Department of Orthopedics, Xinqiao Hospital, Army Military Medical University, Chongqing, 400037, China.
Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Science, Beijing, 100000, China.
Stem Cell Res Ther. 2022 Jul 26;13(1):340. doi: 10.1186/s13287-022-03031-7.
Nucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation.
To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (HO) and explore the therapeutic efficacy of NPMSCs transplantation using HO pretreatment in IVDD.
Rat NPMSCs were pretreated with different concentrations (range from 25 to 300 μM) of HO. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of HO-pretreated NPMSCs in vivo.
NPMSCs pretreated with 75 μM HO demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 μM HO-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 μM HO also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 μM HO-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), which means 75 μM HO-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.
Pretreatment with 75 μM HO was the optimal concentration to improve the proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.
髓核间充质干细胞（NPMSCs）移植是治疗椎间盘退变（IVDD）的一种很有前途的方法。然而，移植的 NPMSCs 表现出较弱的细胞增殖、较高的细胞凋亡以及对退变椎间盘恶劣微环境的低抵抗力。迫切需要探索可行的方法来增强 NPMSCs 移植的治疗效果。
确定 NPMSCs 用过氧化氢（HO）预处理的最佳浓度，并探讨 HO 预处理 NPMSCs 移植治疗 IVDD 的疗效。
用不同浓度（范围为 25 至 300μM）的 HO 预处理大鼠 NPMSCs。通过细胞计数试剂盒-8（CCK-8）检测、5-乙炔基-2'-脱氧尿苷（EdU）染色和流式细胞术，在体外检测 NPMSCs 的增殖、活性氧（ROS）水平和凋亡。利用 Western blot 探索潜在的信号通路。建立大鼠针刺刺激的 IVDD 模型。X 射线、组织学染色和多模式小动物活体成像系统用于体内评估 HO 预处理 NPMSCs 的治疗效果。
75μM HO 预处理的 NPMSCs 通过抑制 Hippo 通路表现出最强的细胞增殖增强作用（P<0.01）。同时，75μM HO 预处理的 NPMSCs 表现出明显增强的抗氧化应激能力（P<0.01），这与 Brd4 和 Keap1 的下调以及 Nrf2 的上调有关。75μM HO 预处理的 NPMSCs 的凋亡也明显减少（P<0.01）。体内实验证实，75μM HO 预处理的 NPMSCs 移植大鼠的椎间盘高度指数（DHI%=90.00±4.55，P<0.01）和组织学形态（组织学评分=13.5±0.5，P<0.01）均得到增强，这意味着 75μM HO 预处理的 NPMSCs 可以更好地适应退变椎间盘的环境并促进 IVDD 的修复。
75μM HO 的预处理是提高移植 NPMSCs 增殖、抗氧化应激和抗凋亡能力的最佳浓度，有望为提高 IVDD 的干细胞治疗效果提供一种新的可行方法。
