【作者】 李雪；

【导师】 杨俊玲；

【作者基本信息】 吉林大学 ， 内科学， 2022， 硕士

【摘要】 目的:肺纤维化是许多疾病的转归和终末期改变,其发病机制复杂,预后极差。肾素血管紧张素系统(renin angiotensin system,RAS)在肺纤维化疾病发生发展中发挥重要作用,我们前期研究结果已经证实血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)可能是促进肺纤维化发生发展的独立因素,但机制仍不明确。氧化应激是肺纤维化形成的重要机制,研究表明,肺泡上皮细胞(alveolar epithelial cell,AEC)的损伤是肺纤维化形成的始动因素。本实验采用AngⅡ诱导II型肺泡上皮细胞损伤,探讨Nrf2(nuclear factor erythroid 2 related factor 2,Nrf2)在AngⅡ诱导的AEC损伤的调控机制,有助于在肺纤维化治疗中提供新的方向。方法:通过培养A549细胞,构建AngⅡ诱导的AEC损伤的模型,应用CCK8方法检测A549细胞活性,应用Western blot及RT q PCR方法检测IL-6、TGF-β1及Nrf2蛋白及m RNA水平,筛选合适AngⅡ、TBHQ刺激浓度。将细胞分为Control、AngⅡ、AngⅡ+TBHQ、TBHQ 4组,应用RT q PCR、Western blot方法检测炎症、氧化应激、凋亡等相关蛋白及m RNA指标;流式细胞术检测各组细胞凋亡情况;应用细胞转染技术,在A549细胞中沉默Nrf2,实验分为未感染组(Control)、阴性对照组(NC)、转染组(Nrf2-si RNA)。转染目的基因后,应用RT q PCR及Western blot方法检测Nrf2基因及蛋白表达水平,同时检测各组炎症、氧化应激、凋亡等相关基因及蛋白表达。结果:1.TBHQ刺激A549细胞,随着TBHQ作用时间及浓度的增加细胞存活率下降,TBHQ浓度在0-30umol/L之间细胞存活率无明显变化;TBHQ浓度在0-30umol/L之间A549细胞Nrf2蛋白表达增加,差异具有统计学意义,P值<0.05。2.AngⅡ刺激A549细胞,随着AngⅡ作用时间及浓度增加细胞存活率下降,AngⅡ浓度在0-1umol/L时TGF-β1蛋白及m RNA表达增加,差异具有统计学意义,P值<0.05。3.AngⅡ诱导A549细胞出现氧化应激,与对照组相比,AngⅡ组IL-6、TGF-β1、Caspases-3蛋白及m RNA表达增加,差异具有统计学意义,P值<0.05;AngⅡ+TBHQ组与AngⅡ组相比IL-6、TGF-β1、Caspases-3蛋白及m RNA表达降低,差异具有统计学意义,P值<0.05。4.转染A549细胞沉默Nrf2,Nrf2-si RNA组与对照组相比,Nrf2及下游抗氧化基因H01、NQO1蛋白及m RNA表达降低,差异具有统计学意义,P值<0.05。Nrf2-si RNA组与空白组相比,Nrf2-si RNA组L-6、TGF-β1、Caspases-3蛋白及m RNA表达增加,差异具有统计学意义,P值<0.05。5.流式细胞术结果显示,AngⅡ组与Control组相比,细胞凋亡率增加,AngⅡ+TBHQ组与AngⅡ组相比,细胞凋亡率减低,差异具有统计学意义,P值<0.05。转染A549细胞沉默Nrf2后Nrf2-si RNA组与Control组相比,细胞凋亡率明显增加,差异具有统计学意义,P值<0.05。结论:1.AngⅡ诱导A549细胞出现氧化应激,加重A549细胞炎症反应及细胞凋亡。2.TBHQ通过上调Nrf2及下游抗氧化因子,减轻AngⅡ诱导的肺泡上皮损伤和凋亡,沉默Nrf2加重AngⅡ诱导的AEC炎症反应及细胞凋亡。更多还原

【Abstract】 Objective:Pulmonary fibrosis is a regression and end-stage change of many diseases with complex pathogenesis and extremely poor prognosis.The renin-angiotensin system(RAS)plays an important role in the development of pulmonary fibrosis disease.Our previous findings have confirmed that angiotensinⅡ(AngⅡ)may be an independent factor in promoting the development of pulmonary fibrosis,but the mechanism remains unclear.Oxidative stress is an important mechanism in the formation of pulmonary fibrosis,and studies have shown that damage to alveolar epithelial cells(AEC)is the initiating factor in the formation of pulmonary fibrosis.In this experiment,we used AngⅡ to induce typeⅡ alveolar epithelial injury and explored thenuclear factor erythroid 2 related factors 2(Nrf2)in AngⅡ-induced AEC injury,which could help provide new directions in the treatment of pulmonary fibrosis.Methods:A model of AngⅡ-induced AEC injury was constructed by culturing A549 cells,applying the CCK8 method to detect A549 cell activity,applying Western blot and RT q PCR method to detect IL-6,TGF-β1,and Nrf2 protein and m RNA levels,and screening appropriate AngⅡ and TBHQ stimulation concentrations.The cells were divided into four groups: Control,AngⅡ,AngⅡ+TBHQ,and TBHQ,and RT q PCR and Western blot methods were applied to detect inflammation,oxidative stress,apoptosis,and other related proteins and m RNA indicators;flow cytometry was used to detect apoptosis in each group;cell transfection technology was applied to silence Nrf2 in A549 cells,and the experiment was divided into uninfected group(Control),negative control group(NC),and transfected group(Nrf2-si RNA).After transfection of the target gene,RT q PCR and Western blot were applied to detect Nrf2 gene and protein levels,and the expression of genes and proteins related to inflammation,oxidative stress,and apoptosis was also detected in each group.Results:1.TBHQ stimulated A549 cells,with the increase of TBHQ action time and concentration cell survival rate decreased,TBHQ concentration between 0-30umol/L cell survival rate did not change significantly;TBHQ concentration in the 0-30umol/L concentration of A549 cells Nrf2 protein expression increased,the difference is statistically significant(P <0.05).2.AngⅡ stimulated A549 cells,the cell survival rate decreased with the increase of AngⅡ action time and concentration,and the expression of TGF-β1 protein and m RNA increased at the concentration of AngⅡ in 0-1umol/L,the difference was statistically significant(P<0.05).3.AngⅡ-induced oxidative stress in A549 cells and the expression of IL-6,TGF-β1,Caspases-3 protein,and m RNA increased in the AngⅡ group compared with the control group,and the difference was statistically significant(P<0.05);the expression of IL-6,TGF-β1,Caspases-3 decreased in the AngⅡ+TBHQ group compared with the AngⅡ group,and the difference was statistically significant(P<0.05).protein and m RNA expression was decreased in the AngⅡ+TBHQ group compared with the AngⅡ group,and the difference was statistically significant(P<0.05).4.Transfected A549 cells with Nrf2 silencing,Nrf2-si RNA group compared with the control group,Nrf2,and downstream antioxidant genes H01,NQO1 protein,and m RNA expression was reduced,the difference was statistically significant(P<0.05).Nrf2-si RNA group compared with the blank group,Nrf2-si RNA group L-6,TGF-β1,Caspases-3 protein,and m RNA expression was reduced,the difference was statistically significant(P<0.05).Caspases-3 protein and m RNA expression were increased in the Nrf2-si RNA group compared with the blank group,and the difference was statistically significant(P<0.05).5.The results of flow cytometry showed that the apoptosis rate increased in the AngⅡ group compared with the Control group and decreased in the AngⅡ+TBHQ group compared with the AngⅡ group,and the difference was statistically significant(P<0.05).The apoptosis rate increased in the Nrf2-si RNA group compared with the Control group after transfection of A549 cells with Nrf2 silencing,and the difference was statistically significant(P<0.05).The apoptosis rate increased in the Nrf2-si RNA group compared with the Control group after transfection of A549 cells with Nrf2 silencing.The difference was statistically significant(P<0.05).Conclusion:1.AngⅡ-induced oxidative stress in A549 cells aggravated the inflammatory response and apoptosis of A549 cells.2.TBHQ reduced AngⅡ-induced alveolar epithelial damage and apoptosis by upregulating Nrf2 and downstream antioxidant factors,and silencing Nrf2 aggravated AngⅡ-induced inflammatory response and apoptosis in AEC.更多还原