卒中是一种高发病率，高死亡率，高致残率，高复发率疾病，而缺血性卒中 主要占卒中病例的 60%-80%。缺血性中风的特点是同时存在缺血性和再灌注相 关的脑伤，导致神经元功能障碍和死亡。脑缺血再灌注损伤（Cerebral ischemicreperfusion injury，CIRI）是一种复杂的病理生理过程，会导致神经细胞通过释放 信号激活神经胶质活化、外周免疫细胞浸润以及多种炎性介质的释放等免疫反应， 并且 CIRI 导致的神经炎症会加速血脑屏障破坏、加重脑水肿并导致微循环障碍， 从而造成继发性脑损伤。鉴于脑缺血再灌注损伤病理机制复杂，单成分、单靶点 的干预药物往往难以取得满意效果。而在中医药强调的“整体观”的指导下，以 中药防治脑缺血再灌注损伤具有“多成分、多靶点、多效应”的优势。

龙血竭是百合科剑叶龙血树 Dracaena cochinchinensis (Lour.) S. C. Chen 的含 脂木材经提取得到的干燥树脂，可以活血祛瘀、软坚散结。现代药理研究表明其具有抗血小板聚集、提高造血能力、抗炎镇痛、抗氧化、疏通并软化血管等能力， 在卒中治疗上具有良好的效果。然而有研究表明龙血竭的生物利用度及脑组织靶向性较低，所以本研究拟用具有引药上行特点的冰片与龙血竭联用来减轻 CIRI， 以期阐明其药效物质基础及作用机制，为缺血性脑卒中预防策略提供新药研发思路。

通过 TTC 染色、HE 染色、ELISA、免疫荧光、蛋白印迹分析、脑血流实时 成像等药理学实验验证，本研究首次证实了与单独使用龙血竭相比，龙血竭与冰 片联用对 CIRI 的预防作用更好。经龙血竭冰片联合治疗后，tMCAO 大鼠运动神 经功能明显改善，脑组织梗死体积减少，氧化应激和炎症反应减轻，局部脑血流 量明显恢复，神经元坏死及凋亡丢失得到明显抑制。

基于转录组学分析，本研究发现龙血竭冰片联用预防 CIRI 主要与炎症反应 及免疫应答等生物过程有关，涉及到细胞因子-细胞因子受体相互作用、IL-17 信号通路、神经活性配体-受体相互作用、趋化因子信号通路和 TNF-ɑ 信号通路等 相关机制的变化。最后，本研究进一步通过蛋白印迹分析证实了龙血竭冰片联用 通过 IL-17 通路拮抗 CIRI。

基于 LC-MS 技术对大鼠血清及脑脊液代谢组学进行分析。初步探究出龙血 竭冰片联用预防 CIRI 的潜在血清生物标志物可能包括肌酸、3-吲哚丙酸、溶血 磷脂酰胆碱（15：0）、溶血磷脂酰胆碱（18：2）、溶血磷脂酰胆碱（18：0）、 溶血磷脂酰胆碱(18:3)、溶血磷脂酰乙醇胺（22：4）、溶血磷脂酰乙醇胺（18： 1）、溶血磷脂酰乙醇胺（18：0）、溶血磷脂酰乙醇胺（20：0）、溶血磷脂酰乙 醇胺（20：1）、十四碳酸、γ-亚麻酸、棕榈油酸、十五烷酸、1-十七烷酰甘油磷 脂酰乙醇胺、N-(2-呋喃基)甘氨酸、胞苷、花生四烯酸、11.14.17-二十碳三烯酸、油酸、棕榈酸、十七烷酸、阿糖胞苷、硬脂酸。本研究基于非靶向代谢组学技术 探究了龙血竭冰片联用保护 CIRI 的脑脊液差异代谢物。在脑脊液中，L-亮氨酰L-丙氨酸、二十六烷酸、γ-谷氨酰半胱氨酸、13-二十二碳烯胺、N-甲酰甲硫氨酸、 尿苷二磷酸半乳糖、磷脂酰丝氨酸(18:0/18:0)和苜蓿酸 3-O-葡萄糖醛酸苷可能是 龙血竭冰片联用预防 CIRI 的潜在生物标志物。

通过 LC-MS 对大鼠血浆及脑脊液中的外源性成分进行分析。结果显示，与 单用龙血竭相比，在龙血竭冰片联用的情况下，大鼠血浆中得到差异显著的 155 个原型成分及 568 个转化成分；大鼠脑脊液中得到差异显著的 73 个原型成分及 127 个转化成分。这些结果说明，冰片可以提高龙血竭的生物利用度及脑组织靶 向性，进而能起到协同增效的作用，提高对 CIRI 的预防作用。

本研究通过药效学评价结合转录组学、代谢组学及血清药物化学等分析，为 寻找新的靶向治疗药物开发提供理论依据，并期望推动中药复方的质量提升和组 成的二次开发从而拓展龙血竭的临床应用，促进龙血竭产业的健康发展。

Stroke is characterized by high morbidity and mortality rates leading to long-term disability. The middle cerebral artery accounts for 60%–80% of stroke cases. Ischemic stroke is characterized by neuronal dysfunction and death as a result of both ischemic and reperfusion injury to the brain. As a complex pathophysiological process, cerebral ischemic-reperfusion injury (CIRI) results are associated with targeting excitotoxicity, calcium overload, oxidative stress, autophagy, inflammation, apoptosis, and bloodbrain barrier damage, resulting in secondary brain damage. Because of the complex pathological mechanism of CIRI, single-component and single-target intervention drugs are often difficult to achieve satisfactory results. Under the guidance of "holism", the prevention and treatment of CIRI with traditional Chinese medicine has the advantage of "multi-component, multi-target and multi-effect".

 Dragon’s Blood (DB), the red resin obtained from Dracaena cochinchinensis (Lour.) Chen has been shown to have a significant role in the clinical therapy of blood stasis in China. Previous studies have demonstrated that Dragon’s blood has the potential to inhibit platelet aggregation, attenuate apoptosis, exert anti-inflammatory and antioxidant effects, reduce microglial activation, and inhibit atherosclerosis. It has a good effect on the treatment of stroke. Because of the low bioavailability and brain targeting of Dragon’s Blood, this study intends to use Borneol, which has the characteristics of guiding drugs to target organs, to reduce CIRI in combination with Dragon’s Blood, to clarify its pharmacodynamic material basis and mechanism, and provide new ideas for ischemic stroke prevention strategies.

 This study has found for the first time that the combination of Dragon’s Blood and Borneol enhanced the efficacy of CIRI compared to Dragon’s Blood or Borneol alone through pharmacological experiments such as TTC staining, HE staining, ELISA, immunofluorescence staining, Western blot, and laser speckle contrast imaging. After the combination of Dragon’s Blood and Borneol treatment, motor nerve function was significantly improved, infarct area and neuronal necrosis and loss were significantly reduced, and regional cerebral blood flow significantly improved.

Through RNA-seq analysis, it has been found that the combination of Dragon’s Blood and Borneol is mainly concentrated in the biological processes of inflammatory response and immune response, in terms of cytokine-cytokine receptor interaction, hematopoietic cell lineage, neuroactive ligand-receptor interaction, cell adhesion molecules, chemokine signaling pathway, TNF signaling pathway, NF-κB signaling pathway, and IL-17 signaling pathway. Finally, this study has analyzed that the combination of Dragon’s Blood and Borneol can antagonize CIRI through the IL-17 pathway based on Western blot.

Serum and cerebrospinal fluid metabolites were assessed by LC-MS. This study has found that 24 potential biomarkers in serum were significantly regulated by the combination of Dragon’s Blood and Borneol, including creatine, 3-indole propionic acid, lysoPC (15:0), lysoPC (18:2), , lysoPC (18:0), tetradecanoic acid,γ-linolenic acid, palmitoleic acid, lysoPE(18:1), pentadecanoic acid, 1-heptadecanoylglycerophosphoethanolamine, lysoPC (18:3), , N-(2-Furoyl)glycine, cytidine, lysoPC (22:4), arachidonic acid, cytarabine, lysoPE (18:0), 11.14.17-eicosatrienoic acid, oleic acid, palmitic acid, lysoPE (20:1), heptadecanoic acid, lysoPE (20:0). And the potential biomarkers in cerebrospinal fluid including eucylalanine, hexacosanoic acid, γglutamylcysteine, 13-docosenamide, N-formyl-L-methionine, uridine diphosphategalactose, phosphatidylserine(18:0/18:0) and medicagenic acid 3-O-b-D-glucuronide.

 The compounds of Dragon’s Blood in plasma and cerebrospinal fluid were analyzed by the means of LC-MS. The result has showed that 155 prototypes and 568 metabolites of Dragon’s Blood were identified in rat plasma, and 73 prototypes and 127 metabolites in cerebrospinal fluid in the the combination of Dragon’s Blood and Borneol group, compared to the Dragon’s Blood group. This result has indicated the role of Borneol in promoting Dragon’s Blood bioavailability and brain tissue targeting. Thus, the combination of Dragon’s Blood plays a synergistic role in improving the prevention of CIRI.

Through pharmacological experiments, RNA-seq, metabonomics, and serum pharmacochemistry analysis, this study provides a theoretical basis for finding new targeted therapeutic drugs to promote the quality improvement and secondary development of Traditional Chinese Medicine compound prescription. This study maybe expand the clinical application of Dragon’s Blood and promote the healthy development of Dragon’s Blood industry.