Study On The Role Of DACH1 In Chronic Obstructive Pulmonary Disease

Objective: DACH1,as a cell fate determinant,is involved in a variety of cellular functions.At present,the role of DACH1 in chronic obstructive pulmonary disease(COPD)remains unclear.This study aims to explore whether DACH1 is involved in the occurrence and development of COPD and its possible mechanism.Methods: Normal lung tissues of patients with or without COPD were collected and the expression of DACH1 were detected.The expression of DACH1 in primary airway epithelia(SAEs)of COPD smokers and non-smoking were analyzed.Whole body smoke exposure was used to establish a mouse model of COPD.Lung function,airway inflammation and alveolar septum destruction were detected in airway epithelial DACH1 knockdown mice and lung DACH1 overexpressed mice after cigarette smoke exposure.Bronchial epithelial cells(16HBEs)were cultured in vitro to detect the effect of knockdown or overexpression of DACH1 on airway epithelial inflammation induced by cigarette smoke extract(CSE)and the nuclear factor erythroid 2-related factor 2(NRF2)signaling pathway.Results: DACH1 was widely expressed in lung tissue.DACH1 expression was decreased in lung tissue of COPD patients compared with non-smokers and non-COPD smokers.DACH1 expression in SAEs in COPD patients was significantly lower than that in nonsmoking.Whole body smoke exposure induced reduced lung function,airway inflammation,and destruction of alveolar septum tissue in mice.Airway epithelial DACH1 knockout and lung tissue overexpression of DACH1 can aggravate and alleviate cigarette smoke exposeinduced lung hypofunction and lung tissue pathological changes in mice,respectively.CSE promoted the secretion of inflammatory cytokines Interleukin-6(IL-6)and Interleukin-8(IL-8)in 16 HBE,and overexpression of DACH1 in 16 HBEs inhibited the inflammatory response induced by CSE.In contrast,knockdown of DACH1 in 16 HBE promoted the inflammatory response caused by CSE.NRF2 may be a potential downstream target of DACH1,and DACH1 can promote the expression of NRF2 by directly binding to the NRF2 promoter.DACH1 is involved in regulating the inflammatory response of airway epithelium through the activation of NRF2/Heme Oxygenase 1(HO-1)signaling pathway.Conclusions: DACH1 expression in lung tissue decreased in smokers and patients with COPD compared with non-smokers.DACH1 mitigated cigarette smoke-induced inflammatory responses by activating the NRF2 signaling pathway.DACH1 may be a potential target for treating airway inflammation in patients with COPD.Part Ⅰ: Expression of DACH1 in lung tissue and its correlation with lung function in patients with chronic obstructive pulmonary diseaseObjective: To investigate the expression level of DACH1 in lung tissues of COPD patients.Methods: Normal lung tissue was collected from patients with COPD,smoking non-COPD and non-smoking non-COPD.The m RNA expression level of DACH1 in lung tissues of three groups was analyzed by RT-PCR,and the correlation between DACH1 m RNA and lung function was analyzed.SAEs of smokers with COPD and non-smoking controls were collected and DACH1 expression in airway epithelial cells was evaluated by Western Blot.At the same time,DACH1 expression in COPD lung tissues was analyzed in series sets of gene expression database(GSE76925,GSE11784).To evaluate the expression of DACH1 in lung tissue of COPD model mice.The effect of cigarette smoke extract on DACH1 expression in airway epithelium was determined in vitro.Results: DACH1 was expressed in airway epithelium of lung tissue in COPD patients.The expression of DACH1 in airway epithelium decreased significantly in COPD patients compared with non-COPD patients.Cigarette smoke exposure reduces DACH1 expression in mouse lung tissue,especially airway epithelium.Cigarette smoke extract can reduce DACH1 expression in airway epithelium in vitro.Conclusion: DACH1 expression is decreased in COPD airway epithelium.Cigarette smoke exposure reduces epithelial DACH1 expression.Part Ⅱ: Effects of DACH1 on airway inflammation and emphysema formation in a mouse model of chronic obstructive pulmonary diseaseObjective: To investigate whether abnormal DACH1 expression has an effect on airway inflammation and emphysema formation in mice with COPD.Methods: Airway epithelial DACH1 knockdown mice were constructed by gene editing technique.Adeno-associated virus was intratracheal injected to construct DACH1 overexpressed mice in lung tissue.The COPD model of mice was established after six months of whole body cigarette smoke exposure.Intratracheal injection of porcine trypsin was used to construct a mouse model of emphysema.After the modeling,the lung function of mice in each group was detected by mouse lung function instrument,and the airway inflammation level,the aggregation degree of airway inflammatory cells,the secretion level of inflammatory factors and the destruction degree of lung tissue interval were evaluated.Meanwhile,airway injection of porcine trypsin induced emphysema model in mice.Results: The knockdown of airway epithelial DACH1 aggravated cigarette smoke-induced pulmonary dysfunction in mice,increased the inflammatory cell infiltration of macrophages and neutrophils around the airway,promoted the secretion of inflammatory factor IL-6 and mouse chemokine 1(CXCL1)and the destruction of alveolar septal tissue.Conversely,overexpression of DACH1 in lung tissue alleviated cigarette smoke-induced lung dysfunction,the infiltration of inflammatory cell to airway,secretion of inflammatory cytokines,and destruction of alveolar septum tissue in mice.Conclusion: DACH1 is involved in airway inflammation and emphysema formation in COPD.DACH1 plays a protective role in COPD by reducing inflammation and destruction of alveolar septal tissue.Part ⅡI: Effect of DACH1 on airway epithelial inflammation induced by cigarette smoke extract and its mechanismObjective: To further explore whether DACH1 is involved in regulating airway epithelial inflammation induced by CSE and its possible pathway of action.Methods: 16 HBEs were cultured in vitro.DACH1 was overexpressed in 16 HBEs by lentivirus transfection.DACH1 expression was decreased by small interfering RNA knockdown.The effects of overexpression and knockdown of DACH1 on CSE-induced secretion of airway epithelial inflammatory cytokines IL-6 and IL-8 were determined by enzymed-linked immunosorbent assay.To evaluate the regulatory effect and mechanism of DACH1 on the expression of its potential downstream protein NRF2.To explore the effect of DACH1 on NRF2/HO-1 signaling pathway.To further elucidate the relationship between DACH1/NRF2/HO-1 and inflammation.Results: Overexpression of DACH1 in 16 HBEs decreased the secretion of IL-6 and IL-8 induced by CSE,and knockdown of DACH1 in 16 HBEs promoted the secretion of IL-6 and IL-8 induced by CSE.NRF2 is a downstream target molecule of DACH1,and DACH1 can bind to the promoter sequence of NRF2.DACH1 can increase the expression of NRF2,promote the activation of NRF2/HO-1 signal,and reduce the production of reactive oxygen species.Conclusion: DACH1 inhibits epithelial inflammatory response by promoting NRF2/HO-1 signaling pathway.DACH1 may be a potential therapeutic target for COPD airway inflammation.