摘要
目的探讨补骨脂素对白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)诱导的人髓核细胞(HNPCs)退变的影响及机制。方法体外培养HNPCs,用10 ng/ml IL-1β和50 ng/ml TNF-α诱导HNPCs 24 h使其发生退变,设置对照组、IL-1β+TNF-α组、补骨脂素(10 μmol/L、25 μmol/L和50 μmol/L)组、补骨脂素(25 μmol/L)+S期激酶相关蛋白2(SKP2)干扰或过表达组、补骨脂素+甲状旁腺激素相关蛋白(PTHrP)重组蛋白(100 nmol/L)组、补骨脂素+PTHrP重组蛋白(100 nmol/L)+沉默信号调节子1(SIRT1)激活剂SRT1720(1 μmol/L)组。采用流式细胞术检测髓核细胞凋亡情况;ELISA法检测髓核细胞中IL-6、基质金属蛋白酶3(MMP-3)和MMP-13的表达水平;DCFDA探针法检测髓核细胞中活性氧(ROS)的水平;Western blotting检测髓核细胞中SKP2、PTHrP、SIRT1、细胞外基质(ECM)相关蛋白[二型胶原蛋白(COL Ⅱ)和蛋白聚糖蛋白]的表达水平;蛋白质免疫共沉淀(Co-IP)实验检测SKP2与PTHrP蛋白的相互作用;泛素化实验检测补骨脂素对SKP2介导的PTHrP泛素化水平的影响。结果补骨脂素以剂量依赖的方式抑制炎症环境下髓核细胞凋亡以及IL-6、MMP-3、MMP-13和ROS的生成(P<0.05或P<0.01),促进SKP2和ECM相关蛋白的表达(P<0.05)。与补骨脂素+杂乱干扰组比较,补骨脂素+SKP2干扰组SKP2和ECM相关蛋白的表达水平明显降低(P<0.05),髓核细胞凋亡率、IL-6、MMP-3、MMP-13和ROS水平明显增高(P<0.05或P<0.01)。Co-IP实验结果显示,SKP2与PTHrP蛋白存在相互作用。泛素化实验结果显示,与补骨脂素+空载体组比较,补骨脂素+SKP2过表达组PTHrP蛋白表达水平明显降低,SKP2蛋白表达水平明显升高(P<0.05)。与补骨脂素组比较,补骨脂素+PTHrP重组蛋白组SIRT1和ECM相关蛋白表达水平明显降低(P<0.05),髓核细胞凋亡率、IL-6、MMP-3、MMP-13和ROS水平明显升高(P<0.05);与补骨脂素+PTHrP重组蛋白组比较,补骨脂素+PTHrP重组蛋白+SRT1720组PTHrP蛋白表达水平�
Objective To investigate the effect of psoralen on the degeneration of human nucleus pulposus cells(HNPCs)induced by interleukin-1β(IL-1β)and tumor necrosis factorα(TNF-α)and its mechanism.Methods HNPCs were cultured in vitro,and HNPCs were stimulated by 10 ng/ml IL-1βand 50 ng/ml TNF-αfor 24 hours to induce degeneration.Cells were divided into control group,IL-1β+TNF-αgroup,psoralen(10μmol/L,25μmol/L and 50μmol/L)group,psoralen(25μmol/L)+S-phase kinase-associated protein 2(SKP2)interference or over-expression group,psoralen+parathyroid hormone-related protein(PTHrP)recombinant protein(100 nmol/L)group,psoralen+PTHrP recombinant protein(100 nmol/L)+silent information regulator 1(SIRT1)activator SRT1720(1μmol/L)group.The apoptosis of nucleus pulposus cells was detected by flow cytometry;the levels of IL-6,MMP-3 and MMP-13 in nucleus pulposus cells were detected by ELISA;the level of reactive oxygen species(ROS) in nucleus pulposus cells was detected by the DCFDA probe method. Western blotting was used to detect the expression levels of SKP2, PTHrP, SIRT1 and extracellular matrix (ECM) related protein [type II collagen (COL II) and proteoglycan protein]. Co- immunoprecipitation (Co-IP) experiment was used to verify the binding of SKP2 and PTHrP, ubiquitination experiment was used to analyze the effect of psoralen on SKP2-mediated PTHrP ubiquitination. Results Psoralen significantly inhibited cell apoptosis, the production of IL-6, MMP-3, MMP-13 and ROS (P<0.05 or P<0.01), and promoted the expression of SKP2 and ECM-related proteins of HNPCs after IL-1β and TNF-α induction in a dose-dependent manner (P<0.05). Compared with psoralen + scramble group, the expression levels of SKP2 and ECM-related proteins were significantly reduced (P<0.05), and the apoptosis rate, the levels of IL-6, MMP-3, MMP-13 and ROS were significantly increased in psoralen + SKP2 interference group (P<0.05 or P<0.01). The results of the Co-IP experiment showed that SKP2 is directly bound to PTHrP. The results of the ubiquitinatio
作者
岳宗进
刘汝银
于露
王新立
冯仲锴
王西彬
Yue Zong-Jin;Liu Ru-Yin;Yu Lu;Wang Xin-Li;Feng Zhong-Kai;Wang Xi-Bin(Department of Spine,Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450000,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2021年第7期657-665,共9页
Medical Journal of Chinese People's Liberation Army
基金
河南省中医药科学研究专项课题(2016ZY2001)。
关键词
补骨脂素
S期激酶相关蛋白2
泛素化
人髓核细胞
退变
psoralen
S-phase kinase-associated protein 2
ubiquitination
human nucleus pulposus cells
degeneration
