Amurensin H对香烟凝集物刺激的巨噬细胞炎症反应的作用及其机制研究  

Role and mechanisms of Amurensin H inhibiting inflammation in cigarette smoke condensate induced macrophages

作　　者：谭春婷[1] 林芳[1] 赵然然[1] 翟惠芬[1] 姚春所[2] 徐波[1] 林明宝[2] TAN Chun-ting;LIN Fang;ZHAO Ran-ran(Department of Respiratory Medicine,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China.)

机构地区：[1]首都医科大学附属北京友谊医院呼吸内科,北京100050 [2]中国医学科学院北京协和医学院药物研究所,北京100050

出　　处：《临床和实验医学杂志》2021年第4期347-351,共5页Journal of Clinical and Experimental Medicine

基　　金：国家自然基金面上项目(编号:81973539);医科院创新工程项目(编号:2016-I2M-2-006)、新药创制重大专项(编号:2018ZX09711001-003-001)。

摘　　要：目的探讨Amurensin H对香烟烟雾凝集物(CSC)刺激的巨噬细胞炎症反应的作用及其机制。方法以佛波酯(PMA)诱导人单核细胞(THP-1)分化为巨噬细胞样细胞,给予不同浓度(0.2、1.0、5.0μmol/L)的Amurensin H共孵育后,加入CSC刺激。四甲基偶氮唑盐比色(MTT)法测定CSC对THP-1分化的巨噬细胞活性的影响;酶联免疫吸附实验(ELISA)测定Amurensin H(0.2、1.0、5.0μmol/L)各组细胞培养上清液白细胞介素-8(IL-8)及肿瘤坏死因子-α(TNF-α)的水平;荧光免疫沉淀法测定Amurensin H(0.2、1.0、5.0μmol/L)各组细胞组蛋白去乙酰化酶(HDAC)的活性;蛋白质印迹(Western blotting)法及免疫荧光法测定Amurensin H(1.0、5.0μmol/L)2组细胞HDAC2蛋白的表达;Western blotting法测定Amurensin H(1.0、5.0μmol/L)2组细胞磷酸化蛋白激酶B(pAKT)的表达。结果与对照组比较,CSC 100μg/mL在12、24、36 h对细胞活性无影响(P>0.05),在48 h可抑制细胞活性(P<0.05);CSC 150μg/mL在24、48 h可抑制细胞活性(P<0.05);CSC 200μg/mL在12 h即可抑制细胞活性(P<0.01))。CSC 100μg/mL刺激THP-1来源的巨噬细胞24 h后,CSC组释放IL-8、TNF-α明显升高(P<0.01)。Amurensin H 1.0、5.0μmol/L可降低CSC刺激巨噬细胞释放IL-8、TNF-α水平(P<0.05、P<0.01),而Amurensin H 0.2μmol/L对CSC刺激巨噬细胞释放IL-8无明显作用(P>0.05)。CSC组巨噬细胞HDAC活性较对照组明显降低(P<0.01),Amurensin H 1.0、5.0μmol/L可部分恢复CSC刺激巨噬细胞HDAC活性降低(P<0.05、P<0.01),Amurensin H 0.2μmol/L对CSC刺激巨噬细胞HDAC活性降低无明显改善作用(P>0.05)。免疫荧光显示CSC组HDAC2表达降低(P<0.01),Amurensin H 1.0、5.0μmol/L组可部分恢复HDAC2表达(P<0.05、P<0.01)。Western blotting结果显示,CSC组与对照组相比pAKT表达明显升高(P<0.01),给予Amurensin H 1.0、5.0μmol/L可抑制CSC刺激的pAKT蛋白表达(P<0.05、P<0.01)。结论Amurensin H可抑制香烟凝集物刺激引起的巨噬细胞炎症,其作用机制可能是�Objective To investigate the role and mechanisms of Amurensis H in cigarette smoke condensate(CSC)induced inflammation in macrophages.Methods Differentiated macrophages from THP-1 cells incubated with different concentrations of Amurensin H(0.2,1.0,5.0μmol/L)and followed by exposing to cigarette smoke condensate(CSC).Proinflammatory cytokines including interleukin-8(IL-8)and tumor necrosis factor-α(TNF-α)levels in different groups of Amurensin H(0.2,1.0,5.0μmol/L)were measured by enzyme-linked immunosorbent assay(ELISA).HDAC activity in different groups of Amurensin H(0.2,1.0,5.0μmol/L)were assessed by HDAC fluorometric immunoprecipitation.HDAC2 expression in different groups of Amurensin H(1.0,5.0μmol/L)were measured by Western blotting and immunofluorescence.PAKT expression in different groups of Amurensin H(1.0,5.0μmol/L)were measured by Western blotting.Results Compared with the control group,CSC 100μg/mL had no effect on cell viability at 12,24,and 36 h(P>0.05),and could inhibit cell viability at 48 h(P<0.05);CSC 150μg/mL at 24,48 h could inhibit cell viability(P<0.05);CSC 200μg/mL could inhibit cell viability in 12 h(P<0.01).CSC 100μg/mL stimulated THP-1-derived macrophages for 24 h,the levels of IL-8 and TNF-αwere significantly increased in the CSC group(P<0.01).Amurensin H 1.0,5.0μmol/L could reduce the level of IL-8 and TNF-αreleased by CSC stimulated macrophages(P<0.05,P<0.01),while Amurensin H 0.2μmol/L had no significant effect on CSC stimulated macrophages to release IL-8(P>0.05).The HDAC activity of macrophages in the CSC group was significantly lower than that of the control group(P<0.01),Amurensin H 1.0,5.0μmol/L could partially restore the HDAC activity of CSC-stimulated macrophages(P<0.05,P<0.01),Amurensin H 0.2μmol/L had no significant effect on improving the reduction of HDAC activity in CSC-stimulated macrophages(P>0.05).Immunofluorescence showed that the expression of HDAC2 in the CSC group was reduced(P<0.01),and the expression of HDAC2 in the Amurensin H 1.0,5.0μmol/L gro

关 键 词：Amurensin H 香烟烟雾凝集物 组蛋白去乙酰化酶2 蛋白激酶B 慢性阻塞性肺疾病 

分 类 号：R285[医药卫生—中药学]