髓核间充质干细胞(NPMSCs)移植是一种很有前途的椎间盘退变(IVDD)治疗方法。然而,移植的NPMSCs表现出微弱的细胞增殖,高细胞凋亡,和低的能力,以抵抗恶劣的微环境的退化椎间盘。迫切需要探索可行的方法来增强NPMSCs移植的治疗效果。
确定过氧化氢(H2O2)预处理NPMSCs的最佳浓度,并探讨IVDD中使用H2O2预处理NPMSCs移植的治疗效果。
用不同浓度(25-300μM)的H2O2预处理大鼠NPMSCs。扩散,活性氧(ROS)水平,细胞计数试剂盒-8(CCK-8)法检测NPMSCs的凋亡,5-乙炔基-2'-脱氧尿苷(EdU)染色,和体外流式细胞术。利用Western印迹探索了潜在的信号传导途径。建立大鼠针刺刺激IVDD模型。X光片,组织学染色,和多模式小动物活体成像系统用于评估H2O2预处理的NPMSCs的体内治疗效果。
用75μMH2O2预处理的NPMSCs通过抑制Hippo途径表现出最强的细胞增殖升高(P<0.01)。同时,75μMH2O2预处理的NPMSCs表现出显著增强的抗氧化应激能力(P<0.01),这与下调的Brd4和Keap1以及上调的Nrf2有关。用75μMH2O2预处理的NPMSCs也表现出明显减少的细胞凋亡(P<0.01)。体内实验证实,75μMH2O2预处理的NPMSCs移植大鼠椎间盘高度指数增强(DHI%=90.00±4.55,P<0.01),组织学形态改善(组织学评分=13.5±0.5,P<0.01)。说明75μMH2O2预处理的NPMSCs能更好地适应退变椎间盘的环境,促进IVDD的修复。
用75μMH2O2预处理是改善增殖的最佳浓度,抗氧化应激,和移植的NPMSCs的抗凋亡能力,有望为提高IVDD的干细胞治疗疗效提供新的可行方法。
BACKGROUND: 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.
OBJECTIVE: To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of
NPMSCs transplantation using H2O2 pretreatment in IVDD.
METHODS: Rat
NPMSCs were pretreated with different concentrations (range from 25 to 300 μM) of H2O2. 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 H2O2-pretreated NPMSCs in vivo.
RESULTS: NPMSCs pretreated with 75 μM H2O2 demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 μM H2O2-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 H2O2 also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 μM H2O2-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 H2O2-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.
CONCLUSIONS: Pretreatment with 75 μM H2O2 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.