知母HPLC-UVD-ELSD指纹图谱的建立与多特征成分含量测定的质控方法研究

目的建立知母指纹图谱结合多特征成分含量测定的HPLC-UVD-ELSD分析方法,并应用于多批次市售药材的质量控制。方法采用UltimateTMXB-C18色谱柱(4.6 mm×250 mm,4.5μm),检测波长258 nm,ELSD漂移管温度65℃,载气流速1.0 L/min,建立知母指纹图谱与主含黄酮及皂苷类成分含量测定的HPLC-UVD-ELSD分析方法,运用相似度软件结合化学计量学分析对多批次药材进行质量评价并同时完成多特征成分的含量测定。结果建立了高效、快捷的知母HPLC-UVD-ELSD指纹图谱与多特征成分含量测定的方法;18批市售药材的指纹图谱相似度在0.786~0.999,并指认了10个色谱峰。化学计量学结果表明造成18批药材差异较大的成分为新芒果苷、知母皂苷BⅡ和知母皂苷BⅢ;进而建立了同时测定新芒果苷、芒果苷、知母皂苷N、知母皂苷BⅡ和知母皂苷BⅢ5个成分含量的方法,结果表明知母中主含知母皂苷BⅡ。结论建立的指纹图谱结合多特征成分含量测定的HPLC-UVD-ELSD分析方法,更全面而准确地描述了知母药材化学成分整体轮廓与各类特征成分含量贡献,为提升市售知母药材的质量控制方法奠定了基础。

Glutathione peroxidase 4 deficit-mediated fer⁃roptosis contributes to dopaminergic neuron degeneration under synucleinopathy

OBJECTIVE Intracellular aggre⁃gation ofα-synuclein(SNCA)is one of the core pathological features of neurodegenerative disor⁃ders including Parkinson disease,whilst the detailed mechanism for consequently neuron loss has not been fully illustrated.Since the altered phospholipid homeostasis has been suggested to play a role in synucleinopathy,this study aims to depict the fully-featured status of phospholip⁃ids and the targets reposingα-synuclein-related neurotoxicity.METHODS SNCAA53T transgenic mice were utilized as the model of Parkinson disease.Behavioral tests including pole test,rotarod test and gait analysis were conducted to assess the motor features of Parkinsonism.Tyro⁃sine hydroxylase were determined by immunohis⁃tochemistry.Glutathione,dopamine,3,4-dihy⁃droxyphenylacetic acid and homovanillic acid were determined by HPLC-ECD analysis.Assess⁃ment of lipid peroxidation included MDA assay and Liperfluo staining.Phospholipid-omics was analyzed based on LC-MS/MS.Investigation on mechanism was relied on Western blotting and qPCR assay.The injection of AAV into midbrain was achieved by ultra-micro injection pump to obtain the target genotype.RESULTS SNCAA53T transgenic mice displayed progres⁃sively deteriorated motor coordination functions and the mechanisms were related with lipid per⁃oxidation and ferroptosis,which might help to explain the parkinsonism.These hydroperoxides were observed on plasm membrane and were further characterized by LC-MS/MS-based phos⁃pholipid-omics analysis.α-synucleinA53T trans⁃genic mice displayed distinct patterns of phos⁃pholipid peroxidation in midbrain regions com⁃pared to wild type littermates.Among different subtypes of oxidized phospholipids,oxidative phosphatidylcholine(PC-ox)was more promi⁃nently elevated.Phospholipid peroxidation is believed as a biomarker of ferroptosis,which is largely a specialized death program caused by insufficiency of glutathione peroxidase-4(GPX4),the only known enzyme that can reduce lipid hydroperoxides within biological membranes.The deficiency of Gpx4 was demonstrated to be responsible forα-synuclein-induced lipid peroxi⁃dation,and the cell lines and mouse models underwent genetic Gpx4 downregulation showed exacerbated dopaminergic neuron loss and par⁃kinsonism.On the other hand,the potentiation of Gpx4 expression remarkably inhibited dopami⁃nergic ferroptotic death and behavioral deficits in a mouse model with synucleinopathy.CONCLU⁃SION A cellular pathway that Gpx4 deficit-medi⁃ated phospholipid peroxidation and behavioral consequence participated in synucleinopathy,suggesting a potential strategy targeting Gpx4 to alleviate PD symptoms.

2种不同药性中药激活潜伏HSV-1引起“上火”的对比研究

中医理论认为“上火”属于阴阳失衡后的综合临床症状群,已有大量临床实践证明服用温热药性中药可引起“上火”,但是其生物学机制和物质基础有待进一步阐明。该研究利用课题组前期已建立的情志应激诱导潜伏Ⅰ型单纯疱疹病毒(HSV-1)复发的“上火”小鼠模型,以平性中药枸杞子Lycii Fructus和温热性中药人参Ginseng Radix et Rhizoma为研究对象,对比和探讨不同药性中药是否会激活HSV-1诱发“上火”,并探讨相关物质基础。结果显示,与枸杞子不同,人参给药14 d能诱导小鼠HSV-1再激活呈现“上火”症状。进一步,该研究制备了人参不同流分包括多糖富集部位(GS1)、水溶性皂苷富集部位(GS2)和非水溶性皂苷富集部位(GS3),并制备了枸杞子不同富集部位枸杞多糖富集部位(LF1)和枸杞多酚富集部位(LF2)。结果发现,与枸杞子给药结果一致,枸杞子2种富集部位LF1和LF2也不能诱发潜伏HSV-1再复发;但是,人参皂苷富集部位GS2和GS3均可引起HSV-1再激活,其中GS3组病毒激活率为100%,由此推测人参引起HSV-1复发可能与其中所含的皂苷成分有关。该研究从HSV-1潜伏再激活角度解析了药源性“上火”的科学内涵,为温热性中药的合理使用提供依据。

磷脂过氧化:神经退行性疾病“易感”的关键因素

大脑的生化完整性对于维持大脑的正常运转十分重要,氧化损伤是导致大脑生化完整性破坏的重要原因之一。大脑富含磷脂和多不饱和脂肪酸,由于高脂质和高耗氧量的特殊性,大脑比其他器官更容易受到氧化应激的影响,氧化应激诱导的磷脂过氧化是大脑的氧化-抗氧化系统失衡的结果。当抗氧化系统不足以抵御氧化损伤时,神经元膜磷脂易受自由基活性氧的攻击发生磷脂过氧化,进而生成多种毒性氧化产物、细胞膜损伤、线粒体障碍和淀粉样蛋白的急剧累积,且神经元的大多数蛋白和核酸可以被过氧化产物共价修饰,导致蛋白原有功能的丧失,进而引发大脑程序性细胞死亡和神经炎症,最终导致神经退行性疾病的易感性的增加。本文在总结磷脂过氧化机制的基础上,重点综述了磷脂过氧化作为关键因素在神经退行性疾病的发生发展中的特点,为靶向干预磷脂过氧化用于预防神经退行性疾病的潜在策略提供理论依据。