参附注射液调控HDAC3抑制LPS诱导的巨噬细胞HMGB1核转位的机制  

Mechanistic on the Inhibition of LPS-induced Nuclear Translocation of HMGB1 in Macrophages by Modulation of HDAC3 by Shenfu Injection

作　　者：杨晓龙 苟玮 艾飞 刘霞 褚春薇 陈向云 郭俊峰 YANG Xiao-long;GOU Wei;AI Fei;LIU Xia;CHU Chun-wei;CHEN Xiang-yun;GUO Jun-feng(School of Basic Medical Sciences,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;The Second Clinical Medical College,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;The Yangsheng College of Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China)

机构地区：[1]贵州中医药大学基础医学院,贵阳550025 [2]贵州中医药大学第二临床医学院,贵阳550025 [3]贵州中医药大学中医养生学院,贵阳550025

出　　处：《科学技术与工程》2025年第6期2265-2273,共9页Science Technology and Engineering

基　　金：国家自然科学基金地区基金(82260874);贵州中医药大学博士启动基金[(2018)36]。

摘　　要：为观察脂多糖(lipopolysaccharides,LPS)诱导的RAW264.7细胞中组蛋白去乙酰化酶3(histone deacetylase 3,HDAC3)对高迁移率组蛋白B1(high mobility group box-1 protein,HMGB1)表达和核移位的影响及参附注射液(Shenfu injection,SFI)的干预作用。通过LPS诱导RAW264.7细胞建立细胞炎症损伤模型,分别用3、6、12μL/mL剂量SFI干预细胞24 h。实时荧光PCR法(RT-qPCR)检测细胞中HDAC3、HMGB1、白介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)转录水平;Western-blot法检测HMGB1和HDAC3蛋白表达;免疫荧光法观察SFI对HMGB1亚细胞定位的影响;ELISA法检测细胞上清HMGB1、IL-1β和TNF-α分泌水平;小干扰RNA(small interfering RNA,siRNA)靶向沉默RAW264.7细胞中HDAC3后,免疫荧光法观察SFI对HMGB1亚细胞定位的影响。结果表明模型组与对照组比较,模型组RAW264.7细胞中HDAC3的转录和表达均显著降低(P<0.01),HMGB1表达显著升高(P<0.01)且同时从核内向胞浆迁移;细胞上清中炎症因子HMGB1、IL-1β和TNF-α明显升高(P<0.01);与模型组比较,SFI(6、12μL/mL剂量组)上调RAW264.7细胞中HDAC3的转录和表达水平,下调炎性因子HMGB1的转录、表达、核移位,抑制HMGB1、IL-1β和TNF-α的分泌;靶向沉默HDAC3后,大量HMGB1定位于胞浆,经LPS刺激后蛋白定位无明显变化,且SFI不能逆转HMGB1的异常定位。可见SFI可能通过上调HDAC3表达从而抑制LPS诱导的RAW264.7细胞中HMGB1核外迁移,进而抑制了其下游的炎症反应。To observe the effects of LPS(lipopolysaccharide)-HDAC3(induced histone deacetylase 3)on the expression of HMGB1(high mobility histone B1)and nuclear translocation in RAW264.7 cells,and the intervention effect of SFI(Shenfu injection).RAW264.7 cells were induced by LPS to establish a cellular inflammatory injury model,and the cells were intervened with SFI at doses of 3,6,and 12μL/mL for 24 h.RT-qPCR(real-time fluorescence PCR)was used to detect the transcriptional levels of HDAC3,HMGB1,IL-1β,and TNF-αin the cells,and Western-blot was used to detect the protein expression of HMGB1 and HDAC3,and immune immunoassay to detect the protein expression of HMGB1 and HDAC3.HDAC3 protein expression,immunofluorescence to observe the effect of SFI on the subcellular localization of HMGB1.ELISA to detect the secretion levels of HMGB1,IL-1β,and TNF-αin the cell supernatant.And small interfering RNA(siRNA)after targeting to silence the HDAC3 in RAW264.7 cells.to observe the effect of SFI on HMGB1 subcellular localization.Compared with the control group,the transcription and expression of HDAC3 in RAW264.7 cells in the model group were significantly reduced(P<0.01),and the expression of HMGB1 was significantly elevated(P<0.01)and simultaneously migrated from the nucleus to the cytoplasm.The inflammatory factors in the supernatant of the cells,such as HMGB1,IL-1βand TNF-α,were significantly elevated(P<0.01).And compared with the model group,SFI(6,12μL/mL dose group)up-regulated the transcription and expression levels of HDAC3,down-regulated the transcription,expression,and nuclear translocation of the inflammatory factor HMGB1,and inhibited the secretion of HMGB1,IL-1β,and TNF-αin RAW264.7 cells.After targeted silencing of HDAC3,a large amount of HMGB1 was localized in the cytoplasm,and there was no significant change in protein localization after LPS stimulation,and SFI could not reverse the abnormal localization of HMGB1.SFI may inhibit LPS-induced extra-nuclear migration of HMGB1 in RAW264.7 cells by up-regulating HD

关 键 词：参附注射液 HMGB1 HDAC3 巨噬细胞 炎症 内毒素休克 

分 类 号：R259[医药卫生—中西医结合]