通腑理肺汤通过调控miR-146a抑制THP-1细胞PD-1/PD-L1信号通路的作用机制  

Mechanism of Tongfu Lifei decoction inhibiting the programmed death-1/programmed death-ligand 1 signaling pathway in THP-1 cells by regulating microRNA-146a

作　　者：吕波 李兰 黄瑞峰 周霞辉 韩立鹏 Lyu Bo;Li Lan;Huang Ruifeng;Zhou Xiahui;Han Lipeng(Department of Critical Care Medicine,the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550001,Guizhou,China;Guizhou University of Traditional Chinese Medicine,Guiyang 550001,Guizhou,China)

机构地区：[1]贵州中医药大学第一附属医院重症医学科,贵阳550001 [2]贵州中医药大学,贵阳550001

出　　处：《中华危重病急救医学》2024年第10期1038-1043,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81860846);贵州省中医药、民族医药重点学科建设项目(QZYYZDXK(JS)-2023-02)。

摘　　要：目的探讨通腑理肺汤(TFL)对脂多糖(LPS)诱导人单核细胞白血病细胞THP-1的保护作用与机制。方法①体外培养THP-1细胞,与1 mg/L LPS共孵育18 h构建体外THP-1细胞炎症模型;另取THP-1细胞作为空白对照组。采用酶联免疫吸附试验(ELISA)检测细胞分泌肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平。②将THP-1炎症细胞分为7组,分别给予0、0.005、0.01、0.02、0.04、0.08、0.16 mL/mL TFL(每毫升培养液加入不同剂量TFL溶液,生药含量为1 kg/L)干预24 h。采用四甲基偶氮唑盐(MTT)比色法检测细胞存活率,筛选出TFL对THP-1炎症细胞无毒性作用的干预剂量。③另取THP-1炎症细胞,依据MTT比色法筛选出的TFL干预剂量,将细胞分为炎症模型组及0.01、0.02、0.04 mL/mL TFL组。各组干预24 h后采用ELISA法测定细胞分泌TNF-α和IL-6水平;采用蛋白质免疫印迹试验(Western blotting)检测细胞中程序性死亡受体-1/程序性死亡受体配体-1(PD-1/PD-L1)信号通路蛋白表达;采用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测细胞中微小RNA(miR-146a、miR-146b、miR-155)表达。④以TFL对THP-1炎症细胞无毒性作用最大剂量0.04 mL/mL作为干预剂量,将THP-1炎症细胞分为炎症模型组、TFL组、TFL+miR-146a抑制剂组、TFL+miR-146b抑制剂组和TFL+miR-155抑制剂组。炎症模型组不给予任何药物干预;各抑制剂组在0.04 mL/mL TFL干预的基础上,加入相应抑制剂100 nmol/L。各组干预24 h后采用ELISA法测定细胞分泌TNF-α和IL-6水平;采用Western blotting检测细胞中PD-1/PD-L1信号通路蛋白表达。结果①与空白对照组比较,炎症模型组细胞分泌TNF-α和IL-6的水平明显升高,说明体外THP-1炎症细胞模型构建成功。②0~0.04 mL/mL TFL均未对THP-1炎症细胞产生毒性作用,而0.08 mL/mL和0.16 mL/mL TFL组细胞存活率明显低于炎症模型组,说明当TFL剂量超过0.04 mL/mL时对THP-1炎症细胞具有毒性作用。③与炎症模型�Objective To explore the protective effect and mechanism of Tongfu Lifei decoction(TFL)on human monocytic leukemia cell THP-1 induced by lipopolysaccharide(LPS).Methods ①THP-1 cells were cultured in vitro,and incubated with 1 mg/L LPS for 18 hours to construct an in vitro THP-1 cell inflammation model.Other THP-1 cells were taken as blank control group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)secreted by cells.②THP-1 cells were divided into seven groups and treated with 0,0.005,0.01,0.02,0.04,0.08,and 0.16 mL/mL TFL for 24 hours(added different dosages of TFL solution per milliliter of culture medium,with a crude drug content of 1 kg/L).The cell survival rate was detected using methyl thiazolyl tetrazolium(MTT)colorimetric method,and the intervention dosage of TFL for its non-toxic effect on THP-1 cells was screened.③Another THP-1 cells were divide into inflammatory model group and 0.01,0.02,and 0.04 mL/mL TFL groups according to the intervention dosage of TFL screened by MTT colorimetry.After 24 hours of intervention,the levels of TNF-αand IL-6 secreted by cells were measured using ELISA.Western blotting was used to detect the expressions of programmed death-1/programmed death-ligand 1(PD-1/PD-L1)signaling pathway proteins in cells.Real time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expressions of microRNAs(miR-146a,miR-146b,miR-155)in cells.④The maximum non-toxic concentration of TFL(0.04 mL/mL)on the THP-1 cell was selected as the intervention dose.THP-1 cells were divided into inflammation model group,TFL group,TFL+miR-146a inhibitor group,TFL+miR-146b inhibitor group,and TFL+miR-155 inhibitor group.The inflammation model group was not given any drug intervention.The other inhibitor groups were added 100 nmol/L corresponding inhibitor.After 24 hours of intervention,the levels of TNF-αand IL-6 secreted by cells were measured using ELISA.Western blotting

关 键 词：脓毒症 通腑理肺汤 微小RNA-146a 程序性死亡受体-1/程序性死亡受体配体-1信号通路 

分 类 号：R285[医药卫生—中药学]