Wang Ziyu, Han Letian, Chen Haoyu, Zhang Shengquan, Zhang Sumei, Zhang Hua, Li Yuhao, Tao Hui, Li Jie
Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Department of Orthopedics and Spine Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Front Bioeng Biotechnol. 2022 Jan 28;10:816362. doi: 10.3389/fbioe.2022.816362. eCollection 2022.
Sa12b is a wasp peptide that can inhibit acid-sensitive ion channels (ASICs). The biological effects of nucleus pulposus mesenchymal stem cells (NP-MSCs) have not been investigated. Therefore, this study investigated the effect of Sa12b on the biological activity of NP-MSCs through ASICs in the acidic environment of intervertebral disc degeneration (IVDD). In this study, NP-MSCs were isolated from the nucleus pulposus (NP) in patients who underwent lumbar disc herniation surgery, identified by flow cytometry and tertiary differentiation, and cultured in an acidic environment model of IVDD with a pH of 6.2. Proliferation, and apoptosis were observed after different Sa12b concentrations were added to P2 generation NP-MSCs. The Ca influx was detected using flow cytometry and laser confocal scanning microscopy, and qPCR was used to detect the relative expression of stem cell-associated genes (Oct4, Nanog, Jag1, and Notch1), the relative expression of extracellular matrix (ECM)-associated genes (collagen II, aggrecan, and SOX-9), and the relative expression of genes encoding ASICs (ASIC1, ASIC2, ASIC3, and ASIC4). Western blotting was used to detect the protein expression of collagen II and aggrecan in different treatment groups. Cells isolated and cultured from normal NP were spindle-shaped and adherent, and they exhibited expansion . Flow cytometry results showed that the cells exhibited high expression of CD73 (98.1%), CD90 (97.5%), and CD105 (98.3%) and low expression of HLA-DR (0.93%), CD34 (2.63%), and CD45 (0.33%). The cells differentiated into osteoblasts, adipocytes, and chondrocytes. According to the International Society for Cellular Therapy criteria, the isolated and cultured cells were NP-MSCs. With an increase in Sa12b concentration, the cell proliferation rate of NP-MSCs increased, and the apoptosis rate decreased significantly, reaching the optimal level when the concentration of Sa12b was 8 μg/μl. When the Sa12b concentration was 8 μg/μl and contained the ASIC non-specific inhibitor amiloride, the Ca influx was the lowest, followed by that when the Sa12b concentration was 8 μg/μl. The Ca influx was the highest in the untreated control group. qPCR results showed that as the concentration of Sa12b increased, the relative expression of Oct4, Nanog, Jag1, Notch1, collagen II, aggrecan, and SOX-9 increased, while that of ASIC1, ASIC2, ASIC3, and ASIC4 decreased. The difference was statistically significant ( < 0.05). In conclusion, Sa12b can improve the biological activity of NP-MSCs in severely acidic environments of the intervertebral disc by reducing Ca influx AISC inhibition and, probably, the Notch signaling pathway. This study provides a new approach for the biological treatment of IVDD. Inhibition of AISCs by Sa12b may delay IVDD and improve low back pain.
Sa12b是一种能够抑制酸敏感离子通道（ASICs）的黄蜂肽。尚未对髓核间充质干细胞（NP-MSCs）的生物学效应进行研究。因此，本研究通过ASICs在椎间盘退变（IVDD）的酸性环境中研究了Sa12b对NP-MSCs生物学活性的影响。在本研究中，从接受腰椎间盘突出症手术患者的髓核（NP）中分离出NP-MSCs，通过流式细胞术和三级分化进行鉴定，并在pH为6.2的IVDD酸性环境模型中培养。将不同浓度的Sa12b添加到第2代NP-MSCs后，观察细胞增殖和凋亡情况。使用流式细胞术和激光共聚焦扫描显微镜检测钙内流，并用qPCR检测干细胞相关基因（Oct4、Nanog、Jag1和Notch1）的相对表达、细胞外基质（ECM）相关基因（胶原蛋白II、聚集蛋白聚糖和SOX-9）的相对表达以及编码ASICs的基因（ASIC1、ASIC2、ASIC3和ASIC4）的相对表达。使用蛋白质印迹法检测不同治疗组中胶原蛋白II和聚集蛋白聚糖的蛋白表达。从正常NP中分离并培养的细胞呈纺锤形且贴壁生长，并表现出增殖能力。流式细胞术结果显示，细胞高表达CD73（98.1%）、CD90（97.5%）和CD105（98.3%），低表达HLA-DR（0.93%）、CD34（2.63%）和CD45（0.33%）。这些细胞可分化为成骨细胞、脂肪细胞和软骨细胞。根据国际细胞治疗协会的标准，分离并培养的细胞为NP-MSCs。随着Sa12b浓度的增加，NP-MSCs的细胞增殖率升高，凋亡率显著降低，当Sa12b浓度为8μg/μl时达到最佳水平。当Sa12b浓度为8μg/μl并含有ASIC非特异性抑制剂阿米洛利时，钙内流最低，其次是Sa12b浓度为8μg/μl时的情况。未处理的对照组中钙内流最高。qPCR结果显示，随着Sa12b浓度的增加，Oct4、Nanog、Jag1、Notch1、胶原蛋白II、聚集蛋白聚糖和SOX-9的相对表达增加，而ASIC1、ASIC2、ASIC3和ASIC4的相对表达降低。差异具有统计学意义（<0.05）。总之，Sa12b可通过减少钙内流、抑制AISC以及可能通过Notch信号通路来改善椎间盘严重酸性环境中NP-MSCs的生物学活性。本研究为IVDD的生物治疗提供了一种新方法。Sa12b对AISCs的抑制作用可能会延缓IVDD并改善腰痛。
