基于LC-MS细胞代谢组学技术的环黄芪醇对多柔比星诱导足细胞损伤保护作用机制研究

目的 基于LC-MS细胞代谢组学方法研究环黄芪醇(cycloastragenol, CAG)保护多柔比星(doxorubicin, DOX)诱导小鼠足细胞(mouse podocyte clone 5,MPC5)损伤的作用机制。方法 体外培养永生型MPC5,分为空白对照组、DOX诱导MPC5细胞损伤模型组、CAG治疗组。MTT法检测MPC5的存活率;Western blot分析各组MPC5中关键蛋白Nephrin、Podocin的表达;结晶紫法检测MPC5黏附性;通过LC-MS代谢组学技术结合多元统计分析筛选潜在药效标志物和关键代谢通路,阐明其作用机制。结果 各浓度CAG均能有效改善DOX损伤存活率;12.5 μg·mL^-1的CAG能增加MPC5关键蛋白Nephrin、Podocin的表达,可显著改善DOX损伤MPC5黏附性;细胞代谢组学结果显示,DOX损伤的MPC5内共发现22种差异代谢物发生显著性变化,给予CAG干预后,细胞内20种差异代谢物得到了不同程度的回调,这些代谢物主要涉及甘油磷脂代谢、精氨酸和脯氨酸代谢、谷胱甘肽代谢、精氨酸生物合成、嘧啶代谢通路。结论 CAG对DOX诱导的MPC5损伤有改善作用,表现在能改善MPC5结构、活性和黏附性,其具体机制可能与甘油磷脂代谢、氨基酸代谢、谷胱甘肽代谢、嘧啶代谢密切相关。

Protective Effect of Cycloastragenol on Doxorubicin-Induced Podocytes Injury Based on Metabolomics by LC-MS Technology

OBJECTIVE To investigate the protective mechanism of cycloastragenol(CAG) against doxorubicin(DOX) -induced podocytes(MPC5) injury using LC-MS-based cellular metabolomics. METHODS MPC5 cells were cultured in vitro and divided into the control group, DOX-induced MPC5 cell injury model group and CAG group.MTT assay was used to detect podocytes survival rate. The expressions of nephrin and podocin in podocytes were detected by Western blot.Podocytes adhesion was detected by crystal violet method. LC-MS metabolomics combined with multivariate statistical analysis was used to screen potential efficacy markers and key metabolic pathways, aiming at elucidating the mechanism of action. RESULTS All concentrations of CAG could effectively improve the survival rate of DOX-injured podocytes. CAG(12.5 μg·mL^-1) could increase the expression of the nephrin and podocin protein, which could significantly improve the adhesion of DOX-injured podocytes.The metabolomic results showed that 22 differential metabolites were significantly changed in DOX-injured podocytes. After treatment with CAG, 20 differential metabolites were reversed to different degrees, and these metabolites were mainly involved in glycerophospholipid metabolism, arginine and proline metabolism, glutathione metabolism, arginine biosynthesis and pyrimidine metabolism pathway. CONCLUSION CAG could alleviate DOX-induced podocytes injury, which can improve the structure, activity and adhesion of MPC5, and the mechanism may be closely related to glycerophospholipid metabolism, amino acid metabolism, glutathione metabolism, pyrimidine metabolism.