Abstract：
Intervertebral disc (IVD) degeneration (IDD) is a primary cause of low-back pain in people, which is associated with nucleus pulposus-derived mesenchymal stem cells (NPMSCs). In this study, the involvement of lipopolysaccharide (LPS) in the pyroptosis of NPMSCs was investigated. The effect of RADKPS on the pyroptosis of NPMSCs and the underlying mechanism behind the impact of RADKPS on the proliferative capacity of NPMSCs were also studied. Pyroptosis of NPMSCs was induced with 10 μg/mL LPS and its effects on the downstream signaling pathways were explored. The protective effect of RADKPS on NPMSCs under the action of LPS and its possible mechanism were explored, using different techniques such as immunohistochemical analysis, cell proliferation assay, quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis. Accordingly, caspase1/p20/p10, a protein associated with pyroptosis, was found to be overexpressed in LPS-challenged NPMSCs, Furthermore, the qPCR results demonstrated that LPS promoted the expression of pyroptosis-related gene IL-1β (p < 0.0001), while downregulating the expression of Sox-9 (p < 0.001), which was a gene associated with the extracellular matrix. The immunohistochemical results identified lowered extracellular signal-regulated kinase 1/2 (ERK1/2) expression and phosphorylated (p-)ERK1/2 in the degenerated IVD tissues. In this study, the influence of RADKPS on the proliferative ability of NPMSCs was evaluated using two-dimensional (2D) and three-dimensional (3D) cultures. It was noted that RADKPS promoted the proliferation of NPMSCs in 2D and 3D cultures. The findings of the Western blot experiments revealed that RADKPS inhibited the expression of pyroptosis-related proteins, while it upregulated the p-ERK1/2 (p < 0.001), RhoA (p < 0.01), collagen II (p < 0.01), and Sox-9 (p < 0.01), whereas ERK inhibitor PD98059 and RhoA signaling pathway inhibitor CCG-1423 inhibited their expression. These findings reveal to us that RADKPS hydrogel may protect NPMSCs from pyroptosis. It was also noted that cell proliferation-related signaling pathways may promote the proliferation of NPMSCs. The results revealed that RADKPS hydrogel could be used as a potential therapeutic approach for IDD. Impact Statement RADKPS inhibits the pyroptosis of NPMSCs and promotes the production of extracellular matrix, which has the potential of intervertebral disc biotherapy.
摘要：
椎间盘(IVD)退变(IDD)是导致下腰痛(LBP)的主要原因,与髓核来源的间充质干细胞（NPMSCs）相关。在这项研究中,研究了脂多糖（LPS）参与NPMSCs的焦亡，还研究了RADKPS对NPMSCs焦凋亡的影响以及RADKPS对NPMSCs增殖能力的影响背后的潜在机制。用10μg/mlLPS诱导NPMSCs的焦亡，并探讨其对下游信号通路的影响。探讨了LPS作用下RADKPS对NPMSCs的保护作用及其可能机制。使用不同的技术，如免疫组织化学分析，细胞增殖试验，qPCR,和蛋白质印迹分析。因此，Caspase1/p20/p10，一种与焦凋亡相关的蛋白质，发现在LPS攻击的NPMSCs中过表达，此外，qPCR结果表明,LPS促进了细胞凋亡相关基因IL-1β的表达(P<0.0001),同时下调sox-9的表达（p<0.001），这是一个与细胞外基质（ECM）相关的基因。免疫组织化学结果鉴定在变性IVD组织中降低ERK1/2表达和磷酸化(p-)ERK1/2。在这项研究中,使用二维和三维培养评估RADKPS对NPMSCs增殖能力的影响。注意到RADKPS促进NPMSCs在二维和三维培养中的增殖。westernblot实验结果表明，RADKPS抑制了细胞凋亡相关蛋白的表达，虽然它上调了p-ERK1/2(p<0.001)，RhoA（p<0.01），胶原蛋白II（p<0.01），和sox-9（p<0.01），而ERK抑制剂PD98059和RhoA信号通路抑制剂CCG-1423抑制其表达。这些发现向我们揭示了RADKPS水凝胶可以保护NPMSCs免于焦亡。还注意到细胞增殖相关的信号通路可能促进NPMSCs的增殖。结果表明，RADKPS水凝胶可用作IDD的潜在治疗方法。
