基于谱效关系探讨肉苁蓉水提物抗糖尿病肾病的有效成分及其作用机制

The effective components and mechanism of Cistanche water extract against diabetic nephropathy based on the spectral-effect relationship

关键词:  肉苁蓉  水提物  UPLC-QE-ORBITRAP -MS/MS  谱效关系  糖尿病肾病  

Key words:  Cistanche tubulosa  water extract  UPLC-QE-Orbtrap-MS/MS  spectral-effect relationship  niabetic nephropathy  

摘要:

目的 基于肉苁蓉水提物不同洗脱部位质谱峰面积与其抗糖尿病肾病(Diabetic nephropathy,DN)作用的谱效关系,筛选出肉苁蓉水提物抗DN的药效物质,探寻其治疗DN的药效物质基础及其作用机制。 方法 应用UPLC-Orbitrap-MS/MS技术定性色谱峰；MTT法检测肉苁蓉水提物不同洗脱部位对高糖高脂HK-2细胞增殖的影响；灰色关联分析和偏最小二乘法分析质谱峰面积与抗DN活性的谱效关系。MTT法测定肉苁蓉水提物单体成分的抗DN活性；生化试剂盒检测过氧化指标SOD和GSH-Px水平；ELISA法检测炎症因子IL-1β、TNF-α和TGF-β水平；Western Blot检测凋亡相关蛋白的表达。 结果 UPLC-Orbitrap-MS/MS技术推测鉴定了72个共有化合物；在肉苁蓉水提物中,水、20％乙醇、40％乙醇洗脱部位对高糖高脂环境中的HK-2细胞增殖率分别为29％、5％和15％；偏最小二乘法和灰色关联分析显示肉苁蓉水提物抗DN贡献较大的成分是8-表去氧马钱子酸、京尼平苷酸、松脂素、甜菜碱、丁香苷等；MTT实验发现8-表去氧马钱子酸、京尼平苷酸对高糖高脂环境中的HK-2细胞增殖率分别为62％和44％；过氧化指标及炎症因子检测结果表明8-表去氧马钱子酸和京尼平苷酸能够上调SOD和GSH-pX活力,同时对炎症因子IL-1β、TNF-α和TGF-β表达水平具有抑制作用；Western Blot结果显示8-表去氧马钱子酸和京尼平苷酸能够下调和上调皮间质转化标志物：α-SMA、Collagen Ⅰ和E-cadherin,并且可以通过抑制PI3K-AKT通路发挥抗DN作用。结论 在肉苁蓉所含众多化学成分中通过抗DN的谱效关系筛选的8-表去氧马钱子酸和京尼平苷酸这两种化合物可以通过抑制PI3K-Akt信号通路来影响氧化应激、炎症反应和肾小管细胞上皮间质转化进程,起到改善肾脏病理、纤维化程度及肾脏功能的作用,为肉苁蓉水提物治疗DN后续的深入研究提供参考。

Abstract:

ABSTRACT：OBJECTIVE Based on the spectral relationship between the mass spectral peak areas of different elution sites of Cistanchis sinensis aqueous extracts and their anti-diabetic nephropathy (DN) effects, the pharmacodynamic substances of Cistanchis sinensis aqueous extracts were screened out and the basis of the pharmacodynamic substances and their mechanism of action in the treatment of DN were explored. METHODS UPLC-Orbitrap-MS/MS technique was used to characterise the chromatographic peaks; MTT method was used to detect the effects of different elution sites of Cistanchis sinensis aqueous extract on the proliferation of high glucose and high fat HK-2 cells; grey correlation analysis and partial least squares method were used to analyse the spectral relationship between mass spectrometry peak area and anti-DN activity; MTT method was used to determine the anti-DN activities of the individual components of Cistanchis sinensis aqueous extract; biochemical kit and ELISA were used to determine the levels of oxidative indicators and inflammatory factors; Western blot was used to detect the expression of apoptosis-related proteins. The levels of peroxidative indicators and inflammatory factors were determined by the MTT method; the expression of apoptosis-related proteins was detected by Western blot. RESULTS UPLC-Orbitrap-MS/MS technique speculated and identified 72 common compounds; among the aqueous extracts of Cistanchis, water, 20% ethanol and 40% ethanol elution sites showed good anti-DN effects; Partial least squares and grey correlation analyses showed that the constituents of the aqueous extracts of Cistanchis with greater anti-DN contributions were 8-epideoxymatricinic acid, kyonipin sapogenins, pinosylvin and betulin, MTT assay showed that 8-epideoxystrychnic acid and genipin sapogenins have good anti-DN activity in vitro; Biochemical kit and ELISA showed that 8-epideoxystrychnic acid and genipin sapogenins were able to up-regulate the activity of SOD and GSH-pX, and at the same time they had an inhibitory effect on the expression of inflammatory factors IL-1β, TNF-α and TGF-β. Western blot results showed that 8-epideoxystrychnic acid was able to inhibit the expression of inflammatory factors IL-1β and TGF-α. Western blot results showed that 8-epideoxystrychnic acid and geniposidic acid were able to down-regulate and up-regulate the markers of dermal mesenchymal transition: α-SMA, collagen Ⅰ and E-cadherin, and they could exert anti-DN effects by inhibiting the PI3K-AKT pathway. CONCLUSION The two compounds, 8-epideoxystrychnic acid and kynurenine glycosides, which were screened by the spectroeffective relationship of anti-DN among the many chemical constituents contained in Cistanchiakis, could affect oxidative stress, inflammation and epithelial-mesenchymal transformation of renal tubular cells by inhibiting the PI3K-Akt signalling pathway, inflammation and epithelial-mesenchymal transformation of renal tubular cells by inhibiting the PI3K-Akt signalling pathway, and improve renal pathology, degree of fibrosis and renal function, which will be useful for the in-depth study of aqueous extracts of Cistanchiakis in the treatment of DN.