【作者】 王旭；

【导师】 乞国艳；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2023， 硕士

【摘要】 目的:通过采用代谢组学分析,对甲泼尼龙治疗前后的胸腺瘤伴重症肌无力患者的血清代谢物进行检测,寻找关键代谢物与关键通路,再结合网络药理学分析,对甲泼尼龙的药物靶点进行挖掘,探索甲泼尼龙治疗胸腺瘤伴重症肌无力的作用机制与治疗靶点,从而更好的指导临床用药与治疗。方法:本研究收集了石家庄市人民医院重症肌无力诊疗中心于2021年3月至2022年3月收治的15名胸腺瘤伴重症肌无力患者的血清,通过液相色谱质谱联用技术(Liquid Chromatograph Mass Spectrometer,LC-MS)测定甲泼尼龙治疗前后血清代谢物的含量变化,利用代谢组学数据分析软件Progenesis QI v2.3对得到的原始质谱碎片信息进行整合与解析,并运用单变量分析、多元统计分析等数据分析方法分析组间代谢物差异,筛选差异代谢物的标准是多元统计分析中变量权重值(Variable important in projection,VIP)>1且单变量分析中的P值<0.05。差异代谢物筛选完成后将其输入“京都基因与基因组百科全书”数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)进行通路富集分析,寻找关键通路;之后通过网络药理学寻找甲泼尼龙治疗的潜在靶点并联合代谢组学探索甲泼尼龙治疗胸腺瘤伴重症肌无力的作用机制;最后采用酶联免疫吸附实验(Enzyme Linked Immunosorbent Assay,Elisa)对寻找到的关键靶点及关键代谢物的上游合成酶进行验证。结果:1.在代谢组学研究中共筛选出了差异代谢物148个,其中富集到KEGG通路的有25个,上调的有6个,包括溶血磷脂(LysoPC,LyP)、24-羟基胆固醇(24-Hydroxycholesterol)等;下调的有19个,包括神经酰胺(Ceramide,Cer)、鞘磷脂(sphingomyelin,SM)、尿酸(Uric acid)、磷脂酰胆碱(Phosphatidyl cholines,PC)、肉碱(Creatine)、左旋岩藻糖(L-Fucose)等;KEGG通路富集分析显示涉及坏死性凋亡(necroptosis)、C型凝集素受体(C-type Lectins receptor)、胰岛素抵抗(insulin resistance)、甘油磷脂代谢(Glycerophospholipid metabolism)等通路。其中关键代谢物Cer与SM在细胞免疫调节、炎症反应与肿瘤调控中发挥巨大作用。2.网络药理学分析得出肿瘤坏死因子(tumor necrosis factor,TNF)、白介素-6(Interleukin-6,IL-6)、类固醇受体共激活因子(Steroid Receptor Coactivator,SRC)等可作为甲泼尼龙治疗胸腺瘤伴重症肌无力的潜在靶点。3.Elisa实验验证结果显示,关键靶点TNF与关键代谢物SM和Cer的上游合成酶在甲泼尼龙治疗后均下调,差异具有统计学意义(P<0.05)。结论:1.甲泼尼龙可能通过作用于TNF靶点影响坏死性凋亡通路进一步影响鞘磷脂的代谢,导致鞘磷脂与神经酰胺减少来治疗胸腺瘤伴重症肌无力。2.甲泼尼龙在发挥治疗作用的同时可对脂质代谢、糖代谢产生影响从而产生副作用,与既往研究及临床相关表现一致。更多还原

【Abstract】 Objective: We detect the serum metabolites of thymoma with myasthenia gravis before and after methylprednisolone treatment by metabolomics analysis to find the key metabolites and key pathways,and then combine with the network pharmacology to explore the drug targets and the mechanism of methylprednisolone in the treatment of thymoma with myasthenia gravis,so as to better guide clinical medication and treatment.Methods: The sera of 15 thymoma patients with myasthenia gravis treated in Shijiazhuang people’s Hospital from March 2021 to March 2022 were collected in this study.The changes of serum metabolites before and after methylprednisolone treatment were determined by Liquid Chromatograph Mass Spectrometer(LC-MS).The original mass spectrometry fragment information was integrated and analyzed by metabonomics data analysis software Progenesis QIv2.3,and the differences of metabolites between groups were analyzed by univariate analysis and multivariate statistical analysis.The criteria for screening differential metabolites were Variable important in projection(VIP)> 1 in multivariate statistical analysis and P<0.05 in univariate analysis.After the differential metabolites were screened,they were input into the Kyoto Encyclopedia of Gene and Genome database(KEGG)for pathway enrichment analysis,and then combined with network pharmacology to find potential targets for methylprednisolone therapy.Finally,enzyme-linked immunosorbent assay(Elisa)was used to verify the key targets and the upstream synthetase of key metabolites.Results: 1.A total of 148 differential metabolites were screened in metabonomics study,of which 25 were enriched in KEGG pathway,6 were up-regulated,including hemolytic phospholipid(LysoPC)24-hydroxycholesterol(24-Hydroxycholesterol),19 were down-regulated,including ceramide(Ceramide,Cer),sphingomyelin(sphingomyelin,SM),uric acid(Uricacid),phosphatidylcholine(Phosphatidylcholines,PC),carnitine(Creatine),etc.KEGG pathway enrichment analysis showed that necrotic apoptosis(necroptosis),C-type lectin receptor(C-type Lectins receptor),insulin resistance(insulin resistance)and glycerol phospholipid metabolism(Glycerophospholipidmetabolism)were involved.Among them,the key metabolites Cer and SM play an important role in cellular immune regulation,inflammatory response and tumor regulation.2.Network pharmacological analysis shows that IL-6,TNF,SCR can be used as a potential target of methylprednisolone in the treatment of thymoma with MG.3.The results of Elisa experiment showed that the key target TNF and the upstream synthase of key metabolites SM and Cer were down-regulated after methylprednisolone treatment,and the difference was statistically significant(P<0.05).Conclusions: 1.Methylprednisolone may further affect the metabolism of sphingomyelin by affecting the necrotizing apoptosis pathway by acting on TNF targets,resulting in the decrease of sphingomyelin and ceramide to treat thymoma with myasthenia gravis.2.Methylprednisolone not only plays a therapeutic role,but also affects lipid metabolism and glucose metabolism,resulting in side effects,which is consistent with previous studies and clinical manifestations.更多还原