基于NAMPT/SIRT1的温肺化纤汤含药血清延缓肺间充质干细胞炎性衰老的作用机制  

M echanism of Wenfei Huaxian Decoction-containing Serum in Delaying Inflammatory Senescence of Lung M esenchymal Stem Cells Based on NAM PT/SIRT1

作　　者：胡君霞 许越淇 王军 朱国双 柯诗文 邱明亮[2] 刘良徛[2] 莫丽莎[2] HU Junxia;XU Yueqi;WANG Jun;ZHU Guoshaung;KE Shiwen;QIU Mingliang;LIU Liangji;MO Lisha(Jiangxi University of Chinese Medicine,Nanchang 330004,China;The Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China)

机构地区：[1]江西中医药大学,南昌330004 [2]江西中医药大学附属医院,南昌330006

出　　处：《中国实验方剂学杂志》2024年第12期45-53,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82305162);江西省自然科学基金项目(20232BAB206145,20224BAB216098,20212BAB206061);江西省科技厅应用研究培育计划项目(20212BAG70038);江西省教育厅课题项目(GJJ211220,GJJ2200912);江西中医药大学校级科技创新团队发展计划项目(CXTD22011);江西省中医药管理局科技计划项目(2023B1200)。

摘　　要：目的:通过温肺化纤汤含药血清干预D-半乳糖(D-gal)诱导的肺间充质干细胞(LMSCs),探索温肺化纤汤通过烟酰胺磷酸核糖转移酶/沉默信息调节因子1(NAMPT/SIRT1)信号通路延缓LMSCs衰老的作用机制。方法:制备温肺化纤汤含药血清。采用梯度密度离心法分离LMSCs,体外培养并鉴定LMSCs。通过D-gal刺激细胞24 h建立体外衰老模型。温肺化纤汤含药血清不同体积分数(5%、10%、20%、40%、80%)干预LMSCs细胞24 h后造模并采用噻唑蓝(MTT)比色法检测细胞增殖水平。将细胞随机分为空白血清组、模型组、温肺化纤汤含药血清高、中、低剂量组。采用衰老相关β-半乳糖苷酶(SA-β-gal)染色检测各组LMSCs衰老情况;比色法检测NAD+含量;酶联免疫吸附测定法(ELISA)检测细胞培养上清液中衰老相关因子(p16、p53)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平;蛋白免疫印迹法(Western blot)检测衰老相关蛋白及NAMPT/SIRT1信号通路相关蛋白的相对表达量。结果:与空白血清组比较,D-gal刺激24 h后,LMSCs细胞增殖受到显著抑制(P<0.01);与模型组比较,予以相应的温肺化纤汤含药血清干预后均可促进LMSCs细胞的增殖(P<0.05,P<0.01)。与空白血清组比较,D-gal刺激后的模型组LMSCs细胞SA-β-gal染色增强,NAD+含量增多(P<0.01),细胞培养上清中衰老因子p16、p53及SASP促炎因子IL-6、TNF-α的表达量均显著提高(P<0.01),衰老相关蛋白p16、p21、p53表达上升(P<0.01),NAMPT、SIRT1、过氧化物酶体增殖活化受体γ辅助活化因子1α(PGC-1α)、叉头框蛋白O1(FoxO1)蛋白表达下降(P<0.01)。与模型组比较,温肺化纤汤含药血清各剂量组LMSCs细胞SA-β-gal染色明显减轻,NAD+含量减少(P<0.01);细胞培养上清中衰老因子(p16、p53)和炎性因子(IL-6、TNF-α)均显著下降(P<0.01);衰老相关蛋白(p16、p21、p53)表达明显减少(P<0.05,P<0.01);NAMPT、SIRT1、PGC-1α、FoxO1的蛋白表达明显上调(P<0.05,P<0Objective::The lung mesenchymal stem cells(LMSCs)induced by D-galactose(D-gal)were intervened by Wenfei Huaxian decoction-containing serum to explore the mechanism of Wenfei Huaxian decoction in delaying the senescence of LMSCs through the nicotinamide phosphoribosyltransferase/silent information regulator 1(NAMPT/SIRT1)signaling pathway.M ethod::Wenfei Huaxian decoction-containing serum was prepared.LMSCs were isolated by gradient density centrifugation,and they were cultured and identified in vitro.The senescence model in vitro was established by stimulating cells via D-gal for 24 h.LMSCs cells were modeled after being treated with different volume fractions(5%,10%,20%,40%,and 80%)of Wenfei Huaxian decoction-containing serum for 24 h,and the cell proliferation level was detected by methyl thiazolyl tetrazolium(MTT)method.The cells were randomly divided into blank serum group,model group,and high,medium,and low dose groups of Wenfei Huaxian decoction-containing serum.Senescence-associatedβ-galactosidase(SA-β-gal)staining was used to detect the senescence of LMSCs in each group.The content of NAD+was detected by colorimetry.The levels of senescence-associated factors(p16 and p53),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to detect the relative expression of senescence-associated proteins and NAMPT/SIRT1 signaling pathway-related proteins.Result::Compared with the blank serum group,the proliferation of LMSCs was significantly inhibited after D-gal stimulation for 24 h(P<0.01).Compared with the model group,the proliferation of LMSCs could be promoted after intervention with the corresponding Wenfei Huaxian decoction-containing serum(P<0.05,P<0.01).Compared with the blank serum group,the SA-β-gal staining of LMSCs in the model group after D-gal stimulation was enhanced,and the content of NAD+was increased(P<0.01).The expression levels of senescence factors p16 and p53,as well as SASP pro-infla

关 键 词：温肺化纤汤 肺间充质干细胞 衰老 烟酰胺磷酸核糖转移酶/沉默信息调节因子1(NAMPT/SIRT1)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33