Hou Jiebin, Chen Wei, Lu Hongtao, Zhao Hongxia, Gao Songyan, Liu Wenrui, Dong Xin, Guo Zhiyong
Department of Nephrology, Changhai Hospital, Second Military Medical University, Shanghai, China.
Department of Naval Aeromedicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, China.
Front Pharmacol. 2018 Jun 13;9:620. doi: 10.3389/fphar.2018.00620. eCollection 2018.
As a Chinese medicinal herb, (Osb.) Merr (DS) has been applied clinically to alleviate crystal-induced kidney injuries, but its effective components and their specific mechanisms still need further exploration. This research first combined the methods of network pharmacology and proteomics to explore the therapeutic protein targets of DS on oxalate crystal-induced kidney injuries to provide a reference for relevant clinical use. Oxalate-induced kidney injury mouse, rat, and HK-2 cell models were established. Proteins differentially expressed between the oxalate and control groups were respectively screened using iTRAQ combined with MALDI-TOF-MS. The common differential proteins of the three models were further analyzed by molecular docking with DS compounds to acquire differential targets. The inverse docking targets of DS were predicted through the platform of PharmMapper. The protein-protein interaction (PPI) relationship between the inverse docking targets and the differential proteins was established by STRING. Potential targets were further validated by western blot based on a mouse model with DS treatment. The effects of constituent compounds, including luteolin, apigenin, and genistein, were investigated based on an oxalate-stimulated HK-2 cell model. Thirty-six common differentially expressed proteins were identified by proteomic analysis. According to previous research, the 3D structures of 15 major constituents of DS were acquired. Nineteen differential targets, including cathepsin D (CTSD), were found using molecular docking, and the component-differential target network was established. Inverse-docking targets including p38 MAPK and CDK-2 were found, and the network of component-reverse docking target was established. Through PPI analysis, 17 inverse-docking targets were linked to differential proteins. The combined network of component-inverse docking target-differential proteins was then constructed. The expressions of CTSD, p-p38 MAPK, and p-CDK-2 were shown to be increased in the oxalate group and decreased in kidney tissue by the DS treatment. Luteolin, apigenin, and genistein could protect oxalate-stimulated tubular cells as active components of DS. The potential targets including the CTSD, p38 MAPK, and CDK2 of DS in oxalate-induced kidney injuries and the active components (luteolin, apigenin, and genistein) of DS were successfully identified in this study by combining proteomics analysis, network pharmacology prediction, and experimental validation.
作为一种中草药，豆科植物云实（Caesalpinia decapetala (Roth) Alston (DS)）已在临床上用于减轻晶体诱导的肾损伤，但其有效成分及其具体机制仍需进一步探索。本研究首次结合网络药理学和蛋白质组学方法，探索云实对草酸晶体诱导的肾损伤的治疗性蛋白质靶点，为相关临床应用提供参考。建立了草酸诱导的肾损伤小鼠、大鼠和HK-2细胞模型。使用iTRAQ结合MALDI-TOF-MS分别筛选草酸组和对照组之间差异表达的蛋白质。通过与云实化合物的分子对接进一步分析三种模型的共同差异蛋白质，以获得差异靶点。通过PharmMapper平台预测云实的反向对接靶点。利用STRING建立反向对接靶点与差异蛋白质之间的蛋白质-蛋白质相互作用（PPI）关系。基于云实治疗的小鼠模型，通过蛋白质印迹进一步验证潜在靶点。基于草酸刺激的HK-2细胞模型研究了木犀草素、芹菜素和染料木黄酮等成分化合物的作用。通过蛋白质组学分析鉴定出36种共同差异表达的蛋白质。根据先前的研究，获得了云实15种主要成分的三维结构。通过分子对接发现了包括组织蛋白酶D（CTSD）在内的19个差异靶点，并建立了成分-差异靶点网络。发现了包括p38丝裂原活化蛋白激酶（p38 MAPK）和细胞周期蛋白依赖性激酶2（CDK-2）在内的反向对接靶点，并建立了成分-反向对接靶点网络。通过PPI分析，17个反向对接靶点与差异蛋白质相连。然后构建了成分-反向对接靶点-差异蛋白质的联合网络。结果显示，草酸组中CTSD、磷酸化p38 MAPK（p-p38 MAPK）和磷酸化CDK-2（p-CDK-2）的表达增加，而云实治疗后肾组织中的表达降低。木犀草素、芹菜素和染料木黄酮作为云实的活性成分，可以保护草酸刺激的肾小管细胞。本研究通过结合蛋白质组学分析、网络药理学预测和实验验证，成功鉴定了云实在草酸诱导的肾损伤中的潜在靶点，包括CTSD、p38 MAPK和CDK2，以及云实的活性成分（木犀草素、芹菜素和染料木黄酮）。
