY+budesonide+vorinostat group.Narrow tracheal tissue,serum,and bronchoalveolar lavage fluid(BALF)specimens were collected 10 days after continuous administration as described above.HE staining method was used to detect pathological changes in each group.HDAC2 expression was detected by immunofluorescence.The expression of type I collagen and type III collagen was detected by immunohistochemical method.The expression of TGF-?1,VEGF and IL-8 in serum and alveolar lavage fluid was detected by ELSIA.The expression of HDAC2,TGF-?1,VEGF and IL-8 in bronchi of each group was detected by Western blotting method.Results:The rabbit model of tracheal stenosis was constructed successfully.Tracheal lumen stenosis,fibroblast proliferation,and mucosal epithelial hyperplasia were observed in control group,NS group,budesonide group and vorinostat group.In ERY group,ERY+budesonide group,ERY+vorinostat group and ERY+budesonide+vorinostat group,the degree of tracheal stenosis was alleviated,and the mucosal epithelium was still slightly proliferated.The effect of ERY combined with other drugs was more obvious.In tracheal tissue,compared with control group,the HDAC2 protein expression increased significantly in ERY group,ERY+budesonide group and ERY+budesonide+vorinostat group and decreased significantly in vorinostat group,the expression of collagen I and III decreased significantly in ERY group,ERY+budesonide group and ERY+budesonide+vorinostat group(P<0.05).The HDAC2 protein expression in the ERY+vorinostat group was significantly lower than that in ERY group,and the expression of collagen I and collagen III was significantly higher than that in ERY group,the difference was statistically significant(P<0.05).In serum,compared with control group,the expression levels of TGF-?1 and IL-8 decreased significantly in ERY group,ERY+budesonide group,ERY+vorinostat group and ERY+budesonide+vorinostat group;the expression of VEGF decreased significantly in ERY group and ERY+budesonide group,and the difference was statistically significant(P<0.05).In bronchoalveolar lavage fluid and tracheal tissue,compared with control group,the protein expression of TGF-?1,VEGF and IL-8 decreased significantly in ERY group,ERY+budesonide group,ERY+vorinostat group and ERY+budesonide+vorinostat group,the difference was statistically significant(P<0.05).Conclusions:Erythromycin inhibited the local inflammatory response,systemic inflammatory response,and excessive proliferation of granulation tissue after tracheobronchial mucosal injury by up-regulating the expression of HDAC2,it promoted wound healing and alleviated tracheobronchial stenosis.When combined with budesonide,it had a good synergistic effect.However,vorinostat could attenuate erythromycin's effect by down-regulating the expression of HDAC2.HDAC2 is an important target for controlling inflammation.By upregulating its activity,it can significantly reduce airway inflammation and improve the degree of tracheal stenosis after injury.