摘要
目的探讨黄芪多糖(APS)对脂肪组织巨噬细胞活化的影响及作用机制。方法2018年11月至2019年5月,通过0.4μm孔径大小的Transwell小室共培养方式,将巨噬细胞种于上室,将脂肪细胞种于下室。实验分为三组,空白对照组(NC组)、肿瘤坏死因子-α(TNF-α)诱导干预组(TNF-α组)和APS干预组(APS+TNF-α组),酶联免疫吸附测定(ELISA)检测细胞培养上清液中白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)水平,实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测巨噬细胞诱生型一氧化氮合酶(iNOS)和TNF-αmRNA表达水平,蛋白质印迹法(Westernblotting)检测iNOS和磷酸化AMP活化蛋白激酶α1(p-AMPKα1)和沉默信息调节蛋白1(SIRT1)水平。同时,采用8μm孔径大小的Transwell小室共培养方式,结晶紫染色观察巨噬细胞的趋化情况。结果与NC组比较,TNF-α组细胞培养上清液中IL-6和MCP-1水平显著升高(P<0.05),巨噬细胞中TNF-αmRNA、iNOSmRNA和蛋白水平显著升高(P<0.05),p-AMPKα1和SIRT1蛋白水平显著降低(P<0.05),趋化巨噬细胞数显著升高(P<0.05),其中NC组iNOSmRNA和蛋白水平分别为(1.00±0.17)、(0.43±0.05),TNF-α组分别为(3.57±0.31)、(1.18±0.16)。与TNF-α组比较,APS+TNF-α组细胞培养上清液中IL-6和MCP-1水平显著降低(P<0.05),巨噬细胞中TNF-αmRNA、iNOSmRNA和蛋白水平显著降低(P<0.05),p-AMPKα1和SIRT1蛋白水平显著升高(P<0.05),趋化巨噬细胞数显著降低(P<0.05),其中TNF-α组iNOSmRNA和蛋白水平(3.57±0.31)、(1.18±0.16),APS+TNF-α组分别为(1.87±0.22)、(0.62±0.08)。结论APS可抑制巨噬细胞的活化,其作用机制与激活AMPK/SIRT1信号通路有关。
Objective To investigate the effect of Astragalus polysaccharide(APS) on the activation of adipose tissue macrophages and its mechanism.Methods From November 2018 to May 2019,macrophages were seeded in the upper chamber and fat cells were cultured in the lower chamber by a 0.4 μm pore size Transwell chamber co-culture method.The experiment was assigned into three groups:blank control group(NC group),tumor necrosis factor-α(TNF-α) induction intervention group(TNF-α group) and APS intervention group(APS+TNF-α group).The levels of interleukin-6(IL-6) and monocyte chemotactic protein-1(MCP-1) in the cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Real-time quantitative PCR(qRT-PCR) was used to detect the mRNA expression level of inducible nitric oxide synthase(iNOS) and TNF-α in macrophages.Western blotting was used to detect iNOS and phospho-adenosine monophosphate activated protein kinase alpha 1(p-AMPKα1) and silent information regulator protein 1(SIRT1) levels.At the same time,the chemo taxis of macrophages was observed by crystal violet staining using a Trans well chamber coculture method with a pore size of 8 μm.Results Compared with NC group,the levels of IL-6 and MCP-1 in the culture supernatant of TNF-α group were significantly increased(P<0.05),and the mRNA levels of TNF-α and iNOS and the protein level of iNOS were significantly increased(P<0.05),and the levels of p-AMPKα1 and SIRT1 protein were significantly reduced(P<0.05),and the number of chemotactic macrophages was increased(P<0.05).The level of iNOS mRNA and protein in NC group was(1.00±0.17) and(0.43±0.05),respectively,and those in TNF-α group was(3.57±0.31) and(1.18±0.16),respectively.Compared with TNF-α group,the levels of IL-6 and MCP-1 in APS+TNF-α group were significantly decreased(P<0.05),and the mRNA levels of TNF-α and iNOS and the protein level of iNOS were significantly decreased(P<0.05),and the levels of p-AMPKα1 and SIRT1 protein were significantly increased(P<0.05),and the number of
作者
朱云峰
覃艳
曹萌
翟倩倩
李燕
王涛
ZHU Yunfeng;QIN Yan;CAO Meng;ZHAI Qianqian;LI Yan;WANG Tao(Ward 2 of Endocrinology Department,The First Affiliated Hospital of Xinxiang Medical University,Xinxiang,Henan 453100,China)
出处
《安徽医药》
CAS
2021年第12期2350-2354,I0001,共6页
Anhui Medical and Pharmaceutical Journal
关键词
黄芪
脂肪组织
胰岛素抵抗
黄芪多糖
肿瘤坏死因子-Α
巨噬细胞
Astragalus membranaceus
Adipose tissue
Insulin resistance
Astragalus polysaccharide
TNF-α
Macrophage
