Intracellular differential gene expression detected by transcriptome sequencing after lactate treatment of nucleus pulposus cells

The accumulation of metabolites in the intervertebral disc is regarded as an important inducement of intervertebral disc degeneration (IVDD). Lactate, a metabolite produced by the cellular anaerobic glycolysis process, has been proved to be closely associated with IVDD. However, little was known about the role of lactate in nucleus pulposus cells (NPCs) senescence. This study attempted to investigate the effect and molecular mechanism of lactate on NPCs senescence in vitro and in vivo. A puncture-induced disc degeneration model was established in rats. Metabolomics analysis proved that lactate was significantly increased in the rat degenerated intervertebral disc. Eliminating extra lactate using lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentration lactate could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing (RNA-seq) results and bioinformatic analysis exhibited that lactate-stimulated NPCs senescence may be related to the PI3K/Akt signaling pathway. Further study verified that high concentration lactate could induce NPCs senescence and oxidative stress via inhibiting PI3K/Akt signaling and downstream Akt/p21/p27/cyclinD1 and Akt/Nrf2/HO-1 pathway. Utilizing molecular docking, we found that lactate may suppress the Akt phosphoactivation via binding to Lys39 and Leu52 in the PH domain of Akt. In conclusion, this study illuminates that lactate could promote the senescence of NPCs to aggravate IVDD by suppressing the PI3K/Akt signaling pathway and the expression of its downstream genes. These results highlight the involvement of lactate NPCs senescence, which may become a novel potential therapeutic target for the treatment of IVDD. Comparative gene expression profiling analysis of RNA-seq data for NP cells and NP cells after treated with lactate.

椎间盘内代谢物蓄积被认为是椎间盘退变（intervertebral disc degeneration, IVDD）的重要诱因。乳酸作为细胞无氧糖酵解过程产生的代谢物，已被证实与IVDD密切相关。然而，目前关于乳酸在髓核细胞（nucleus pulposus cells, NPCs）衰老中发挥的作用仍知之甚少。本研究旨在探讨乳酸对体内外髓核细胞衰老的影响及其分子机制。我们在大鼠中构建了穿刺诱导的椎间盘退变模型。代谢组学分析证实，大鼠退变椎间盘内乳酸水平显著升高。通过过表达乳酸氧化酶（lactate oxidase, LOx）的慢病毒清除过量乳酸，可缓解IVDD的疾病进展。体外实验结果显示，高浓度乳酸可诱导髓核细胞衰老及氧化应激。高通量RNA测序（high-throughput RNA sequencing, RNA-seq）结果与生物信息学分析表明，乳酸刺激的髓核细胞衰老可能与PI3K/Akt信号通路相关。进一步研究验证，高浓度乳酸可通过抑制PI3K/Akt信号通路及其下游Akt/p21/p27/cyclinD1与Akt/Nrf2/HO-1通路，诱导髓核细胞衰老及氧化应激。通过分子对接实验，我们发现乳酸可通过结合Akt的PH结构域中的Lys39与Leu52位点，抑制Akt的磷酸化激活。综上，本研究阐明了乳酸可通过抑制PI3K/Akt信号通路及其下游基因的表达，促进髓核细胞衰老进而加重IVDD。上述结果揭示了乳酸在髓核细胞衰老中的作用，其或可成为治疗IVDD的新型潜在治疗靶点。本数据集包含针对髓核细胞经乳酸处理前后的RNA-seq数据的比较基因表达谱分析内容。