【作者】 周健；

【导师】 宋珏；

【作者基本信息】 安徽医科大学 ， 药学， 2021， 硕士

【摘要】 慢性阻塞性肺疾病(COPD)是一种不能完全逆转的炎症性疾病,其特征是气道阻塞和异常的炎症反应。多药耐药相关蛋白1(MRP1)可以排泄各种有毒的外源物质和某些促炎分子,MRP1的上调可以进一步减轻肺部炎症和损害。DJ-1(PARK7)是一种保护神经元免受氧化应激的多功能蛋白,能够稳定转录因子核因子红细胞2相关因子2(Nrf2)。Wnt/β-catenin信号参与胚胎发育、同源自噬和人类疾病发病机制,其中Wnt3a能够抵抗COPD与肺气肿。噻托溴铵(TB)与奥达特罗(O)是目前治疗COPD的首选吸入型药物,除了它们本身的支气管扩张作用,已明确显示出抗炎作用。但是,在肺部炎症中,DJ-1是否通过激活Wnt3a/β-catenin信号通路调节Nrf2介导的MRP1表达和肺抗氧化防御以及TB和O的抗炎机制仍不清楚。目的将肺泡上皮细胞(A549)暴露于香烟烟雾提取物(CSE)和脂多糖(LPS)建立炎症细胞模型,探究CSE与LPS对MRP1蛋白表达的影响、炎症状态下MRP1蛋白表达变化的调控机制以及TB与O对炎症的干预作用。方法1.MTT法检测CSE,LPS,TB以及O对A549细胞活性的影响并结合文献参考来确定适宜的给药浓度。2.Western blot检测单用CSE、单用LPS以及联用CSE与LPS刺激A549细胞后主要标记物的表达。3.Western blot、实时荧光定量PCR检测不同浓度的CSE联合LPS刺激A549细胞后主要标记物的表达。4.Western blot检测炎症状态下过表达或沉默A549细胞中Wnt3a后主要标记物的表达。5.Western blot检测炎症状态下分别过表达和沉默A549细胞中的Wnt3a和Nrf2后主要标记物的表达。6.Western blot和免疫荧光染色检测炎症状态下过表达或沉默A549细胞中DJ-1后主要标记物的表达。7.Western blot检测炎症状态下分别过表达和沉默A549细胞中的DJ-1和Wnt3a后主要标记物的表达。8.Western blot检测炎症状态下单用TB、单用O以及联用TB与O刺激A549细胞后主要标记物的表达。结果单用CSE、单用LPS以及联用CSE与LPS刺激A549细胞,均能引起p-Nrf2、Nrf2和MRP1的蛋白表达下调以及TNF-α和IL-6的蛋白表达上调,且CSE与LPS联合产生影响更强。CSE与LPS联合刺激A549细胞,DJ-1、Wnt3a、β-catenin、p-Nrf2、Nrf2、MRP1、HO-1的表达发生下调,TNF-α,IL-6,IL-18和IL-1β的表达发生上调,且随着CSE浓度的增加,DJ-1、Wnt3a、Nrf2和MRP1的蛋白表达逐渐降低,呈剂量依赖性。将A549细胞暴露于CSE和LPS后,Nrf2蛋白表达显著下降,但是Nrf2的m RNA水平却没有变化。在暴露于CSE和LPS的A549细胞中,DJ-1和Wnt3a的过表达均能激活Nrf2信号,增加MRP1和HO-1蛋白水平,降低IL-6蛋白水平,其中Wnt3a的过表达还能增加p-GSK3β的蛋白表达和降低Axin1和GSK3β的蛋白表达。而DJ-1和Wnt3a的敲低则相反。另外,DJ-1的过表达和DJ-1的敲低分别增加和降低了Wnt3a和β-catenin的蛋白水平。Nrf2和Wnt3a缺乏分别降低Wnt3a和DJ-1对A549细胞的保护作用。但是,DJ-1和Wnt3a的蛋白水平并未因Wnt3a和Nrf2缺失而改变。在暴露于CSE和LPS的A549细胞中,我们发现TB与O均能上调p-Nrf2、Nrf2和MRP1蛋白水平,下调IL-6,TNF-α和IL1β蛋白水平,两者联合使用后表现出更强的作用。结论总之,在经CSE和LPS处理的A549细胞中,DJ-1至少部分地通过激活Wnt3a/β-catenin信号通路来调节Nrf2介导的MRP1表达和抗氧化防御。在炎症治疗过程中,TB和O可能部分是通过上调Nrf2和MRP1的表达发挥抗炎作用。这些发现可能为COPD的干预提供潜在的治疗靶标。更多还原

【Abstract】 Chronic obstructive pulmonary disease(COPD)is an inflammatory disease that cannot be completely reversed and is characterized by airway obstruction and abnormal inflammatory responses.Multidrug resistance associated protein 1(MRP1)exudes a variety of toxic exogenous substances and certain pro-inflammatory molecules,and the up-regulation of MRP1 can further reduce pulmonary inflammation and damage.DJ-1(PARK7)is a multifunctional protein that protects neurons from oxidative stress and stabilizes the transcription factor nuclear factor erythrocyte2-associated factor 2(Nrf2).Wnt/β-catenin signalling is involved in embryonic development,autophagy,and human disease pathogenesis,in which Wnt3 a is resistant to COPD and emphysema.Tiotropium bromide(TB)and odaterol(O)are currently the inhaled drugs of choice for the treatment of COPD,and in addition to their own bronchiectasis,they have clearly demonstrated anti-inflammatory effects.However,it remains unclear whether DJ-1 regulates Nrf2-mediated MRP1 expression and lung antioxidant defense through activation of Wnt3a/β-catenin signalling pathway,as well as the anti-inflammatory mechanisms of TB and O in pulmonary inflammation.AimsAlveolar epithelial cells(A549)were exposed to cigarette smoke extract(CSE)and lipopolysaccharide(LPS)to establish an inflammatory cell model and explore the effects of CSE and LPS on MRP1 protein expression,the regulatory mechanism of MRP1 protein expression changes in the inflammatory state and the intervention effects of TB and O on inflammation.Methods1.MTT assay was used to detect the effects of CSE,LPS,TB and O on the activity of A549 cells,and combined with literature reference to determine the appropriate administration concentration.2.Western blot was used to detect the expression of major markers in A549 cells stimulated by CSE alone,LPS alone or combined use of CSE and LPS.3.Western blot and quantitative real-time PCR were used to detect the expression of major markers in A549 cells stimulated by different concentrations of CSE combined with LPS.4.Western blot was used to detect the expression of major markers after overexpression or silencing of Wnt3 a in A549 cells under inflammatory state.5.Western blot was used to detect the expression of major markers after Wnt3 a and Nrf2 were,respectively,overexpressed and silenced in A549 cells under inflammatory state.6.Western blot and immunofluorescence staining were used to detect the expression of major markers after overexpression or silencing of DJ-1 in A549 cells under inflammatory state.7.Western blot was used to detect the expression of major markers after DJ-1 and Wnt3 A were,respectively,overexpressed and silenced in A549 cells under inflammatory state.8.Western blot was used to detect the expression of major markers in A549 cells stimulated by TB alone,O alone or combined TB and O under inflammatory state.ResultsCSE alone,LPS alone and CSE combined with LPS stimulated A549 cells,all of which could induce down-regulation of the protein expressions of p-Nrf2,Nrf2 and MRP1 and up-regulation of the protein expressions of TNF-α and IL-6,and the combination of CSE and LPS has a stronger effect.CSE combined with LPS stimulated A549 cells,the expressions of DJ-1,Wnt3 a,β-catenin,p-Nrf2,Nrf2,MRP1,and HO-1 were down-regulated while the expressions of TNF-α,IL-6,IL-18 and IL-1βwere up-regulated,and with the increase of CSE concentration,the protein expressions of DJ-1,Wnt3 a,Nrf2 and MRP1 gradually decreased in a dose-dependent manner.After exposing A549 cells to CSE and LPS,Nrf2 protein expressions were significantly decreased,while there was no change in Nrf2 m RNA levels.In A549 cells exposed to CSE and LPS,overexpression of DJ-1 and Wnt3 a can activate Nrf2 signal,increase MRP1 and HO-1 protein levels,and reduce IL-6 protein levels.Overexpression of Wnt3 a can also increase the expressions of p-GSK3β and decrease the expressions of Axin1 and GSK3β.The knockdown of DJ-1 and Wnt3 a is the opposite.In addition,overexpression of DJ-1 and knockdown of DJ-1 increased and decreased the protein levels of Wnt3 a and β-catenin,respectively.The lack of Nrf2 and Wnt3 a reduced the protective effects of Wnt3 a and DJ-1 on A549 cells,respectively.However,the protein levels of DJ-1 and Wnt3 a did not change due to the deletion of Wnt3 a and Nrf2.In A549 cells exposed to CSE and LPS,we found that both TB and O can up-regulate the protein levels of p-Nrf2,Nrf2 and MRP1,and down-regulate the protein levels of IL-6,TNF-α and IL1β.After showing a stronger effect.ConclusionsIn conclusion,in A549 cells treated with CSE and LPS,DJ-1 regulates Nrf2-mediated MRP1 expression and antioxidant defense,at least in part,by activating the Wnt3a/β-catenin signalling pathway.In the treatment of inflammation,Tiotropium and Odaterol may partly play an anti-inflammatory effect by up-regulating the expressions of Nrf2 and MRP1.These findings may provide potential therapeutic targets for COPD intervention.更多还原