Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao, China 266003.
Cancer Institute, The Qingdao University, Qingdao, China 266003.
Oxid Med Cell Longev. 2020 Dec 7;2020:6697577. doi: 10.1155/2020/6697577. eCollection 2020.
This study is aimed at determining the effects of human urine-derived stem cell-derived exosomes (USCs-exos) on pressure-induced nucleus pulposus cell (NPC) apoptosis and intervertebral disc degeneration (IDD) and on the ERK and AKT signaling pathways.
The NPCs were obtained from patients with herniated lumbar discs. Western blot analysis (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine endoplasmic reticulum (ER) stress levels of NPCs under stress. Human USCs were identified using an inverted microscope, three-line differentiation experiments, and flow cytometry. A transmission microscope, nanoparticle size analysis, and WB procedures were used to identify the extracted exosomes and observe NPC uptake. A control group, a 48 h group, and a USCs-exos group were established. The control group was untreated, and the 48 h group was pressure-trained for 48 h, while the USCs-exos group was pressure-trained for 48 h and treated with USCs-exos. WB, qRT-PCR, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis were used to determine the ER stress levels in stress conditions and after exosomal treatment. The AKT and ERK pathways were partially detected. Magnetic Resonance Imaging (MRI) and computed tomography (CT) were used to evaluate cell degeneration while exosomal effects on the intervertebral disc (IVD) tissue were determined by hematoxylin and eosin (HE) staining, Safranin O-fast green staining, immunohistochemical staining (IHC), nuclear magnetic resonance (NMR), spectrometric detection, and total correlation spectroscopy (TOCSY). IVD metabolites were also identified and quantified.
After pressure culture, ER stress markers (GRP78 and C/EBP homologous protein (CHOP)) in the NPCs were significantly elevated with time ( < 0.05). Human USCs are short and spindle-shaped. They can successfully undergo osteogenic, adipogenic, and chondrogenic differentiation. In this study, these stem cells were found to be positive for CD29, CD44, and CD73. The exosomes were centrally located with a diameter of 50-100 nm. CD63 and Tsg101 were highly expressed while the expression of Calnexin was suppressed. The exosomes can be ingested by NPCs. USCs-exos significantly improved ER stress responses and inhibited excessive activation of the unfolded protein response (UPR) as well as cell apoptosis and disc degeneration through the AKT and ERK signaling pathways ( < 0.05).
Through the AKT and ERK signaling pathways, USCs-exos significantly inhibit ER stress-induced cell apoptosis and IDD under pressure conditions. It is, therefore, a viable therapeutic strategy.
本研究旨在探讨人尿源干细胞衍生的外泌体（USCs-exos）对压力诱导的髓核细胞（NPC）凋亡和椎间盘退变（IDD）的影响，以及对细胞外信号调节激酶（ERK）和蛋白激酶 B（AKT）信号通路的影响。
从腰椎间盘突出症患者中获取 NPC。采用 Western blot 分析（WB）和实时定量聚合酶链反应（qRT-PCR）检测 NPC 在应激条件下内质网（ER）应激水平。使用倒置显微镜、三线分化实验和流式细胞术鉴定人 USCs。使用透射电镜、纳米颗粒大小分析和 WB 程序鉴定提取的外泌体并观察 NPC 摄取情况。建立对照组、48 h 组和 USCs-exos 组。对照组未处理，48 h 组压力培养 48 h，USCs-exos 组压力培养 48 h 后给予 USCs-exos 处理。采用 WB、qRT-PCR 和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）分析检测应激条件下和外泌体处理后的 ER 应激水平。部分检测 AKT 和 ERK 通路。磁共振成像（MRI）和计算机断层扫描（CT）用于评估细胞退变，苏木精和伊红（HE）染色、番红 O-快绿染色、免疫组织化学染色（IHC）、核磁共振（NMR）、光谱检测和总相关谱（TOCSY）用于确定外泌体对椎间盘（IVD）组织的影响。还鉴定和定量了 IVD 代谢物。
压力培养后，NPC 中的 ER 应激标志物（GRP78 和 C/EBP 同源蛋白（CHOP））随时间显著升高（<0.05）。人 USCs 呈短梭形，可成功进行成骨、成脂和成软骨分化。在这项研究中，这些干细胞被发现对 CD29、CD44 和 CD73 呈阳性。外泌体呈中央定位，直径为 50-100nm。CD63 和 Tsg101 表达水平较高，Calnexin 表达受到抑制。外泌体可被 NPC 摄取。USCs-exos 通过 AKT 和 ERK 信号通路显著改善 ER 应激反应，抑制过度激活未折叠蛋白反应（UPR）以及细胞凋亡和椎间盘退变（<0.05）。
通过 AKT 和 ERK 信号通路，USCs-exos 显著抑制压力下 ER 应激诱导的细胞凋亡和 IDD。因此，这是一种可行的治疗策略。
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