基于细胞代谢组学的商陆皂苷甲诱导HKC细胞损伤的机制研究

Mechanism research of HKC cell injury induced by esculentoside A based on cell metabolomics

目的基于细胞代谢组学技术,考察商陆皂苷甲(EsA)诱导人肾小管上皮HKC细胞损伤前后对细胞代谢产物的影响,寻找潜在的生物标志物。方法 MTT法检测EsA 0(对照组)、0.125、0.250、0.500、0.750、1.000 mmol/L对HKC细胞活力的影响;试剂盒法检测EsA 0(对照组)、0.25、0.75 mmol/L对HKC细胞上清LDH含量的影响,明确EsA肾细胞毒性;利用UPLC-Q/TOF-MS分析技术,将EsA 0(对照组)、0.25、0.75 mmol/L作用24 h的HKC细胞进行代谢组学分析,采用Progenesis QI软件进行数据处理,将得到的数据导入SIMCA-P12.0 software进行多元统计分析,利用主成分分析(PCA)剔除离群值,进行正交偏最小二乘分析(OPLS-DA)得到变量权重值(VIP),选取VIP>1的差异代谢物,进行检验,获得具有统计学意义的生物标志物。通过HMDB、KEGG、METLIN等代谢物数据库对生物标记物进行鉴定,利用MetPA平台对进行代谢通路分析。结果与对照组比较,EsA在大于0.25 mmol/L时对HKC细胞活力有显著的抑制作用(P<0.05、0.01);细胞上清液中LDH含量显著上升(P<0.05)。通过代谢组学技术和相关数据库分析筛选出了15个生物标志物,主要涉及甘油磷脂代谢、嘧啶代谢、鞘脂代谢、氨基酸代谢和能量代谢途径。结论 EsA可诱导肾细胞损伤,可能与氧化应激损伤,氨基酸代谢失调,能量代谢异常以及脂质代谢紊乱相关。

Objective To investigate the effects of Esculentoside A(EsA) on cellular metabolites before and after injury of human renal tubular epithelial HKC cells, and to find potential biomarkers based on cell metabolomics techniques.Methods MTT method was used to detect the effect of ESA 0(control group), 0.125, 0.250, 0.500, 0.750 and 1.000 mmol/L on the activity of HKC cells;Kit method was used to detect the effect of ESA 0(control group), 0.25 and 0.75 mmol/L on the content of LDH in the supernatant of HKC cells, so as to determine the nephrotoxicity of EsA;By using UPLC-Q/TOF-MS analysis technology, the HKC cells treated with EsA 0(control group), 0.25 and 0.75 mmol/L for 24 hours were analyzed by metabonomics. The data were processed by Progenesis QI software and imported into SIMCA-P12.0 Software carries on multivariate statistical analysis, uses PCA to eliminate outliers, carries on OPLS-DA to obtain variable weight value(VIP), selects VIP > 1 differential metabolite, carries on the test,obtains the biomarker with statistical significance. Biomarkers were identified by HMDB, KEGG, metlin and other metabolite databases, and metabolic pathways were analyzed by metpa platform. Results After stimulation with EsA, HKC cells showed a decrease in cell viability(P < 0.05) and up-regulation of LDH in cell supernatant(P < 0.05), 15 biomarkers were screened by metabolomics technology and related database analysis, mainly related to glycerophospholipid metabolism, pyrimidine metabolism,sphingolipid metabolism, amino acid metabolism and energy metabolism pathway.Conclusion EsA can induce renal cell injury, may be associated with oxidative stress damage, amino acid metabolism disorders, abnormal energy metabolism, and lipid metabolism disorders.