Sirt1通过去乙酰化NF-κB/p65减轻小鼠肺泡Ⅱ型上皮细胞脂多糖损伤  

Sirt1 protects against lipopolysaccharide-induced injury in mouse type Ⅱ alveolar epithelial cells by deacetylating RelA/p65 subunit of nuclear factor-κB

作　　者：刘俊彦[1] 吕学军[1] 赵维[1] 胡明冬[1] 李玉英[1] 王关嵩[1] 徐剑诚 钱桂生[1] 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所全军呼吸病研究重点实验室,重庆400037

出　　处：《第三军医大学学报》2017年第14期1415-1421,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81370167)~~

摘　　要：目的研究酵母沉默信息调节因子2相关酶1(sirtuin type 1,Sirt1)在肺泡Ⅱ型上皮细胞(AECⅡ)脂多糖(LPS)损伤中对NF-κB的作用。方法购买Sirt1过表达(Tg)小鼠和野生型(WT)小鼠,饲养、繁殖、鉴定新生小鼠基因型;通过qRT-PCR和Western blot鉴定Tg小鼠和WT小鼠肺组织Sirt1 mRNA和蛋白表达差异;两种不同Sirt1基因背景的小鼠LPS(15 mg/kg)腹腔注射12 h后,HE染色鉴定肺组织病理变化差异;分离纯化两种小鼠的AECⅡ并进行鉴定;两种不同Sirt1基因背景的AECⅡ用LPS 10μg/mL处理后,分别在0、2、4、6、8、12、24 h用ELISA检测细胞上清液中白细胞介素-6(IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平;用LPS 10μg/mL处理两种AECⅡ,同时设置生理盐水对照组,12 h后用Western blot鉴定细胞NF-κB/p65蛋白和乙酰化NF-κB/p65蛋白的表达差异。结果新生小鼠有Tg和WT两种不同的基因型;Tg小鼠肺组织Sirt1 mRNA和蛋白的表达水平显著高于WT小鼠(P<0.05);小鼠腹腔注射LPS 15 mg/kg 12 h后肺组织病理变化显示,Tg小鼠较WT小鼠肺组织损伤程度轻;两种AECⅡ用LPS 10μg/mL处理后,细胞上清液中IL-6和TNF-α的表达水平随时间推移呈现逐渐升高的趋势,Tg组AECⅡ细胞上清液中IL-6和TNF-α的表达水平在多个时间点都显著低于WT组(P<0.05);两种AECⅡ经LPS(10μg/mL)处理后,与各自的空白对照组相比Sirt1含量下降,且WT组AECⅡ下降更明显,LPS处理后WT组AECⅡ的NF-κB/p65和乙酰化NF-κB/p65的变化倍数均高于Tg组。结论 Sirt1可能通过去乙酰化NF-κB/p65参与调控AECⅡLPS炎症损伤,抑制急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)小鼠肺部炎症反应。Objective To determine whether nuclear factor-κB（ NF-κB） are regulated by Sirtuin type 1（ Sirt1） in mouse type Ⅱ alveolar epithelial cells（ AEC Ⅱs） following exposure to lipopolysaccharide（ LPS）. Methods Genetically modified mice with Sirt1 overexpression（ Tg） and wild-type（ WT） mice were bred and the genotypes of the offspring mice were identified. The expression levels of Sirt1 mRNA and protein in the lung tissues of Tg mice and WT mice were compared using qRT-PCR and Western blotting. Tg mice and WT mice were subjected to a single intraperitoneal injection of LPS（ 15 mg/kg）,and 12 h later,the lung tissues of the mice were harvested for HE staining. AECⅡs were isolated and purified from both Tg mice and WT mice,and at 0,2,4,6,8,12,and 24 h after LPS（ 10 μg/mL） exposure,the cells were examined for interleukin-6（ IL-6） and tumor necrosis factor-α（ TNF-α） in the supernatant using ELISA. The AEC Ⅱ s from Tg and WT mice were challenged with LPS（ 10 μg/mL） or normal saline（ control）,and Western blotting was used to detect the expression of NF-κB/p65 and acetylized NF-κB/p65 in the cells 12 h after the treatment. Results In Tg mice,the expression levels of Sirt1 mRNA and protein in the lung tissue were significantly higher than those in WT mice（ P〈0. 05）. At 12 h of after injection of LPS（ 15 mg/kg）,Tg mice exhibited milder lung injuries than the WT mice. In AECⅡs from Tg and WT mice,LPS（ 10 g/mL）treatment resulted in significant and progressive increases in the expression levels of IL-6 and TNF-α as the treatment time extended,and at each time point of measurement,the levels of IL-6 and TNF-α were significantly lower in AECⅡs from Tg mice than in the cells from the WT mice（ P〈0. 05）. LPS（ 10 μg/mL） treatment reduced the expression of Sirt1 in AEC Ⅱ s from both Tg and WT mice,and the decrease was more pronounced in the cells from the WT mice; LPS also induced more obvious changes in the levels of NF-κB/p65 and acetylized

关 键 词：SIRT1 急性呼吸窘迫综合征 肺泡Ⅱ型上皮细胞 NF-ΚB 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]