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
李静 1何茳萍 2许娟 2赵天雪 2刘思悦 2仇海燕 2詹宇红2
作者信息
- 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
- 折叠
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、LC3B Ⅱ/LC3B Ⅰ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-1 β、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蛋白表达水平较MUS组显著升高(P＜0.05),并且MUS+RAPA组细胞中P62蛋白表达水平较MUS组进一步降低,ATG-5、LC3B蛋白表达水平较MUS组进一步升高(P＜0.05);si-SIRT6转染THP-1细胞中P62蛋白表达水平较空白病毒转染THP-1细胞显著降低,ATG-5、LC3B蛋白表达水平较空白病毒转染THP-1细胞显著升高.结论 SIRT-6基因低表达可促进单核细胞炎症反应和细胞自噬作用,并且尿酸盐和自噬激动剂雷帕霉素可加重炎症反应和细胞自噬作用.
Abstract
Objective To investigate the effect of low expression of silencing information regulator-6(SIRT-6)on inflammatory reaction and autophagy in monocytes.Methods Human 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 LC3B Ⅱ/LC3B Ⅰ in 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.Results The 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-α in 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-α in the supernatant of cells further increased by MUS(P＜0.05).The levels of IL-1β,IL-6 and TNF-α in 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 LC3B in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells significantly increased by MUS(P＜0.05).The gene expression of p62 in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells further decreased by RAPA(P＜0.05),the gene expression of ATG-5,Beclin-1,LAMP-1 and LC3B in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells further increased by RAPA(P＜0.05).The gene expression level of p62 in si-SIRT6 transfected THP-1 cells significantly decreased than that in si-SIRT6 NC transfected THP-1 cells(P＜0.05),and the gene expression level of ATG-5,LC3B,Beclin-1 and LAMP-1 significantly increased than that in si-SIRT6 NC transfected THP-1 cells(P＜0.05).The protein expression of p62 in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells significantly decreased by MUS(P＜0.05),the protein expression of ATG-5 and LC3B Ⅱ/LC3B Ⅰ protein in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells significantly increased by MUS(P＜0.05).The protein expression of p62 in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells further decreased by RAPA P＜0.05)),the protein expression of ATG-5 and LC3B Ⅱ/LC3B Ⅰ in si-SIRT6/si-SIRT6 NC-transfected THP-1 cells further increased by RAPA(P＜0.05).The protein expression level of p62 in si-SIRT6 transfected THP-1 cells significantly decreased than that in si-SIRT6 NC transfected THP-1 cells(P＜0.05),and the protein expression level of ATG-5 and LC3B 11/LC3B Ⅰ significantly increased than that in si-SIRT6 NC transfected THP-1 cells(P＜0.05).Conclusion Low expression of SIRT-6 gene can promote inflammatory reaction and autophagy in monocytes,and Monosodium urate and autophagy agonist rapamycin can aggravate inflammatory reaction and autophagy.
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Key words
Gout/Silent information regulator/Monocytes/Inflammatory reaction/Autophagy/Urate/Rapamycin引用本文复制引用
基金项目
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)
出版年
2024
NETL
NSTL
万方数据