SIRT6对椎间盘退变的作用及机制

Roles and mechanisms of SIRT6 on intervertebral disc degeneration

中文关键词:  椎间盘退变性病变  SIRT6  氧化应激  TGFβ-smad1/5信号通路

英文关键词:Disc degeneration disease  SIRT6  Oxidative stress  TGFβ-smad1/5 signaling pathway

中文摘要:

  【摘要】 目的：探讨Sirtuin（SIRT）6对椎间盘退变的作用及机制。方法：32只C57BL/6小鼠随机分为假手术组、模型组、SIRT6激动剂组、SIRT6抑制剂组，每组8只。造模小鼠采用穿刺法构建Co5/6椎间盘退变模型，造模成功后，SIRT6激动剂组和SIRT6抑制剂组分别腹腔注射SIRT6激动剂（20mg/kg）和SIRT6抑制剂（20mg/kg），假手术组、模型组均腹腔注射等量生理盐水，连续7d。干预结束后，对小鼠进行影像学观察，并采集椎间盘组织，采用免疫组化检测椎间盘组织SIRT6阳性百分比；苏木精-伊红（hematoxylin eosin，HE）染色、Masson染色及甲苯胺蓝染色观察椎间盘组织病理学改变；生化检测椎间盘组织丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）及活性氧（ROS）含量；实时荧光定量逆转录聚合酶链式反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测椎间盘组织SIRT6、转化生长因子（transforming growth factor-β，TGF-β）1、smad1、smad5 mRNA表达；Western blot检测椎间盘组织骨胶原（collagen）Ⅱ、collagen X、TGF-β1、smad1、smad5、p-smad1、p-smad5蛋白表达。结果：影像学观察发现假手术组尾椎排列有序，无骨赘形成；模型组出现骨赘形成，相较于模型组，SIRT6激动剂组骨赘形成受到明显抑制，而SIRT6抑制剂组骨赘形成更明显。与假手术组相比，模型组小鼠椎间盘组织SIRT6表达及GSH-Px、SOD含量明显降低，而MDA、ROS含量及collagen Ⅱ、collagen X、TGF-β1、smad1、smad5、p-smad1、p-smad5表达明显升高（P<0.01）；模型组椎间盘组织软骨终板局部软骨减少，胶原纤维紊乱、致密，形态模糊；与模型组相比，SIRT6激动剂组椎间盘组织SIRT6表达及GSH-Px、SOD含量明显升高，MDA、ROS含量及collagen Ⅱ、collagen X、TGF-β1、smad1、smad5、p-smad1、p-smad5表达明显降低（P<0.05）；SIRT6抑制剂组则呈现与激动剂组相反的结果。结论：SIRT6过表达可有效调节氧化应激，抑制TGFβ-smad1/5信号通路，进而助于减缓椎间盘退变。

英文摘要:

  【Abstract】 Objectives: To investigate sirtuin(SIRT)6′s effects and its mechanisms on intervertebral disc degeneration. Methods: 32 C57BL/6 mice were randomly divided into sham group, model group, SIRT6 agonist group, and SIRT6 inhibitor group, with 8 mice in each group. The mice modeled were constructed as intervertebral disc degeneration models by puncture method, and SIRT6 agonist group and SIRT6 inhibitor group of mice were administered intraperitoneally with SIRT6 agonist and SIRT6 inhibitor(20mg/kg) respectively, and the sham group and model group of mice were administered an equal amount of physiological saline into the abdominal cavity for 7d. Imaging observations were performed and intervertebral disc tissues were collected at the end of the intervention, and the percentage of SIRT6 positive intervertebral disc tissues was detected by immunohistochemistry; Hematoxylin-eosin(HE) staining, Masson staining, and toluidine blue staining were used to observe the histopathological changes of intervertebral discs; The contents of MDA, GSH-Px, SOD and ROS in intervertebral disc tissues were detected biochemically; The expressions of SIRT6, transforming growth fator(TGF-β)1, smad1, and smad5 mRNA in intervertebral disc tissues were detected by quantitative real-time polymerase chain reaction(qRT-PCR); The expressions of collagen Ⅱ, collagen X, TGF-β1, smad1, smad5, p-smad1 and p-smad5 protein were detected by Western blot. Results: Imaging observation result after intervention showed that osteophyte formation was found in the model group, but not the sham group, and comparing with the model group, SIRT6 agonist group was inhibited while SIRT6 inhibitor group was more obvious. Comparing with the sham group, the expression of SIRT6 and the contents of GSH-Px and SOD in the intervertebral disc tissue of mice in the model group were significantly lower, while the contents of MDA and ROS and the expression of collagen Ⅱ, collagen X, TGF-β1, smad1, smad5, p-smad1, p-smad5 were significantly higher(P<0.01); Local cartilage endplates of intervertebral disc tissue of the model group reduced, collagen fibers were disorganized and dense, and blurred in morphology. Compared with the model group, the expression of SIRT6 and the contents of GSH-Px and SOD in the intervertebral disc tissues in the SIRT6 agonist group were significantly increased, and the contents of MDA and ROS and the expression of collagen Ⅱ, collagen X, TGF-β1, smad1, smad5, p-smad1, p-smad5 were significantly decreased (P<0.05), while those in the SIRT6 inhibitor group were the opposite. Conclusions: SIRT6 over-expression can effectively regulate oxidative stress and inhibit the TGFβ-smad1/5 signaling pathway, which in turn can slow down intervertebral disc degeneration.