Table_2_An Integrative Pharmacology-Based Analysis of Refined Qingkailing Injection Against Cerebral Ischemic Stroke: A Novel Combination of Baicalin, Geniposide, Cholic Acid, and Hyodeoxycholic Acid.xlsx

Stroke is the second leading cause of death after heart disease globally and cerebral ischemic stroke accounts for approximately 70% of all incident stroke cases. We selected four main compounds from a patent Chinese medicine, Qingkailing (QKL) injection, including baicalin from Scutellaria baicalensis Georgi (Huang Qin), geniposide from Gardenia jasminoides J. Ellis (Zhizi), and cholic acid and hyodeoxycholic acid from Bovis Calculus (Niuhuang) with a ratio of 4.4:0.4:3:2.6 m/m, to develop a more efficacious and safer modern Chinese medicine injection against ischemic stroke, refined QKL (RQKL). In this study, we investigated multiple targets, levels, and pathways of RQKL by using an integrative pharm\acology combining experimental validation approach. In silica study showed that RQKL may regulate PI3K-Akt, estrogen, neurotrophin, HIF-1, MAPK, Hippo, FoxO, TGF-beta, NOD-like receptor, apoptosis, NF-kappa B, Wnt, chemokine, TNF, Toll-like receptor signaling pathways against ischemic stroke. The experimental results showed that RQKL improved neurological function and prevented infract volume and blood-brain-barrier damage. RQKL inhibited microgliosis and astrogliosis, and protected neurons from ischemic/reperfusion injury. RQKL also inhibited cell apoptosis and affecting the ratio of the anti-apoptosis protein B-cell lymphoma-2 (Bcl2) and pro-apoptosis protein Bcl2-associated X protein (Bax). Western blot analysis showed that RQKL activated AKT/PI3K signaling pathway and antibody array showed RQKL inhibited inflammatory response and decreased proinflammatory factor Tnf, Il6, and Il1b, and chemokines Ccl2, Cxcl2, and Cxcl3, and increased anti-inflammatory cytokine Il10. In conclusion, RQKL protected tissue against ischemic stroke through multiple-target, multiple signals, and modulating multiple cell-types in brain. This study not only promoted our understanding of the role of RQKL against ischemic stroke, but also provided a pattern for the study of Chinese medicine combining pharmaceutical Informatics and system biology methods.

中风是全球继心脏病之后的第二大死因，其中脑缺血性中风约占所有突发中风病例的70%。本研究从专利中药清开灵（QKL）注射剂中选取了四种主要成分，包括自黄芩（Huang Qin）Scutellaria baicalensis Georgi中的黄芩苷，自栀子（Zhizi）Gardenia jasminoides J. Ellis中的栀子苷，以及自牛黄（Niuhuang）Bovis Calculus中的胆酸和鹅脱氧胆酸，以4.4:0.4:3:2.6的质量比进行配比，开发了一种更为有效且安全的中药注射剂——精制QKL（RQKL），以应对缺血性中风。在本研究中，我们通过整合药理学方法结合实验验证手段，对RQKL的多重靶点、水平和通路进行了探究。模拟实验表明，RQKL可能调节PI3K-Akt、雌激素、神经营养因子、HIF-1、MAPK、Hippo、FoxO、TGF-β、NOD样受体、凋亡、NF-κB、Wnt、趋化因子、TNF和Toll样受体等信号通路，以对抗缺血性中风。实验结果显示，RQKL能够改善神经功能，预防梗死体积和血脑屏障损伤，抑制小胶质细胞和星形胶质细胞的活化，并保护神经元免受缺血/再灌注损伤。RQKL还抑制细胞凋亡，并影响抗凋亡蛋白B细胞淋巴瘤-2（Bcl2）与促凋亡蛋白Bcl2相关X蛋白（Bax）的比例。Western印迹分析显示，RQKL激活了AKT/PI3K信号通路，而抗体芯片显示RQKL抑制了炎症反应，并降低了促炎因子Tnf、Il6和Il1b，以及趋化因子Ccl2、Cxcl2和Cxcl3的水平，并增加了抗炎细胞因子Il10。总之，RQKL通过多重靶点、多重信号和调节大脑中多种细胞类型，保护组织免受缺血性中风的侵害。本研究不仅深化了我们对RQKL在对抗缺血性中风中作用的认知，也为将药物信息学与系统生物学方法结合研究中药提供了范例。