补肾活血汤调节rabHK-2外泌体对椎间盘髓核细胞凋亡及Wnt信号通路的影响  

Effect of Bushen Huoxue(补肾活血)Decoction on Apoptosis and Wnt Signaling Pathway of Nucleus Pulposus Cells in Intervertebral Disc by Regulating Exosomes of rabHK-2 Cells

作　　者：冯帅华[1] 吴官保[1] 杨届 万文渊 孙湘云 谢玉城 廖建晖[1] FENG Shuaihua;WU Guanbao;YANG Jie;WAN Wenyuan;SUN Xiangyun;XIE Yucheng;LIAO Jianhui(Department of Spinal Orthopaedic Oncology,Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha 410006;2Hunan University of Chinese Medicine,Changsha 410208)

机构地区：[1]湖南省中医药研究院附属医院脊柱骨肿瘤科,长沙410006 [2]湖南中医药大学,长沙410208

出　　处：《中药药理与临床》2023年第5期2-8,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：湖南省中医药科研计划重点项目(编号:201902);湖南省中医药研究院联合基金重点课题(编号:202016);湖南省自然科学基金面上项目(编号:2022JJ30025)。

摘　　要：目的:观察补肾活血汤对兔退变椎间盘组织细胞凋亡及Wnt信号通路的影响,以及调节兔肾小管上皮细胞(rabHK-2)来源外泌体的分泌,对退变椎间盘髓核细胞(NPCs)增殖、凋亡及Wnt信号通路蛋白表达的影响。方法:建立兔椎间盘退变模型,补肾活血汤干预8 w后,采用TUNEL法检测椎间盘组织凋亡阳性细胞率,Western-blot法检测椎间盘组织中Wnt信号通路相关蛋白的表达;5.41 g/kg补肾活血汤灌胃新西兰白兔制备含药血清,以10%补肾活血汤含药血清作用于rabHK-2细胞,采用超高速离心法提取rabHK-2细胞外泌体,透射电镜观察外泌体形态,Western-blot法检测外泌体标志蛋白集群分化因子9(CD9)和集群分化因子63(CD63)的表达;用PKH67荧光试剂盒标记后的外泌体与退变NPCs共孵育,荧光显微镜观察不同浓度外泌体作用于退变NPCs后,退变NPCs对rabHK-2细胞外泌体的摄取情况,CCK-8法检测退变NPCs活力。rabHK-2外泌体与补肾活血汤含药血清单独或联合干预后,应用流式细胞术检测退变NPCs凋亡情况,RT-PCR检测退变NPCs中II型胶原和蛋白多糖的mRNA表达,Western-blot和RT-RCR检测退变NPCs中Wnt信号通路相关蛋白和mRNA的表达情况。结果:补肾活血汤干预后,明显降低了兔退变椎间盘组织细胞凋亡率,并上调了Wnt通路中GSK-3β的蛋白表达,下调了β-catenin的表达(P<0.05);经补肾活血汤含药血清干预后,rabHK-2外泌体CD9、CD63的表达明显上调(P<0.05);不同浓度rabHK-2外泌体干预后,荧光显微镜可见外泌体被退变NPCs所摄取,退变NPCs细胞活力增强(P<0.05);外泌体与补肾活血汤含药血清联合干预后,退变NPCs凋亡率受到明显抑制,细胞中II型胶原和蛋白多糖的mRNA表达明显上调,同时显著上调了GSK-3β的蛋白和mRNA表达,下调了β-catenin的表达,联合补肾活血汤含药血清处理后效果较单独应用外泌体更为明显(P<0.05)。结论:补肾活血汤可能通过抑制Wnt信号通路的活Objective:To observe the effect of Bushen Huoxue(补肾活血)Decoction(BHD)on cell apoptosis and Wnt signaling pathway in degenerative intervertebral discs of rabbits,and the effect on the proliferation,apoptosis,and Wnt signaling pathway-related protein expression of nucleus pulposus cells(NPCs)in intervertebral disc by regulating the secretion of exosomes from rabHK-2 cells.Methods:Intervertebral disc degeneration was induced in rabbit.After the intervention with BHD for 8 weeks,apoptosis of intervertebral disc tissue was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay,and the expression of Wnt signaling pathway-related proteins by Western blotting.The 5.41 g/kg BHD was given(ig)to New Zealand white rabbits to collect the BHD-containing serum.The rabHK-2 cells were treated with 10%BHD-containing serum,and the exosomes of the cells were extracted by ultra-high-speed centrifugation.The morphology of exosomes was observed under the transmission electron microscope.The expression of exosome marker proteins cluster of differentiation 9(CD9)and cluster of differentiation 63(CD63)was detected by Western blotting.The PKH67-labeled exosomes were incubated with NPCs of the degenerative disc.The uptake of rabHK-2 exosomes by the NPCs was observed under fluorescence microscope and the viability of the NPCs was detected by Cell Counting Kit-8(CCK-8)assay.After the treatment with different concentration of exosomes or combination of exosomes and BHD-containing serum separately,the apoptosis of the NPCs was detected by flow cytometry and the mRNA expression of type II collagen and proteoglycan by RT-RCR.Then,Western blotting and RT-RCR were employed to detect the expression of Wnt signaling pathwayrelated proteins and mRNA in the NPCs.Result:BHD significantly reduced the apoptosis rate of rabbit degenerative intervertebral disc,upregulated the protein expression of glycogen synthase kinase-3β(GSK-3β)in Wnt pathway,and down-regulated the expression ofβ-catenin(P<0.05).After the interven

关 键 词：补肾活血汤 外泌体 椎间盘退变 髓核细胞 WNT信号通路 

分 类 号：R285.5[医药卫生—中药学]