基于TLR4/NF-κB通路研究桃叶珊瑚苷联合ADSCs-exos对TBHP诱导的髓核细胞保护作用  

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway

作　　者：杨雷 李兆勇 马露[2] 郭彦涛[1] 杨少锋[1] 熊辉[2] 伍搏宇 段嘉豪 刘恩旭 张超[1] 聂颖[1] 陈龙 刘林券 YANG Lei;LI Zhao-yong;MA Lu;GUO Yan-tao;YANG Shao-feng;XIONG Hui;WU Bo-yu;DUAN Jia-hao;LIU En-xu;ZHANG Chao;NIE Ying;CHEN Long;LIU Lin-quan(the First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]湖南中医药大学,湖南长沙410208

出　　处：《中国中药杂志》2023年第19期5294-5303,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(8217151084);湖南省自然科学基金项目(2022JJ70029);长沙市自然科学基金项目(kq2202460);湖南省卫健委课题(202204074858);湖南省中医药管理局科研项目(D2022094);湖南省研究生科研创新课题(CX20220785)。

摘　　要：该文研究桃叶珊瑚苷(aucubin)协同脂肪间充干细胞外泌体(adipose-derived stem cells-exosomes,ADSCs-exos)对叔丁基过氧化氢(tert-butyl hydroperoxide,TBHP)诱导的人源髓核细胞(nucleus pulposus cells,NPCs)炎性损伤、衰老及凋亡的保护作用及机制。将TBHP诱导NPCs分为正常组、模型组、aucubin组、ADSCs-exos组及aucubin+ADSCs-exos组。细胞计数试剂-8(cell counting kit-8,CCK-8)检测细胞活力,EdU染色法检测细胞增殖,细胞染色-β-半乳糖苷酶活性染色方法(senescence-associated-β-galactosidase,SA-β-Gal)检测细胞衰老,流式细胞术检测细胞周期与凋亡,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-10(interleukin-10,IL-10)炎症因子的表达,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和蛋白免疫印记(Western blot)法检测聚集蛋白聚糖(aggrecan)、Ⅱ型胶原α1(collagen typeⅡalpha 1,COL2A1)、Toll样受体4(Toll-like receptor 4,TLR4)及核因子-κB(nuclear factor kappa-B,NF-κB)的mRNA和蛋白表达情况。结果显示,与模型组比较,aucubin或ADSCs-exos组NPCs活性和增殖上升,G_(0)/G_(1)期细胞比例显著降低,S期细胞比例显著增加,凋亡减少,细胞衰老比例降低,IL-1β和TNF-α水平下降,TLR4和NF-κB mRNA及蛋白表达显著减少,IL-10水平上升,aggrecan和COL2A1 mRNA及蛋白表达均升高;与aucubin或ADSCs-exos单独处理组比较,aucubin+ADSCs-exos组NPCs活性和增殖进一步上升,G_(0)/G_(1)期细胞比例进一步降低,S期细胞比例进一步增加,凋亡水平和细胞衰老比例都进一步降低,IL-1β和TNF-α水平,TLR4及NF-κB mRNA和蛋白表达水平均进一步下降,而IL-10和aggrecan、COL2A1 mRNA及蛋白水平均进一步上升。综上所述,aucubin和ADSCs-exos均能通过抑制炎症反应,减少NPCs凋亡和衰老,促进细胞活性、增殖以及细胞外基质(ECM)的合成而发�This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury,senescence,and apoptosis.The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal,model,aucubin,ADSCs-exos,and aucubin+ADSCs-exos groups.The cell viability was examined by cell counting kit-8(CCK-8),cell proliferation by EdU staining,cell senescence by senescence-associated-β-galactosidase(SA-β-Gal),and cell cycle and apoptosis by flow cytometry.Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β),IL-10,and tumor necrosis factor-α(TNF-α).Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan),type Ⅱ collagen alpha 1(COL2A1),Toll-like receptor 4(TLR4),and nuclear factor-kappa B(NF-κB).The results showed that compared with the model group,the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs,decreased proportion of G_0/G_(1) phase cells,increased proportion of S phase cells,reduced apoptosis and proportion of cells in senescence,lowered IL-1β and TNF-α levels,elevated IL-10 level,down-regulated mRNA and protein levels of TLR4 and NF-κB,and up-regulated mRNA and protein levels of aggrecan and COL2A1.Compared with the aucubin or ADSCs-exos group,the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs,decreased the proportion of G_0/G_(1) phase cells,increased the proportion of S phase cells,reduced the apoptosis and proportion of cells in senescence,lowered the IL-1β and TNF-α levels,elevated the IL-10 level,down-regulated the mRNA and protein levels of TLR4 and NF-κB,and up-regulated the mRNA and protein levels of aggrecan and COL2A1.In summary,both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses,reducing apoptosis and senescence o

关 键 词：脂肪间充干细胞外泌体 髓核细胞 桃叶珊瑚苷 TLR4/NF-κB信号通路 

分 类 号：R285[医药卫生—中药学]