Ren Mihong, Li Yong, Yuan Jianmei, Wang Jiajun, Lu Danni, Xu Zhuo, Xie Qian, Ma Rong, Chen Jiannan, Gong Daoyin, Li Jinxiu, Wang Jian
State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu, 611137, PR China; School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, PR China; College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, PR China.
State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu, 611137, PR China; College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, PR China.
J Ethnopharmacol. 2025 Feb 27;342:119390. doi: 10.1016/j.jep.2025.119390. Epub 2025 Jan 27.
Bovis calculus (BC) has a medicinal history of over 2000 years in treating stroke in China. Bovis Culus Sativus (BCS) has similar pharmacological effects to BC. Due to the scarcity of BC, BCS is often used as a substitute for BC in clinical practice for treating stroke in traditional Chinese medicine.
This study aims to investigate the inhibitory effect of BCS on blood-brain barrier (BBB) damage following stroke, and to elucidate the molecular basis of BCS neuroprotection through network pharmacology and bioinformatics.
The contents of bilirubin and bile acids in BCS were quantified using HPLC. A cerebral ischemia-reperfusion injury (CIRI) rat model was established to assess neurological function, cerebral infarction, pathological damage, and Evans Blue staining. R language was used to analyze GEO public data to identify therapeutic targets for ischemic stroke. Public databases and literature were utilized to screen for active components of BCS, and the Swiss Target Prediction database was used to predict the active drug targets. Network pharmacology analysis was conducted on drug and disease targets, followed by immune infiltration and molecular docking of key targets. Finally, ELISA, RT-PCR, Western blot, IHC, and TEM were employed to validate the effectiveness of the targets.
The content of bile acids and bilirubin in the tested BCS was 6.9% and 37.89%, respectively. The study showed that BCS reduced neurological function scores and cerebral infarction rates in stroke rats, prevented Evans Blue leakage, and mitigated histopathological damage in the ischemic brain region. Additionally, BCS improved the structural and functional integrity of the BBB, enhancing the expression of Occludin, ZO-1, and Claudin-5 while downregulating the expression of MDR1, aquaporin-4, MMP-9, and MMP2. Bioinformatics and network pharmacology analyses indicated that the therapeutic effects of BCS in stroke are primarily associated with the inhibition of inflammatory pathways, including TNF, NFKB, and MAPK. ELISA, RT-PCR, and Western blot results further confirmed that BCS significantly suppressed neuroinflammation in stroke rats.
BCS shows promising efficacy against ischemic stroke, maintaining the function and structural integrity of the BBB. Its protective effect on the BBB may be related to the inhibition of the TNF-NFκB-MAPK signaling pathways.
牛黄在中国治疗中风已有2000多年的药用历史。人工牛黄与牛黄具有相似的药理作用。由于天然牛黄稀缺，在中医临床治疗中风中，人工牛黄常被用作天然牛黄的替代品。
本研究旨在探讨人工牛黄对中风后血脑屏障（BBB）损伤的抑制作用，并通过网络药理学和生物信息学阐明人工牛黄神经保护的分子基础。
采用高效液相色谱法（HPLC）测定人工牛黄中胆红素和胆汁酸的含量。建立脑缺血再灌注损伤（CIRI）大鼠模型，评估神经功能、脑梗死、病理损伤及伊文思蓝染色情况。使用R语言分析基因表达综合数据库（GEO）公共数据，以确定缺血性中风的治疗靶点。利用公共数据库和文献筛选人工牛黄的活性成分，并使用瑞士靶点预测数据库预测活性药物靶点。对药物和疾病靶点进行网络药理学分析，随后对关键靶点进行免疫浸润和分子对接。最后，采用酶联免疫吸附测定（ELISA）、逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western blot）、免疫组织化学（IHC）和透射电子显微镜（TEM）验证靶点的有效性。
受试人工牛黄中胆汁酸和胆红素的含量分别为6.9%和37.89%。研究表明，人工牛黄可降低中风大鼠的神经功能评分和脑梗死率，防止伊文思蓝渗漏，并减轻缺血脑区的组织病理学损伤。此外，人工牛黄改善了血脑屏障的结构和功能完整性，增加紧密连接蛋白（Occludin）、闭合蛋白（ZO-1）和Claudin-5的表达，同时下调多药耐药蛋白1（MDR1）、水通道蛋白4（aquaporin-4）、基质金属蛋白酶9（MMP-9）和基质金属蛋白酶2（MMP2）的表达。生物信息学和网络药理学分析表明，人工牛黄治疗中风的作用主要与抑制包括肿瘤坏死因子（TNF）、核因子κB（NFKB）和丝裂原活化蛋白激酶（MAPK）在内的炎症信号通路有关。ELISA、RT-PCR和Western blot结果进一步证实，人工牛黄可显著抑制中风大鼠的神经炎症。
人工牛黄对缺血性中风显示出良好的疗效，可维持血脑屏障的功能和结构完整性。其对血脑屏障的保护作用可能与抑制TNF-NFκB-MAPK信号通路有关。
