中国修复重建外科杂志

中国修复重建外科杂志

白藜芦醇通过 Wnt/β-catenin 信号通路调控髓核细胞细胞外基质表达

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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。

ObjectiveTo investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism.MethodsTen 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.ResultsImmunohistochemical 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).ConclusionRES regulates the ECM expression of NPC via Wnt/β-catenin signaling pathway, which provide a new idea for intervertebral disc degeneration disease treatment.

关键词: 白藜芦醇; 髓核细胞; 细胞外基质; SIRT1; Wnt/β-catenin 信号通路

Key words: Resveratrol; nucleus pulposus cells; extracellular matrix; SIRT1; Wnt/β-catenin signaling pathway

引用本文: 刘沪喆, 沈皆亮, 周浩, 许圣茜, 胡侦明. 白藜芦醇通过 Wnt/β-catenin 信号通路调控髓核细胞细胞外基质表达. 中国修复重建外科杂志, 2018, 32(4): 476-483. doi: 10.7507/1002-1892.201709097 复制

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