Sirtuins在慢性鼻窦炎伴鼻息肉中的表达和调控机制

发布时间：2019-02-18 20:28

【摘要】：尽管近年来对慢性鼻窦炎伴鼻息肉的发病机制和分子机制方面取得重大研究进展,但是关于慢性鼻窦炎伴鼻息肉中复杂的炎症网络以及对其调控仍认识的不充分。越来越多研究显示Sirtuins家族成员(Sirt1-Sirt7 )在各种急性或慢性炎性疾病,免疫疾病和代谢性疾病的病理进展的衰老,炎症,细胞死亡和氧化应激过程中发挥保护作用。前期的研究表明Sirt1和Sirt6参与脂多糖诱导鼻黏膜上皮细胞核表达的HMGB1易位和主动释放。然而,Sirtuins在鼻息肉中的作用和分子机制尚不清楚,是否调控慢性鼻窦炎伴鼻息肉中各种促炎细胞因子(包括IL-1β,IL-5,IL-8, IL-10,IL-17和HMGB1)和活性氧(ROS)。因此,Sirtuins在鼻息肉中是否存在与炎症和氧化应激相关的特定分子机制和其调节机制需要深入研究。目的:观察Sirt1-Sirt6在慢性鼻窦炎伴鼻息肉与正常鼻黏膜组织中的表达差异,以及探讨Sirt1在鼻黏膜上皮细胞炎症反应和氧化应激中相关的分子作用机制。方法:收集52例鼻息肉组织及15例正常的中鼻甲组织作为对照。通过实时荧光定量PCR,免疫印迹和免疫组织化学方法检测Sirtuins,炎症细胞因子(IL-1β, IL5,IL-8, IL-10, IL-17和HMGB1等),氧化应激相关抗氧化酶GPX4的表达水平。用LPS刺激鼻息肉的原发性人鼻腔上皮(HNE)细胞。用基因克隆技术过表达HNE细胞中的Sirt1表达水平,用siRNA技术敲低HNE细胞中的Sirt1表达。通过MTT检测敲低和过表达的Sirt1对HNE增殖的影响。通过流式细胞术(FACS)观察HNE细胞的分化。运用FACS技术,通过膜联蛋白V/7AAD染色测定细胞死亡,通过BODIPY染色评估ROS产生。结果:在本研究中,我们发现Sirt1, Sirt3和Sirt6 mRNA和蛋白表达水平在CRSwNP组织中比正常鼻粘膜组织中低。在CRSwNP中HMGB1, Nrf2, NF-kB和pIkB的蛋白水平增加。然而,GPX4和p-STAT在CRSwNP中表达降低。此外,在鼻息肉中GPX4mRNA表达与IL-1β,IL-5, IL-8和IL-17mRNA表达呈负相关。在体外细胞实验中,我们建立了 Sirt1过表达的人鼻黏膜上皮(HNE )细胞,并通过RNA干扰技术敲低Sirt1。我们发现Sirt1敲低后明显抑制HNE细胞生长,并诱导细胞死亡。并且Sirt1敲低能影响HNE细胞的纤毛分化,以及HNE细胞中的CK14表达水平增加。Sirt1通过调控GPX4, NF-kB和STAT通路来调节HNE细胞的氧化应激能力。此外,在LPS刺激下Sirt1敲低的HNE细胞发生ROS积累和ferroptosis细胞死亡。结论:这些研究结果揭示了 Sirtuins在鼻息肉中的表达异常。Sirt1在鼻黏膜上皮细胞的生长、分化、和炎症氧化应激反应中扮演了重要角色,提供了对免疫应答的表观遗传调节机制的了解。因此,它可以作为控制和预防鼻息肉潜在的治疗靶点。
[Abstract]:Although great progress has been made in the pathogenesis and molecular mechanism of chronic sinusitis and nasal polyps in recent years, the complex inflammatory network and its regulation in chronic sinusitis and nasal polyps are still not well understood. More and more studies have shown that Sirtuins family members (Sirt1-Sirt7) play a protective role in the progression of aging, inflammation, cell death and oxidative stress in various acute or chronic inflammatory diseases, immune diseases and metabolic diseases. Previous studies have shown that Sirt1 and Sirt6 are involved in HMGB1 translocation and active release induced by lipopolysaccharide. However, the role and molecular mechanism of Sirtuins in nasal polyps are unclear, and whether the regulation of various pro-inflammatory cytokines (including IL-1 尾, IL-5,IL-8, IL-10,IL-17 and HMGB1) and reactive oxygen (ROS). (Ros) in chronic sinusitis and nasal polyps is unclear. Therefore, the existence of specific molecular mechanisms and regulatory mechanisms related to inflammation and oxidative stress in nasal polyps requires further study. Aim: to investigate the expression of Sirt1-Sirt6 in chronic sinusitis with nasal polyps and normal nasal mucosa, and to explore the molecular mechanism of Sirt1 in the inflammatory response of nasal epithelial cells and oxidative stress. Methods: 52 cases of nasal polyp and 15 cases of normal middle turbinate were collected as control group. The expression of Sirtuins, inflammatory cytokines (IL-1 尾, IL5,IL-8, IL-10, IL-17, HMGB1, etc.) and the expression of oxidative stress-related antioxidant enzymes (GPX4) were detected by real-time quantitative PCR, blotting and immunohistochemistry. Primary human nasal epithelial (HNE) cells of nasal polyps were stimulated by LPS. The expression level of Sirt1 in HNE cells was overexpressed by gene cloning technique, and Sirt1 expression in HNE cells was reduced by siRNA technique. The effect of knockout and overexpression of Sirt1 on HNE proliferation was detected by MTT. The differentiation of HNE cells was observed by flow cytometry (FACS). FACS technique was used to detect cell death by V/7AAD staining and ROS production was evaluated by BODIPY staining. Results: in this study, we found that the expression levels of Sirt1, Sirt3 and Sirt6 mRNA and protein were lower in CRSwNP than in normal nasal mucosa. The protein levels of HMGB1, Nrf2, NF-kB and pIkB increased in CRSwNP. However, the expression of GPX4 and p-STAT decreased in CRSwNP. In addition, the expression of GPX4mRNA was negatively correlated with the expression of IL-1 尾, IL-5, IL-8 and IL-17mRNA in nasal polyps. In vitro, we established human nasal epithelial (HNE) cells with overexpression of Sirt1, and knocked down Sirt1. by RNA interference technique. We found that Sirt1 knockout significantly inhibited the growth of HNE cells and induced cell death. Furthermore, Sirt1 knockout can affect the ciliated differentiation of HNE cells and increase the expression of CK14 in HNE cells. Sirt1 regulates the oxidative stress ability of HNE cells by regulating GPX4, NF-kB and STAT pathways. In addition, ROS accumulation and ferroptosis cell death occurred in HNE cells with low Sirt1 knockout stimulated by LPS. Conclusion: these results reveal the abnormal expression of Sirtuins in nasal polyps. Sirt1 plays an important role in the growth, differentiation, and inflammatory oxidative stress of nasal epithelial cells. It provides an understanding of the epigenetic regulation mechanism of immune response. Therefore, it can be used as a potential therapeutic target for the control and prevention of nasal polyps.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R765

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