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背景:研究表明,烟酰胺能够对白细胞介素1β或肿瘤坏死因子α致椎间盘退变起到保护作用,但烟酰胺对椎间盘细胞凋亡与增殖的保护机制尚不很清楚。目的:观察烟酰胺对兔髓核细胞增殖及凋亡的调控作用及其机制。设计、时间及地点:随机对照分组设计,于2007-04/10在华中科技大学同济医学院附属协和医院骨科实验室及协和医院干细胞中心完成。材料:2～3月龄日本大白兔10只,体质量1.5～2.0kg,取L1～L6节段椎间盘内髓核细胞,体外培养髓核细胞凋亡模型。方法:实验分为6组,正常对照组:不加任何药物;烟酰胺组:加入0.5g/L烟酰胺;白细胞介素1β组:加入10μg/L白细胞介素1β;白细胞介素1β+Caspase抑制剂组:加入10μg/L白细胞介素1β及非特异性Caspase抑制剂Z-VAD-FM;白细胞介素1β+小剂量烟酰胺组:加入10μg/L白细胞介素1β及0.05g/L烟酰胺组;白细胞介素1β+大剂量烟酰胺组:10μg/L白细胞介素1β及0.5g/L烟酰胺。3d后对各组细胞行AnnexinV-PI染色,Caspase-3、Caspase-8、Caspase-9功能染色检测及MTT检测。主要观察指标:各组细胞凋亡率、caspase-3、8、9功能染色阳性细胞率及各组细胞的吸光度。结果:①白细胞介素1β+Caspase抑制剂组和白细胞介素1β+大剂量烟酰胺组较白细胞介素1β组细胞凋亡率明显下降(P<0.01)。②白细胞介素1β+Caspase抑制剂组、白细胞介素1β+大、小剂量烟酰胺组Caspase-3、Caspase-8、Caspase-9功能染色阳性细胞率较白细胞介素1β组明显下降(P<0.01或P<0.05)。③与白细胞介素1β组相比,白细胞介素1β+Caspase抑制剂组和白细胞介素1β+大剂量烟酰胺组吸光度升高(P<0.01)。结论:烟酰胺可以促进髓核细胞的增殖并抑制白细胞介素1β诱导的髓核细胞凋亡,对凋亡的抑制作用主要通过抑制凋亡的线粒体途径实现。
BACKGROUND: Niacinamide has been shown to protect interleukin-1β or tumor necrosis factor-α-induced disc degeneration. However, the protective mechanism of nicotinamide on the apoptosis and proliferation of the intervertebral disc remains unclear. Objective: To observe the regulatory effect of nicotinamide on the proliferation and apoptosis of rabbit nucleus pulposus cells and its mechanism. DESIGN, TIME AND SETTING: A randomized controlled study was performed at the Orthopedic Laboratory of Peking Union Medical College Hospital and the Stem Cell Center of Peking Union Medical College Hospital, Tongji Medical College, Huazhong University of Science and Technology from April 2007 to October 10, 2007. MATERIALS: Ten Japanese rabbits aged 2-3 months were enrolled in this study. The body weight was 1.5-2.0 kg. The nucleus pulposus cells of L1 ~ L6 segments were obtained and the apoptotic models of nucleus pulposus were cultured in vitro. Methods: The experiment was divided into 6 groups, the normal control group: without any drug; nicotinamide group: adding 0.5g / L nicotinamide; IL-1β group: adding 10μg / L IL-1β; IL- Inhibitor group: adding 10μg / L interleukin-1β and non-specific inhibitor of Caspase Z-VAD-FM; interleukin 1β + low dose nicotinamide group: adding 10μg / L interleukin 1β and 0.05g / L nicotinamide Group; Interleukin 1β + high dose nicotinamide group: 10μg / L interleukin 1β and 0.5g / L nicotinamide. Annexin V-PI staining, Caspase-3, Caspase-8, Caspase-9 functional staining and MTT assay were performed on the 3 groups. MAIN OUTCOME MEASURES: The apoptosis rate of each group, the positive rate of caspase-3, 8, 9 and the absorbance of each group of cells. Results ① The apoptosis rate of IL-1β + Caspase inhibitor group and IL-1β + high-dose nicotinamide group was significantly lower than that of IL-1β group (P <0.01). ② The positive rates of Caspase-3, Caspase-8 and Caspase-9 positive cells in IL-1β + Caspase inhibitor group and interleukin-1β + large and small dose nicotinamide group were significantly lower than that in IL- 0.01 or P <0.05). ③ Compared with IL-1β group, the absorbance of IL-1β + Caspase inhibitor group and IL-1β + high-dose nicotinamide group increased (P <0.01). Conclusion: Niacinamide can promote the proliferation of nucleus pulposus cells and inhibit the apoptosis of nucleus pulposus cells induced by interleukin - 1β. The inhibitory effect on apoptosis is mainly through the mitochondrial pathway of inhibiting apoptosis.