补肾活血方调节ADSCs-Exos对椎间盘退变髓核细胞凋亡及ERK信号通路的影响  

Bushen Huoxue Decoction regulates ADSCs-Exos to affect nucleus pulposus cell apoptosis and ERK signaling pathway in intervertebral disc degeneration

作　　者：李兆勇 李玲慧[2] 杨雷 杨少锋[1] 郭彦涛[1] 陈龙 段嘉豪 谢求恩[1] 刘恩旭 孙钰 孙飞 LI Zhao-yong;LI Ling-hui;YANG Lei;YANG Shao-feng;GUO Yan-tao;CHEN Long;DUAN Jia-hao;XIE Qiu-en;LIU En-xu;SUN Yu;SUN Fei(the First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Wangjing Hospital,China Academy of Chinese Medical Sciences,Bejing 100102,China;Xiangtan Hospital of Chinese Medicine,Xiangtan 411199,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]中国中医科学院望京医院,北京100102 [3]湘潭市中医医院,湖南湘潭411199

出　　处：《中国中药杂志》2024年第21期5704-5712,共9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(2024JJ9420,2023JJ40504,2022JJ70029);湖南省教育厅科研项目(23B0351,21C0233);长沙市自然科学基金项目(kq2202460,kq2208203);湖南省中医药科研计划项目(B2023029);湖南省卫生健康委卫生科研课题(W20243273,20231630,202204074858);湖南中医药大学校级科研项目(2022XYLH007)。

摘　　要：研究补肾活血方调节脂肪间充质干细胞(adipose-derived stem cells,ADSCs)-外泌体(exosome,Exos)对椎间盘髓核细胞凋亡及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号通路的影响。将叔丁基过氧化氢(tert-butyl hydrogen peroxide,TBHP)诱导髓核细胞(nucleus pulposus cells,NPCs)分为正常组、模型组、含药血清组、空白外泌体组、正常血清外泌体组及含药血清外泌体组。使用CCK-8检测细胞活力,EdU染色法检测细胞增殖,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-6(interleukin-6,IL-6)等炎症因子的表达,qRT-PCR法检测蛋白聚糖(aggrecan)、Ⅱ型胶原基因(collagen typeⅡalpha 1 chain,COL2A1)和ERK mRNA表达,Western blot法检测aggrecan、COL2A1、p-ERK蛋白表达。结果显示,与模型组比较,单独给予含药血清、空白外泌体及正常血清外泌体处理后,3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,IL-1β、TNF-α和IL-6水平下降,aggrecan、COL2A1 mRNA及蛋白表达水平上升,ERK mRNA及p-ERK蛋白表达水平下降;与含药血清组、空白外泌体组及正常血清外泌体组比较,含药血清外泌体处理后,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,凋亡进一步减少,IL-1β、TNF-α和IL-6水平进一步下降,aggrecan、COL2A1 mRNA及蛋白水平显著上升,ERK mRNA及p-ERK蛋白表达水平显著下降。结论证实,补肾活血方含药血清处理后ADSCs分泌的Exos能够促进退变NPCs的增殖,抑制凋亡和炎症因子的表达,促进蛋白多糖、胶原生成,进而延缓椎间盘退变,其机制与调节ERK信号通路有关。This study aims to investigate the effects of Bushen Huoxue Decoction regulating adipose-derived stem cells(ADSCs)-exosomes(Exos)on the apoptosis of intervertebral disc nucleus pulposus cells(NPCs)and extracellular signal-regulated kinase(ERK)signaling pathway.Tert-butyl hydrogen peroxide(TBHP)-induced NPCs were divided into control,model,drug-containing serum,blank Exos,normal serum Exos,and drug-containing serum Exos groups.Cell viability and proliferation were examined by the CCK-8 assay and EdU staining,respectively.The cell cycle and apoptosis were evaluated by flow cytometry.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin(IL)-1β,tumor necrosis factor(TNF)-α,and IL-6.The mRNA levels of aggrecan,collagen typeⅡalpha 1 chain(COL2A1),and ERK were determined by qRT-PCR,and the protein levels of aggrecan,COL2A1,and p-ERK were determined by Western blot.The results showed that compared with the model group,the treatments with drug-containing serum,blank Exos,and normal serum Exos enhanced the viability and proliferation of NPCs,decreased the proportion of cells in the G_(0)/G_(1)phase,increased the proportion of cells in the S phase,reduced apoptosis,lowered the levels of IL-1β,TNF-α,and IL-6,up-regulated the mRNA and protein levels of aggrecan and COL2A1,and down-regulated the mRNA level of ERK and the protein level of p-ERK.Compared with the drug-containing serum,blank Exos,and normal serum Exos groups,the treatment with drug-containing serum Exos enhanced the viability and proliferation of NPCs,decreased the proportion of cells in the G_(0)/G_(1)phase,increased the cells in the S phase,reduced apoptosis,lowered the levels of IL-1β,TNF-α,and IL-6,up-regulated the mRNA and protein levels of aggrecan and COL2A1,and down-regulated the mRNA level of ERK and the protein level of p-ERK.The results confirmed that the Exos secreted by ADSCs after treatment with Bushen Huoxue Decoction-containing serum promoted the proliferation of degenerated NPCs,inhibited apoptosis and the ex

关 键 词：椎间盘退变 补肾活血方 ERK信号通路 外泌体 脂肪间充质干细胞 

分 类 号：R285[医药卫生—中药学]