目的 探究蒙药扎冲十三味丸（ZC13）抗血管性痴呆（VaD）的作用机制。方法 通过完全随机分组法将大鼠分为假手术组、模型组、多奈哌齐（Don，阳性药，0.45 mg·kg^-1）组和ZC13高、低剂量（0.324、0.162 g·kg^-1）组，利用双侧颈总动脉结扎术构建血管性痴呆大鼠模型，假手术组仅分离双侧颈总动脉不结扎，于造模完成24 h首次ig给药，每天1次，连续28 d。采用Morris水迷宫实验考察大鼠的学习和记忆能力；伊文思蓝（EB）染色评估大鼠血脑屏障（BBB）通透性；尼氏（Nissl）染色及苏木精-伊红（HE）染色检测脑组织病理损伤；转录组学分析结合基因本体（GO）功能和京都基因与基因组百科全书（KEGG）富集揭示ZC13可能调控的关键信号通路。并采用Western blotting对其中的核心靶点进行实验验证，ELISA检测通路下游炎症因子水平，免疫组化检测细胞凋亡水平。结果 与假手术组比较，模型组大鼠游泳路径紊乱，穿越平台次数减少（P＜0.01），平均游泳速度下降（P＜0.001），目标象限停留时间显著缩短（P＜0.001），逃避潜伏期也明显延长；神经细胞明显受损，BBB通透性显著增加，胞质紧密黏连蛋白1（ZO-1）、闭合蛋白（Occludin）、紧密连接蛋白-1（Claudin-1）表达显著下降（P＜0.001），尼氏体数量大幅下降（P＜0.01）；差异基因（DEGs）功能主要集中在少突胶质细胞发育，氧化还原酶活性等方面，主要富集的通路途径包括丝裂原活化蛋白激酶（MAPK）、核因子（NF）-κB、白细胞介素（IL）-17等信号通路；肿瘤坏死因子受体相关因子6（TRAF6）、环氧化酶-2（COX-2）、核因子κB激活剂1（ACT1）、c-Jun氨基末端激酶（JNK）、c-Jun、p38、IκBα及p65等蛋白表达水平显著上调（P＜0.001）； IL-1β、TNF-α、IL-6、IL-8及IL-17促炎因子水平也显著升高（P＜0.001）； Bax蛋白表达水平显著升高（P＜0.001），Bcl-2蛋白表达水平显著降低（P＜0.001）。与模型组相比，不同剂量ZC13干预后，大鼠的学习记忆能力得不同程度的改善；神经元损伤减轻，BBB通透性下降； DEGs功能主要富集在T细胞活化的正调控、对Ⅱ型干扰素的应答等方面，主要富集的通路途径包括MAPK、NF-κB等信号通路；TRAF6、COX-2、ACT1、JNK、c-Jun、p38、IκBα及p65等蛋白表达水平显著下调（P＜0.001）； IL-1β、TNF-α、IL-6、IL-8、IL-17促炎因子水平也显著下降（P＜0.001）； Bax蛋白表达水平显著降低（P＜0.01、0.001），Bcl-2蛋白表达水平显著升高（P＜0.001）。结论 ZC13能够有效抑制VaD发生与发展进程中神经炎症反应及神经元凋亡，其作用机制可能与负向调控IL-17/NF-κB/MAPK信号通路，下调促炎因子水平，调控Bax/Bcl-2表达有关。

Objective To investigate the therapeutic effects and underlying molecular mechanisms of Zhachong 13 Pills (ZC13) against vascular dementia (VaD). Methods Rats were divided into a sham operation group, a model group, a donepezil (Don, positive control, 0.45 mg·kg^-1) group, and ZC13 high and low dose (0.324, 0.162 g·kg^-1) groups using a completely randomized grouping method. A vascular dementia rat model was established by bilateral common carotid artery ligation. The sham operation group only underwent separation of bilateral common carotid arteries without ligation. The first ig administration was performed 24 hours after the completion of model establishment, once daily for 28 consecutive days. A rat model of vascular dementia was generated by bilateral common carotid artery occlusion. Cognitive performance was assessed using the Morris water maze test. Blood-brain barrier integrity was evaluated by Evans blue extravasation, and histopathological changes in brain tissues were analyzed by Nissl and HE staining. Transcriptomic profiling of rat hippocampal tissue was conducted using RNA sequencing to screen differentially expressed genes, followed by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses to explore signaling pathways potentially regulated by ZC13. Representative molecules within these pathways were subsequently examined by Western blotting and enzyme-linked immunosorbent assay. Results Compared with the sham operation group, the model group exhibited disordered swimming paths, reduced number of platform crossings (P<0.01), decreased average swimming speed (P<0.001), significantly shortened time spent in the target quadrant (P<0.001), and markedly prolonged escape latency. Neuronal damage was evident, with significantly increased blood-brain barrier (BBB) permeability and notably reduced expression of cytoplasmic tight junction proteins including zonula occludens-1 (ZO-1), occludin, and claudin-1 (P<0.001), as well as a substantial decrease in Nissl body count (P<0.01). The functions of differentially expressed genes (DEGs) were primarily associated with oligodendrocyte development and redox enzyme activity, with enriched pathways including mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB, and interleukin (IL)-17 signaling pathways. Protein levels of tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), NF-κB activator 1 (ACT1), c-Jun N-terminal kinase (JNK), c-Jun, p38, IκBα, and p65 were significantly upregulated (P<0.001). Levels of pro-inflammatory cytokines IL^-1β, TNF-α, IL-6, IL-8, and IL^-17 were also significantly elevated (P<0.001). Bax protein expression was significantly increased (P<0.001), while Bcl-2 protein expression was significantly decreased (P<0.001). Compared to the model group, rats treated with different doses of ZC13 showed varying degrees of improvement in learning and memory abilities; neuronal injury was alleviated and BBB permeability decreased. DEG functions were mainly enriched in positive regulation of T-cell activation and response to type II interferons, with key pathways including MAPK and NF-κB signaling. Expression levels of TRAF6, COX-2, ACT1, JNK, c-Jun, p38, IκBα, and p65 were significantly downregulated (P<0.001). Proinflammatory cytokine levels of IL^-1β, TNF-α, IL-6, IL-8, and IL^-17 were also significantly reduced (P<0.001). Bax protein expression was significantly decreased (P<0.01, 0.001), whereas Bcl-2 protein expression was significantly increased (P<0.001). Conclusion ZC13 negatively regulates the IL^-17/NF-κB/MAPK signaling pathways by modulating key signaling molecules, including TRAF6, COX-2, ACT1, JNK, c-Jun, p38, IκBα, and p65, thereby alleviating neuroinflammation and intervening in the progression of vascular dementia.

R965

内蒙古自治区高等学校科学研究项目（NJZY21054，NJZY20177）