基于广泛靶向代谢组学技术探究延胡索乙素对慢性疼痛大鼠脊髓代谢谱的影响

Exploring Effect of Levo-tetrahydropalmatine on Spinal Metabolic Profiles of Rats with Chronic Pain Based on Widely-targeted Metabolomics

摘要:

目的 从代谢水平阐释延胡索乙素（l-THP）缓解慢性疼痛的潜在机制，辨识l-THP调控的关键代谢物和代谢通路。方法 在大鼠体内构建经典的慢性压迫性坐骨神经损伤（CCI）模型，通过测定机械缩足阈值评价动物的疼痛程度。从造模后第6天开始灌胃给药l-THP（64 mg·kg^-1）和阳性药普瑞巴林（Pre， 30 mg·kg^-1），连续给药5 d，于末次给药后1 h收集各组的造模侧脊髓组织进行广泛靶向代谢组学分析，每组8只大鼠。以变量投影重要性（VIP）>1.0且P<0.05为标准筛选差异代谢物，并进行京都基因与基因组百科全书（KEGG）功能富集分析和相互作用分析，得到与l-THP镇痛作用相关的关键代谢物和代谢通路。结果 行为学上，给药l-THP和Pre均显著改善了CCI大鼠的机械痛敏（P<0.01），减轻了疼痛。代谢组分析结果显示，给药l-THP有效纠正了CCI大鼠脊髓中异常的代谢轮廓，回调了53个差异代谢物，其中包括多个经典的疼痛生物标志物，如鞘氨醇-1-磷酸（S1P）、环磷酸腺苷（cAMP）、乙酰胆碱和谷氨酸。对差异代谢物的功能富集分析结果显示，铁死亡和自噬相关细胞过程，神经活性配体-受体相互作用、磷脂酶D和cAMP相关信号通路，谷胱甘肽代谢和辅因子生物合成等代谢途径可能是l-THP影响脊髓代谢谱的关键环节。进一步对代谢物相对丰度和代谢途径的分析表明，l-THP可能通过显著降低脊髓中谷氨酸、甘氨酸的相对水平（P<0.01），促进还原型谷胱甘肽（GSH）的合成，升高还原型/氧化型谷胱甘肽的比值（P<0.05），减轻CCI大鼠脊髓中的氧化应激，显著降低乙酰辅酶A水平（P<0.01），从而抑制铁死亡发生。结论 l-THP可能通过调节谷胱甘肽代谢、铁死亡、辅因子生物合成、氨基酸合成等多条代谢途径，纠正CCI大鼠脊髓中异常的代谢轮廓，发挥镇痛作用。铁死亡和谷胱甘肽代谢可能是l-THP调控的关键途径，谷氨酸、甘氨酸、谷胱甘肽和乙酰辅酶A可能是其中的关键代谢物。

Abstract:

Objective To elucidate the underlying mechanism of the efficacy of Levo-tetrahydropalmatine （l-THP） in alleviating chronic pain and identify the key metabolites and metabolic pathways for l-THP regulation.Method A classical chronic constrictive injury （CCI） model was built in rats’ bodies， and the pain intensity was evaluated by detecting the mechanical withdrawal threshold. On the sixth day after surgery， oral administration of l-THP （64 mg·kg^-1） and positive control drug pregabalin （Pre， 30 mg·kg^-1） was performed on rats. After the last administration following consecutive five times of administration， ipsilateral spinal cord tissues were collected for widely-targeted metabonomics， with eight rats in each group. Differential metabolites （DEMs） were identified according to the standard of VIP>1.0 and P<0.05， and functional enrichment and interaction analyses of the Kyoto Encyclopedia of Genes and Genomes （KEGG） were performed to obtain the key metabolites and metabolic pathways associated with the analgesic effects of l-THP.Result In behavioral science， administration of both l-THP and Pre significantly improved mechanical hyperalgesia in CCI rats （P<0.01）， thus mitigating pain. Metabonomic analysis results revealed that l-THP administration corrected the aberrant metabolic profile in the spinal cord of CCI rats. Meanwhile， 53 DEMs were called back， including several classical pain biomarkers such as sphingosine-1-phosphate （S1P）， cyclic adenosine monophosphate （cAMP）， acetylcholine， and glutamate. Functional enrichment analysis of the DEMs indicated the involvement of metabolic pathways such as ferroptosis， autophagy， neuroactive ligand-receptor interactions， phospholipase D and cAMP-related signaling pathways， glutathione metabolism， and cofactor biosynthesis in mediating the effects of l-THP on the metabolic profile of the spinal cord. Further analyses on the relative metabolite abundance and metabolic pathways indicated that by significantly decreasing the relative levels of glutamate （P<0.01） and glycine （P<0.01） in the spinal cord， l-THP can promote the synthesis of reduced glutathione （GSH） and increase the ratio of reduced/oxidized GSH （P<0.05）. Additionally， it can relieve oxidative stress in the spinal cord of CCI rats and significantly reduce the acetyl-CoA level （P<0.01） to finally inhibit ferroptosis occurrence.Conclusion l-THP may exert analgesic effects by regulating multiple metabolic pathways including GSH metabolism， ferroptosis， cofactor biosynthesis， and amino acid synthesis to correct the aberrant metabolic profile in the spinal cord of CCI rats. Ferroptosis and GSH metabolism may be the key pathways for l-THP regulation， with glutamate， glycine， glutathione， and acetyl-CoA as the key metabolites.