Alterations of Brain Quantitative Proteomics Profiling Revealed the Molecular Mechanisms of Diosgenin against Cerebral Ischemia Reperfusion Effects

Diosgenin (DIO), the starting material for the synthesis of steroidal anti-inflammatory drugs in the pharmaceutical industry, has been previously demonstrated to display pharmaceutical effects against cerebral ischemic reperfusion (I/R). However, the alterations of brain proteome profiles underlying this treatment remain elusive. In the present study, the proteomics analysis of the brain tissues from I/R rats after DIO treatment was performed using an integrated TMT-based quantitative proteomic approach coupled with the liquid chromatography with tandem mass spectrometry technology. A total of 5043 proteins (ProteomeXchange identifier: PXD016303) were identified, of which 58 common differentially expressed proteins were significantly dysregulated in comparison between sham versus I/R and I/R versus DIO. The eight validated proteins including EPG5, STAT2, CPT1A, EIF2AK2, GGCT, HIKESHI, TNFAIP8, and EMC6 by quantitative polymerase chain reaction and western blotting consistently supported the TMT-based proteomic results, which were mainly associated with autophagy and inflammation response. Considering the anti-inflammatory characters of DIO, the biological functions of STAT2 and HIKESHI that are the probable direct anti-inflammatory targets were further investigated during the course of I/R treated with DIO. In addition, the combination of verified STAT2 and HIKESHI in peripheral blood samples from stroke patients resulted in the area under the curve value of 0.765 with P < 0.004 to distinguish stroke patients from healthy controls. Taken together, the current findings first mapped comprehensive proteomic changes after I/R was treated with DIO to better decipher the molecular mechanisms mainly based on the anti-inflammatory aspect underlying this therapeutic effect, providing a foundation for developing potentially therapeutic targets of anti-I/R of DIO and clinically prognostic biomarkers of stroke.薯蓣皂苷元（Diosgenin, DIO）是制药工业中合成甾体类抗炎药物的起始原料，既往研究已证实其可发挥抗脑缺血再灌注（cerebral ischemic reperfusion, I/R）损伤的药理活性。然而，该药物发挥治疗作用背后的脑蛋白质组谱变化仍有待阐明。本研究采用基于串联质量标记（Tandem Mass Tag, TMT）的定量蛋白质组学策略，结合液相色谱-串联质谱（liquid chromatography with tandem mass spectrometry, LC-MS/MS）技术，对脑缺血再灌注模型大鼠经薯蓣皂苷元处理后的脑组织进行蛋白质组学分析。本研究共鉴定到5043种蛋白质（ProteomeXchange数据库收录编号：PXD016303）；在假手术组与脑缺血再灌注组、脑缺血再灌注组与薯蓣皂苷元处理组的对比分析中，共有58种共同差异表达蛋白出现显著表达失调。通过实时定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）与蛋白质免疫印迹（Western Blotting, WB）验证的8种蛋白质（包括EPG5、STAT2、CPT1A、EIF2AK2、GGCT、HIKESHI、TNFAIP8及EMC6）的表达趋势与基于TMT的蛋白质组学结果高度一致，这些蛋白主要参与自噬与炎症应答过程。鉴于薯蓣皂苷元的抗炎特性，本研究进一步探讨了STAT2与HIKESHI的生物学功能——二者被推测为薯蓣皂苷元抗脑缺血再灌注损伤的直接抗炎靶点。此外，在脑卒中患者外周血样本中验证的STAT2与HIKESHI的联合检测模型，其区分脑卒中患者与健康对照受试者的曲线下面积（AUC）值达0.765（P<0.004）。综上，本研究首次绘制了薯蓣皂苷元干预脑缺血再灌注损伤后的全脑蛋白质组变化图谱，以更好地解析该治疗作用背后主要基于抗炎通路的分子机制，为开发薯蓣皂苷元抗脑缺血再灌注损伤的潜在治疗靶点以及脑卒中临床预后生物标志物提供了坚实的理论基础。