Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

线粒体功能障碍在椎间盘退变（IDD）的发展中起着重要作用。Sirtuin 5 (SIRT5) 通过其去琥珀酰酶活性参与线粒体稳态的维持。然而，目前尚不清楚琥珀酰化或SIRT5是否参与了过度机械应力引起的线粒体损伤和IDD的发生。我们的 4D 无标记定量蛋白质组学结果显示，机械负荷下大鼠髓核 (NP) 组织中去琥珀酰酶 SIRT5 的表达降低。Sirt5的过表达有效缓解了机械应激下NP细胞的凋亡和功能障碍，而Sirt5的敲除则加剧了NP细胞的凋亡和功能障碍，这与Sirt5 KO小鼠比接受腰椎不稳（LSI）手术的野生型小鼠更严重的IDD表型一致。此外，免疫沉淀偶联质谱 (IP-MS) 结果表明 AIFM1 是 SIRT5 的下游靶标，这一点已通过 Co-IP 测定得到验证。我们进一步证明，SIRT5表达减少导致AIFM1琥珀酰化增加，进而消除AIFM1和CHCHD4之间的相互作用，从而导致NP细胞中电子传递链（ETC）复合物亚基减少。ETC 复合体亚基减少导致线粒体功能障碍，并随后在机械应力下发生 IDD。最后，我们通过在压迫诱导的大鼠 IDD 模型中上调 SIRT5 表达或亚甲蓝 (MB) 给药来验证针对线粒体蛋白输入中断的治疗的功效。总之，我们的研究为 IDD 的发生和发展提供了新的见解，并为 IDD 提供了有前途的治疗方法。