Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou, China.
Department of Orthopedics Center, Ningbo No.2 Hospital, Ningbo, Zhejiang, China.
J Cell Physiol. 2021 Sep;236(9):6441-6456. doi: 10.1002/jcp.30319. Epub 2021 Feb 9.
Senescence of nucleus pulposus (NP) cells (NPC) is a major cause of intervertebral disc degeneration (IVDD), so delay NPC senescence may be beneficial for mitigating IVDD. We studied the effect and mechanism of silent information regulator 2 homolog 3 (SIRT3) on NPC senescence in vivo and in vitro. First, we observed SIRT3 expression in normal and degenerated NPC with immunohistochemical and immunofluorescence staining. Second, using SIRT3 lentivirus transfection, reactive oxygen species probe, senescence-associated β-galactosidase staining, polymerase chain reaction, and western blot to observe the oxidative stress, senescence, and degeneration degree among groups. Subsequently, pretreatment with adenosine monophosphate-activated protein kinase (AMPK) agonists and inhibitors, observing oxidative stress, senescence, and degeneration degree among groups. Finally, the IVDD model was constructed and divided into Ctrl, Vehicle, LV-shSIRT3, and LV-SIRT3 groups. X-ray and magnetic resonance imaging scans were performed on rat's tails after 1 week; hematoxylin and eosin and safranin-O staining were used to evaluate the degree of IVDD; immunofluorescence staining was used to observe SIRT3 expression; immunohistochemical staining was used to observe oxidative stress, senescence, and degeneration degree of NP. We found that SIRT3 expression is reduced in degenerated NP tissues but increased in H O -induced NPC. Moreover, SIRT3 upregulation decreased oxidative stress, delayed senescence, and degeneration of NPC. In addition, activation of the AMPK/PGC-1α pathway can partially mitigate the NPC oxidative stress, senescence, and degeneration caused by SIRT3 knockdown. The study in vivo revealed that local SIRT3 overexpression can significantly reduce oxidative stress and ECM degradation of NPC, delay NPC senescence, thereby mitigating IVDD. In summary, SIRT3 mediated by the AMPK/PGC-1α pathway mitigates IVDD by delaying oxidative stress-induced NPC senescence.
核髓核细胞（NP）衰老（NPC）是椎间盘退变（IVDD）的主要原因，因此延缓 NPC 衰老可能有利于减轻 IVDD。我们研究了沉默信息调节因子 2 同源物 3（SIRT3）在体内和体外对 NPC 衰老的影响及其机制。首先，我们通过免疫组化和免疫荧光染色观察正常和退变 NP 中的 SIRT3 表达。其次，使用 SIRT3 慢病毒转染、活性氧探针、衰老相关β-半乳糖苷酶染色、聚合酶链反应和 Western blot 观察各组间的氧化应激、衰老和退变程度。随后，用腺苷单磷酸激活蛋白激酶（AMPK）激动剂和抑制剂预处理，观察各组间的氧化应激、衰老和退变程度。最后，构建 IVDD 模型，分为对照、载体、LV-shSIRT3 和 LV-SIRT3 组。1 周后对大鼠尾巴进行 X 射线和磁共振成像扫描；用苏木精和伊红以及番红 O 染色评估 IVDD 程度；用免疫荧光染色观察 SIRT3 表达；用免疫组化染色观察 NP 中氧化应激、衰老和退变程度。我们发现 SIRT3 在退变 NP 组织中的表达减少，但在 H2O2 诱导的 NPC 中增加。此外，SIRT3 上调可降低 NPC 的氧化应激，延缓其衰老和退变。此外，AMPK/PGC-1α 通路的激活可部分减轻 SIRT3 敲低引起的 NPC 氧化应激、衰老和退变。体内研究表明，局部 SIRT3 过表达可显著降低 NPC 的氧化应激和 ECM 降解，延缓 NPC 衰老，从而减轻 IVDD。综上所述，AMPK/PGC-1α 通路介导的 SIRT3 通过延缓氧化应激诱导的 NPC 衰老来减轻 IVDD。
