Abstract：
This study investigated the role of apoptosis signal-regulated kinase-1 (ASK1) in intervertebral disc degeneration (IDD). The nucleus pulposus (NP) tissues of non-IDD and IDD patients were subjected to hematoxylin and eosin, Safranin O-fast green, and immunohistochemical staining. Quantitative real-time PCR was used to assess the ASK1 mRNA level within NP tissue samples and cells. The Cell Counting Kit-8 assay, senescence-associated β-galactosidase staining, and then flow cytometry were conducted, respectively, to assess the viability, senescence, and apoptosis of NP cells. The extracellular matrix-related factors were detected using Western blot analysis. Furthermore, the effect of ASK1 on the IDD rat model was evaluated through nuclear magnetic resonance imaging analysis, hematoxylin and eosin, Safranin O-fast green staining, and immunohistochemical staining. Finally, c-Jun N-terminal kinase (JNK) inhibitors were used to verify the effect of the JNK/p38 signaling on IDD. ASK1 mRNA and protein were up-regulated within NP tissue samples from the IDD group, IL-1β-stimulated NP cells, and IDD rats. ASK1 inhibition promoted cell viability and repressed the senescence and apoptosis of NP cells; promoted collagen II and aggrecan; inhibited matrix metalloproteinase 3, matrix metalloproteinase 9, a disintegrin and metalloproteinase with thrombospondin motifs 4, and a disintegrin and metalloproteinase with thrombospondin motifs 5 protein levels; and increased NP cells in rat intervertebral disc tissues. ASK1 overexpression exerted the opposite effects of ASK1 inhibition on NP cells. Additionally, JNK/p38 signaling suppression could reverse the ASK1 up-regulation-induced dysfunction. In conclusion, ASK1 facilitated the senescence and apoptosis of NP cells in promoting IDD progression, which may be mediated by the JNK/p38 pathway.
摘要：
本研究探讨了凋亡信号调节激酶1（ASK1）在椎间盘退变（IDD）中的作用。非IDD和IDD患者的髓核（NP）组织进行H＆E，Safranin-O-fast绿色，和IHC染色。qRT-PCR用于评估NP组织样品和细胞内的ASKlmRNA水平。CCK-8测定，SA-β-gal染色,然后进行流式细胞术，分别,为了评估生存能力，衰老，和NP细胞凋亡。使用Westernblot分析检测细胞外基质(ECM)相关因子。此外，通过核磁共振成像（MRI）分析评估ASK1对IDD大鼠模型的影响，他,Safranin-O-fast绿色染色，和IHC染色。最后,使用JNK抑制剂来验证JNK/p38信号传导对IDD的作用。来自IDD组的NP组织样本中ASK1mRNA和蛋白上调,IL-1β刺激的NP细胞,IDD大鼠ASK1抑制促进细胞活力并抑制NP细胞的衰老和凋亡；促进胶原蛋白II和Aggrecan；抑制MMP3，MMP9，ADAMTS4和ADAMTS5蛋白水平；并增加大鼠IVD组织中的NP细胞。ASK1过表达对NP细胞发挥ASK1抑制的相反作用。此外,JNK/p38信号传导抑制可以逆转ASK1上调诱导的功能障碍。总之,ASK1在促进IDD进展中促进NP细胞衰老和凋亡，这可能是由JNK/p38途径介导的。
