【摘要】 目的 探究冬凌草甲素（ORI）对人食管鳞状细胞癌（ESCC）细胞KYSE-150内谷胱甘肽代谢网络（GMN）中13个主要代谢物的影响，包括半胱氨酸（Cys）、高半胱氨酸（HCys）、谷胱甘肽（GSH）、甲硫氨酸（Met）、S-腺苷高半胱氨酸（SAH）、胱硫醚（Cysta）、甘氨酸（Gly）、谷氨酸（Glu）、谷氨酰胺（Gln）、谷氨酰半胱氨酸（GC）、半胱氨酰甘氨酸（CG）、O-乙酰丝氨酸（OAS）和丝氨酸（Ser）。方法 首先，采用细胞计数试剂盒（CCK-8）法检测ORI对人ESCC细胞KYSE-150活性的影响，以半抑制浓度（IC50）作为后续细胞培养的干预浓度。将KYSE-150细胞按照不同的培养时间分为0 h、12 h、24 h、36 h组，以0 h组作为对照（空白培养液），12 h、24 h、36 h组采用含ORI的培养液（12.5μmol/L）对细胞进行相应时间的干预。然后，采用超声提取法提取细胞中的内容物，利用液相色谱-质谱（LC-MS）法直接检测ORI与GMN中巯基代谢物结合形成的缀合物，利用LC-MS结合化学衍生的方法检测13个目标GMN代谢物。最后，通过活性氧（ROS）检测试剂盒检测细胞提取物中ROS水平。结果 (1)ORI对人ESCC细胞KYSE-150有明显的抑制作用，IC50值为12.5μmol/L。(2)与0 h组相比，ORI干预后的3组KYSE-150细胞内GMN中的大部分代谢物（CG、GSH、GC、Cly、Glu、SAH、Cys、OAS和Cysta），尤其是含巯基的代谢物（CG、GSH、GC和Cys），浓度呈现先升高后下降的趋势。(3)在细胞提取物中检测到ORI与Cys、HCys、GSH这3个巯基内源性代谢物反应生成的缀合物。(4)ORI干预后KYSE-150细胞内ROS水平持续升高。结论 ORI可能通过影响KYSE-150细胞内GMN中代谢物水平引起ROS的蓄积，进而降低细胞对抗脂质过氧化和氧化应激的能力。更多还原

【Abstract】 Objective To investigate the effects of oridonin(ORI) on the 13 metabolites within the glutathione metabolic network(GMN) in human esophageal squamous cell carcinoma(ESCC) KYSE-150 cells, including cysteine(Cys), homocysteine(HCys), glutathione(GSH), methionine(Met), S-adenylyl homocysteine(SAH), cystathionine(Cysta), glycine(Gly), glutamic acid(Glu), glutamine(Gln), glutamylcysteine(GC), cysteinylglycine(CG), O-acetyl-serine(OAS) and serine(Ser). Methods Firstly, the Cell Counting Kit-8(CCK-8) assay was utilized to assess the effect of ORI on the viability of KYSE-150 cells, and the half-maximal inhibition concentration(IC50) was used as the intervention concentration for subsequent cell culture. KYSE-150 cells were then divided into 0 h, 12 h, 24 h, and 36 h groups based on different culture time. The 0 h group was set as control(blank culture medium), and the 12 h, 24 h and 36 h groups were treated with culture medium containing ORI(12.5 μmol/L) for corresponding time. Subsequently, cell contents from each group were extracted via ultrasonication. Liquid chromatography-mass spectrometry(LC-MS) method was employed for the identification of ORI and GMN thiol adducts, while LC-MS combined with chemical derivatization method was utilized for detecting the 13 target metabolites. Finally, reactive oxygen species(ROS) levels of cellular extracts were quantified using a ROS assay kit. Results(1)ORI had a significant inhibitory effect on human ESCC KYSE-150 cells with an IC50 value of 12.5 μmol/L.(2)Compared with the 0 h group, the concentrations of most metabolites within the GMN of KYSE-150 cells(CG, GSH, GC, Cly, Glu, SAH, Cys, OAS and Cysta), especially thiol-containing metabolites(CG, GSH, GC and Cys), showed a trend of initial increase followed by decrease after intervention with ORI.(3)Three conjugates resulting from the binding of ORI to thiol-containing metabolites(Cys, HCys and GSH) were identified in cellular extracts.(4)The level of ROS in KYSE-150 cells continued to increase after ORI intervention. Conclusion ORI may induce ROS accumulation by modulating metabolite levels within the GMN of KYSE-150 cells, consequently impairing the cells’ capacity to counteract lipid peroxidation and oxidative stress.更多还原