Nrf2活化在萝卜硫素减轻博莱霉素所致肺纤维化中的作用

发布时间：2018-05-01 00:17

本文选题：肺纤维化 + 博莱霉素　；参考：《吉林大学》2017年博士论文

【摘要】：肺纤维化在病理上表现为I型肺泡上皮细胞持续性受损、大量炎性细胞在肺组织内浸润、成纤维细胞增生和分泌细胞外基质,过多分泌的细胞外基质沉积在肺间隔内,最终出现正常肺组织结构异常改变。研究者目前认为失控的氧化应激,氧化/抗氧化平衡失调是诱发肺组织出现纤维化的分子学基础,参与肺纤维化发生、发展的各个阶段。因此,通过上调内源性抗氧化通路可能在治疗肺纤维化中起到一定作用。核因子Nrf2能够通过转录活化其下游多种抗氧化剂的表达,增加体内抗氧化剂水平和活性。许多研究表明Nrf2在肺纤维化的发病中发挥作用,敲除Nrf2基因可增加博莱霉素诱导肺纤维化的易感性。大量实验发现萝卜硫素(SFN),一种Nrf2激动剂,在多种疾病模型中发挥间接抗氧化、抗毒性作用。本研究通过体内和体外实验,目的是探讨萝卜硫素通过Nrf2活化发挥对博莱霉素所致小鼠肺纤维化的保护作用。为探讨SFN对博莱霉素诱导氧化应激所致肺纤维化的作用机制,本实验采用一次性气管内注射博莱霉素,建立小鼠肺纤维化体内模型,并于造模后每隔一天给予皮下注射SFN干预处理。分别于第7、28天处死小鼠,收集肺组织标本。应用病理学检查进行HE染色、Masson染色、TUNEL和免疫组化染色,用于观察肺组织炎症及纤维化程度,细胞凋亡和Nrf2的表达情况;利用Western blot法测定促炎症因子、促凋亡因子、促纤维化因子和氧化物3NT、4HNE的表达水平;使用羟脯氨酸测定试剂盒检测肺组织中羟脯氨酸含量;分别采用实时定量PCR和Western blot法检测Nrf2及其下游HO-1、NQO1、SOD1、CAT基因和蛋白水平。此外,我们还在体外培养人肺成纤维细胞,通过血管紧张素II(Ang II)模拟体内氧化应激,通过给予SFN预处理,观察SFN对Ang II诱导人肺成纤维细胞TGF-β分泌和ROS产生的影响;并应用Nrf2特异性si RNA沉默人肺成纤维细胞的Nrf2基因,从而探讨Nrf2在SFN减轻氧化应激所致肺纤维化过程中的作用。SFN干预博莱霉素诱导肺纤维化体内实验结果显示:给予SFN后,7、28天时肺泡炎症改变明显减轻;7天时因博莱霉素所致肺泡上皮细胞的凋亡减少;28天时SFN干预组肺纤维化程度、评分和羟脯氨酸胶原含量明显减轻。给予SFN处理,还能降低促炎因子IL-1β、TNF-α、促凋亡因子caspase-3和促纤维化因子TGF-β的表达,减少肺组织氧化标志物3NT、4HNE含量,同时伴随着Nrf2及其下游基因表达增强。通过进行体外实验,观察到SFN能减少因Ang II所致人肺成纤维细胞TGF-β分泌和ROS产生,且发现这一保护作用是通过上调Nrf2及HO-1表达所致。利用特异性si RNA沉默Nrf2基因可以阻断SFN保护Ang II诱导成纤维细胞分泌TGF-β的作用。结合以上实验结果,我们发现SFN通过活化Nrf2,增加抗氧化酶活性,减轻博莱霉素所致小鼠肺纤维。该研究为SFN用于预防和治疗因氧化应激所致肺部炎症和肺纤维化提供理论研究基础。
[Abstract]:The pathological manifestations of pulmonary fibrosis are persistent damage of type I alveolar epithelial cells, infiltration of a large number of inflammatory cells in lung tissue, proliferation and secretion of extracellular matrix of fibroblasts, and excessive secretion of extracellular matrix in the interpulmonary septum. Finally, abnormal changes of normal lung tissue structure appeared. Researchers believe that out of control oxidative stress, oxidative / antioxidant imbalance is the molecular basis of pulmonary fibrosis, involved in the occurrence and development of various stages of pulmonary fibrosis. Therefore, up-regulation of endogenous antioxidant pathway may play a role in the treatment of pulmonary fibrosis. Nuclear factor Nrf2 can activate the expression of many antioxidants downstream through transcription and increase the antioxidant level and activity in vivo. Many studies have shown that Nrf2 plays a role in the pathogenesis of pulmonary fibrosis. Knockout of Nrf2 gene can increase the susceptibility of bleomycin induced pulmonary fibrosis. A large number of experiments have found that sulforaphane, a Nrf2 agonist, plays an indirect antioxidant and antitoxic effect in various disease models. The aim of this study was to investigate the protective effect of sulforaphane on bleomycin induced pulmonary fibrosis in mice by Nrf2 activation in vitro and in vivo. In order to investigate the mechanism of SFN on bleomycin induced pulmonary fibrosis induced by oxidative stress, a mouse model of pulmonary fibrosis was established by single intratracheal injection of bleomycin. The model was treated by subcutaneous injection of SFN every other day. The mice were killed on the 7th day and the lung tissues were collected. In order to observe the degree of inflammation and fibrosis, the expression of apoptosis and Nrf2 in lung tissue, the expression of pro-inflammatory factors and pro-apoptotic factors were determined by Western blot method, and the immunohistochemical staining and Tunel staining with HE staining were used to observe the degree of inflammation and fibrosis, the expression of apoptosis and the expression of Nrf2 in lung tissue. The expression levels of fibrogenic factor and oxide 3NTO4HNE, hydroxyproline assay kit were used to detect the content of hydroxyproline in lung tissue, and Nrf2 and its downstream HO-1NQO1SOD1 cat gene and protein were detected by real-time quantitative PCR and Western blot respectively. In addition, we cultured human lung fibroblasts in vitro, simulated oxidative stress by angiotensin II (II(Ang II) in vivo, and observed the effect of SFN on TGF- 尾 secretion and ROS production induced by Ang II by SFN pretreatment. The Nrf2 gene of human lung fibroblasts was silenced by Nrf2 specific si RNA. To explore the role of Nrf2 in alleviating oxidative stress induced pulmonary fibrosis induced by SFN in vivo, the results showed that the alveolar inflammation was significantly reduced at 728 days after SFN administration. Apoptosis of alveolar epithelial cells induced by mycin was reduced at 28 days after treatment with SFN. Scores and hydroxyproline collagen content were significantly reduced. SFN treatment could also reduce the expression of IL-1 尾 -TNF- 伪, caspase-3 and TGF- 尾, and reduce the content of 3NTN ~ 4HNE, which was accompanied by the increased expression of Nrf2 and its downstream genes. Through in vitro experiments, it was observed that SFN could reduce the secretion of TGF- 尾 and the production of ROS in human lung fibroblasts induced by Ang II, and the protective effect was caused by up-regulating the expression of Nrf2 and HO-1. Silencing Nrf2 gene by specific si RNA can block the protective effect of SFN on the secretion of TGF- 尾 in fibroblasts induced by Ang II. Combined with the above results, we found that SFN can increase the activity of antioxidant enzymes by activating Nrf2, and reduce the lung fiber induced by bleomycin in mice. This study provides a theoretical basis for the use of SFN in the prevention and treatment of pulmonary inflammation and pulmonary fibrosis due to oxidative stress.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R563

【相似文献】