组蛋白去乙酰化酶2调节Nrf2乙酰化水平在脂多糖诱导Ⅱ型肺泡上皮细胞损伤中的抗氧化作用机制  

Antioxidant mechanism of HDAC2 regulating Nrf2 acetylation in LPS-induced type II alveolar epithelial cell injury

作　　者：陈隆望[1] 罗以楠 蔡文超 李萌芳[1] 连洁[1] 赵光举[1] 洪广亮[1] 卢中秋[1] 邱俏檬[1] Chen Longwang;Luo Yin an;Cai Wenchao;Li Mengfang;Lian Jie;Zhao Guangju;Hong Guangliang;LuZhongqiu;Qiu Qiaomeng(Emergency Department, the First A ffiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China)

机构地区：[1]温州医科大学附属第一医院急诊医学中心,325000

出　　处：《中华急诊医学杂志》2019年第3期328-334,共7页Chinese Journal of Emergency Medicine

基　　金：浙江省自然科学基金(LY15H150006, LQ15H150003);国家自然科学基金(81772112, 81571937, 81871583);浙江省医药卫生科研项目(2015KYA152);浙江省中医药管理局项目(2015ZA120);温州市科技计划项目(Y20170177, Y20170185, Y20150182).

摘　　要：目的探讨组蛋白去乙酰化酶2(histone deacetylases,HDAC2)调节核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)乙酰化水平在脂多糖(lipopolysaccharides,LPS)诱导的Ⅱ型肺泡上皮细胞损伤中的抗氧化作用机制。方法常规培养小鼠Ⅱ型肺泡上皮细胞,用不同浓度的LPS(10、100、1 000 ng/mL)刺激,分别在0、6、12、24及48 h时用CCK-8检测细胞活性,选择合适的浓度和时间点。常规培养小鼠Ⅱ型肺泡上皮细胞,随机分为对照组、LPS组、HDAC2慢病毒干扰组(siRNA-HDAC2组)和HDAC2慢病毒过表达组(LV-HDAC2组)。蛋白免疫印迹法(Western blot)检测HDAC2和Nrf2蛋白表达水平,免疫共沉淀法检测Nrf2乙酰化水平,放线菌酮作用后检测Nrf2稳定性。化学比色法检测细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)活性。采用SPSS 23.0统计软件,多组间比较采用单因素方差分析检验,组间两两比较采用LSD-t检验。结果与对照组相比,LPS组HDAC2的蛋白表达水平升高(t=5.974,P=0.027),Nrf2乙酰化水平降低(t=7.223,P=0.002),Nrf2蛋白水平升高(t=2.929,P=0.043),Nrf2的蛋白稳定性升高,SOD活性下降(t=121,P<0.01),MDA含量升高(t=10.45,P=0.000 5)。与LPS组相比,过表达HDAC2后Nrf2乙酰化水平降低(t=11.29,P=0.000 4),Nrf2蛋白表达水平升高明显(t=3.194,P=0.033),Nrf2的蛋白稳定性升高,SOD活性升高(t=4.678,P=0.009),MDA含量下降(t=5.417,P=0.005 6),而下调HDAC2后呈现相反的趋势。结论LPS刺激后Ⅱ型肺泡上皮细胞氧化应激加重,HDAC2可降低Nrf2乙酰化水平,提高Nrf2蛋白表达,减轻LPS引起的氧化应激。Objective To explore the antioxidant mechanism of histone deacetylase 2 (HDAC2) regulating Nrf 2 acetylation in lipopolysaccharide (LPS)-induced type Ⅱ alveolar epithelial cell injury. Methods The experiment was divided into two parts. The first part was the routine culture of type Ⅱ alveolar epithelial cells of mice. The cells were stimulated with different concentrations of LPS (10 ng/ mL, 100 ng/mL and 1 000 ng/mL). CCK-8 was used to detect the cell activity at 0 h, 6 h, 12 h, 24 h and 48 h, respectively. The second part: Alveolar epithelial cells of typeⅡwere cultured and divided into the normal control group (control group), LPS group, HDAC2 lentivirus interference group (siRNA-HDAC2 group) and HDAC2 lentivirus overexpression group (LV-HDAC2 group). The expression of HDAC2 and Nrf2 were detected by Western blot, the acetylation of Nrf2 was detected by immunoprecipitation, and the stability of nrf2 was detected after actinidone action. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by chemical colorimetry. SPSS 23.0 statistical software was used. LSD-t test was used for comparison between two groups, and one-way ANOVA test was used for comparison among multiple groups. Results Compared with the control group, the expression of HDAC2 protein in the LPS group increased (t=5.974, P=0.027), the acetylation level of Nrf2 decreased (t=7.223, P=0.002), the Nrf2 protein level increased (t=2.929, P=0.043), the protein stability of Nrf2 increased, the SOD activity decreased (t=121. P<0.01), and the MDA content increased (t=10.45, P=0.000 5). Compared with the LPS group, Nrf2 acetylation level decreased in the LV-HDAC2 group (t=11.29, P=0.000 4), Nrf2 protein expression increased (t=3.194, P=0.033), Nrf2 protein stability increased, SOD activity increased (t=4.678, P=0.009), and MDA content decreased in the LV-HDAC2 group (t=5.417, P=0.005 6). While the opposite trend was observed in the siRNA-HDAC2 group. Conclusion After LPS stimulation, oxidative stress of type Ⅱ alveolar e

关 键 词：组蛋白去乙酰化酶2 核因子E2相关因子2 脓毒症 脂多糖 氧化应激 丙二醛 超氧化物歧化酶 

分 类 号：R459.7[医药卫生—急诊医学]