沉默信息调节因子6基因低表达对单核细胞炎症反应细胞自噬的调控作用及对痛风炎症的影响  

Effect of low expressed SIRT-6 gene on inflammatory reaction and autophagy in monocytes

作　　者：李静 何茳萍 许娟 赵天雪 刘思悦 仇海燕 詹宇红 Li Jing;He Jiangping;Xu Juan;Zhao Tianxue;Liu Siyue;Qiu Haiyan;Zhan Yuhong(Department of Endocrinology,Hangzhou Geriatric Hospital,Hangzhou 310022,China;Department of Endocrinology,Affiliated Hangzhou First People's Hospital,School of Medicine,Westlake University,Hangzhou 310006,China)

机构地区：[1]杭州市老年病医院内分泌科,杭州310022 [2]西湖大学医学院附属杭州市第一人民医院内分泌科,杭州310006

出　　处：《中华风湿病学杂志》2024年第8期558-565,共8页Chinese Journal of Rheumatology

基　　金：浙江省医药卫生科技计划项目(2021KY879);杭州市医药卫生科技项目(A20200753)。

摘　　要：目的探讨沉默信息调节因子6(SIRT-6)低表达对单核细胞炎症反应及细胞自噬作用的调控作用。方法人急性单核细胞白血病细胞株(THP-1)通过转染SIRT6基因感染慢病毒(si-SIRT6)建立SIRT-6低表达THP-1细胞株,将细胞分为对照组、尿酸盐(MUS)组和MUS+雷帕霉素(RAPA)组,对照组细胞加入PBS,MUS组细胞加入MUS,MUS+RAPA组细胞加入MUS和RAPA,各组细胞培养48 h,采用ELISA检测各组细胞上清液IL-1β、IL-6和TNF-α水平,采用定量聚合酶链反应(Q-PCR)检测各组细胞自噬相关蛋白-5(ATG-5)、重组人自噬效应蛋白(Beclin-1)、溶酶体相关膜蛋白-1(LAMP-1)、微管相关蛋白1轻链3B(LC3B)和p62基因表达水平,采用蛋白免疫印迹法检测各组细胞p62、ATG-5、LC3BⅡ/LC3BⅠ蛋白表达水平。计量资料通过单因素方差分析(ANOVA)分析多组间数据的差异,组间比较采用LSD-t检验。结果si-SIRT-6转染组THP-1细胞SIRT-6基因和蛋白表达水平较空白病毒转染组显著降低(基因:1.09±0.08与0.57±0.03,t=14.91,P<0.001;蛋白:0.21±0.04与0.12±0.03,t=4.41,P=0.070)。MSU组si-SIRT6/空白病毒转染THP-1细胞上清液IL-1β、IL-6和TNF-α水平较对照组显著升高,并且MUS+RAPA组细胞上清液IL-1β、IL-6和TNF-α水平较MUS组进一步升高;si-SIRT6转染THP-1细胞上清液IL-1β、IL-6和TNF-α水平较空白病毒转染THP-1细胞显著升高。MSU组si-SIRT6/空白病毒转染THP-1细胞中p62基因表达水平较对照组显著降低,ATG-5、Beclin-1、LAMP-1和LC3B基因表达水平较对照组显著升高,并且MUS+RAPA组细胞中p62基因表达水平较MUS组进一步降低,ATG-5、Beclin-1、LAMP-1和LC3B基因表达水平较MUS组进一步升高;si-SIRT6转染THP-1细胞中p62基因表达水平较空白病毒转染THP-1细胞显著降低,ATG-5、LC3B、Beclin-1和LAMP-1基因表达水平较空白病毒转染THP-1细胞显著升高。MSU组si-SIRT6/空白病毒转染THP-1细胞中P62蛋白表达水平较MUS组显著降低,ATG-5、LC3B蛋白表达水ObjectiveTo investigate the effect of low expression of silencing information regulator-6(SIRT-6)on inflammatory reaction and autophagy in monocytes.MethodsHuman acute monocytic leukemia cell line THP-1 was transfected with si-SIRT6 to establish THP-1 cell line with low expressed SIRT-6.The cells were divided into control group,MUS group and MUS+RAPA group.Cells in control group were cultured with medium added with PBS,cells in MUS Group were cultured with medium added with MUS,and cells in MUS+RAPA Group were added with MUS and Rapamycin.Cells in each group were cultured for 48 hours.The levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the supernatant of each group were detected by enzyme-linked immunosorbent assay(ELISA).The gene expression levels of autophagy-associated protein-5(ATG-5),Beclin-1,lysosomal-associated membrane protein-1(LAMP-1),microtubule-associated protein-1 light chain 3B(LC3B)and p62 in cells of each group were detected by Q-PCR.The protein expression levels of p62,ATG-5 and LC3BⅡ/LC3BⅠin cells of each group.The one-way analysis of variance(ANOVA)was used for the measurement data in multi-groups,and the LSD-t test was used for the measurement data in both groups.ResultsThe gene and protein expression of SIRT-6 in THP-1 cells decreased significantly after si-SIRT6 transfection(Gene:1.09±0.08 vs.0.57±0.03,t=14.91,P<0.001;Protein:0.21±0.04 vs.0.12±0.03,t=4.41,P=0.070).The levels of IL-1β,IL-6,and TNF-αin the supernatant of si-SIRT6/si-SIRT6 NC-transfected THP-1 cells increased significantly by MUS(P<0.05),and the levels of IL-1β,IL-6,and TNF-αin the supernatant of cells further increased by MUS(P<0.05).The levels of IL-1β,IL-6 and TNF-αin the supernatant of si-SIRT6-transfected THP-1 cells increased significantly compared with those of si-SIRT6 NC-transfected THP-1 cells(P<0.05).The gene expression of p62 in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells significantly decreased by MUS(P<0.05),the gene expression of ATG-5,Beclin-1,LAMP-1 and

关 键 词：痛风 沉默信息调节因子 单核细胞 炎症反应 自噬 尿酸盐 雷帕霉素 

分 类 号：R589.7[医药卫生—内分泌]