Objective: To investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.
Methods: Ten patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot.
Results: Immunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group ( t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased ( P<0.05). In cytology experiments, the protein expression of β-catenin in group B was significantly higher than that in groups A and C, and the protein expressions of collagen type Ⅱ and Aggrecan in group B were significantly lower than those in groups A and C ( P<0.05). After transfection of siRNA, the protein expressions of SIRT1 and β-catenin significantly decreased ( P<0.05). The results of cell immunofluorescence staining further confirmed that when compared with group 3, after the SIRT1 was silenced by siRNA in group 4, the attenuated nuclear translocation of β-catenin by RES treatment was aggravated. Western blot results showed that the protein expressions of collagen type Ⅱ and Aggrecan in group Ⅱ were significantly lower than those in group Ⅰ( P<0.05); after transfection of β-catenin-siRNA in group Ⅲ, the degradation of ECM by IL-1β was obviously inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly increased when compared with group Ⅱ ( P<0.05); after transfection of SIRT1-siRNA in group Ⅴ, the protective effect of RES on the degradation of ECM was inhibited, the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased when compared with group Ⅳ ( P<0.05).
Conclusion: RES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.
目的: 探讨白藜芦醇（resveratrol，RES）对退变髓核细胞（nucleus pulposus cells，NPC）细胞外基质（extracellular matrix，ECM）表达的调控作用及其相关分子机制。.
方法: 选取临床上接受椎间盘摘除术的 10 例患者，其中 5 例为年轻脊柱爆裂骨折患者，作为对照组；余 5 例为老年腰椎间盘突出症患者，作为退变组。取两组患者髓核组织，免疫组织化学染色比较 β-catenin 的原位表达，Western blot 检测 β-catenin、Ⅱ型胶原和聚集蛋白聚糖（Aggrecan）蛋白表达。取退变组髓核组织分离、培养 NPC，分别用 IL-1β 单独（B 组）或联合 RES（C 组）刺激第 3 代 NPC，并设未刺激细胞作为空白对照组（A 组），Western blot 检测Ⅱ型胶原和 Aggrecan 蛋白表达。进一步采用小干扰 RNA（small interfering RNA，siRNA）靶向沉默 SIRT1 和 β-catenin 后，采用 Western blot、实时荧光定量 PCR 检测 β-catenin、SIRT1 蛋白和基因表达。用完全培养基（1 组）、IL-1β（2 组）、RES+IL-1β（3 组）、SIRT1-siRNA+RES+IL-1β（4 组）刺激第 3 代 NPC 培养 24 h 后，细胞免疫荧光染色检测 β-catenin 核转位情况；用完全培养基（Ⅰ组）、IL-1β（Ⅱ组）、IL-1β+β-catenin-siRNA（Ⅲ组）、IL-1β+RES（Ⅳ组）、IL-1β+RES+SIRT1-siRNA（Ⅴ组）刺激第 3 代 NPC 培养 24 h 后，采用 Western blot 检测Ⅱ型胶原和 Aggrecan 蛋白表达。.
结果: 免疫组织化学染色及 Western blot 检测示，与对照组比较，退变组髓核组织中 β-catenin 阳性表达的细胞比例显著升高（ t=4.616， P=0.010）；β-catenin 蛋白相对表达量显著升高，Ⅱ型胶原和 Aggrecan 蛋白相对表达量显著下降（ P<0.05）。细胞学实验发现，B 组 β-catenin 蛋白相对表达量显著高于 A、C 组，Ⅱ型胶原和 Aggrecan 相对表达量显著低于 A、C 组（ P<0.05）。siRNA 转染后，Western blot 检测显示 SIRT1、β-catenin 蛋白表达显著降低（ P<0.05）。细胞免疫荧光染色结果进一步提示与 3 组相比，4 组 SIRT1 被 siRNA 沉默后，RES 减弱的 β-catenin 核转位现象加剧。Western blot 检测示，Ⅱ组Ⅱ型胶原和 Aggrecan 蛋白表达较Ⅰ组明显降低（ P<0.05）；Ⅲ组 NPC 转染 β-catenin-siRNA 后，IL-1β 对 ECM 的降解作用被明显抑制，Ⅱ型胶原和 Aggrecan 蛋白表达较Ⅱ组明显升高（ P<0.05）；Ⅴ组 NPC 转染 SIRT1-siRNA 后，抑制了 RES 对 ECM 降解的保护作用，Ⅱ型胶原和 Aggrecan 蛋白表达较Ⅳ组明显降低（ P<0.05）。.
结论: RES 可以通过抑制 Wnt/β-catenin 信号通路维持 NPC ECM 的表达，为椎间盘退行性疾病的治疗提供了新思路。.
Keywords: Resveratrol; SIRT1; Wnt/β-catenin signaling pathway; extracellular matrix; nucleus pulposus cells.