冬凌草甲素对食管鳞状细胞癌细胞内谷胱甘肽代谢网络影响的研究  

Study on effects of oridonin on glutathione metabolism network in esophageal squamous cell carcinoma cells

作　　者：王敏丹 刘莉 崔夏莲 韦懿芯 张雯雯 陈秀萍 张芳[1] 冯陈国 WANG Mindan;LIU Li;CUI Xialian;WEI Yixin;ZHANG Wenwen;CHEN Xiuping;ZHANG Fang;FENG Chenguo(Innovation Research Institute of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学创新中药研究院,上海201203

出　　处：《上海中医药杂志》2024年第7期77-82,94,共7页Shanghai Journal of Traditional Chinese Medicine

基　　金：上海市自然科学基金项目(23ZR1460900)。

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①ORI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。④ORI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。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(12.5μmol/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.Results①ORI had a significant inhibitory effect on human ESCC KYSE-150 cells with an IC50 value of 12.5μmol/L.②Compared 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.③Three conjugates resulting from the binding of ORI to thiol-containing metabolites(Cys,HCys and GSH)were identified in cellular extracts.④The level of ROS in KYSE-150 cells continued to increase after ORI intervention.Conclusion ORI may induce ROS accumulation by modulating m

关 键 词：食管鳞状细胞癌 冬凌草甲素 谷胱甘肽 活性氧 代谢组学 中药研究 

分 类 号：R285[医药卫生—中药学]