Anhui Province Key Laboratory of Chinese Medicinal Formula, Anhui University of Chinese Medicine, Hefei 230012, PR China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, PR China; Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Traditional Chinese Medicine, Hefei 230012, PR China.
Anhui Province Key Laboratory of Chinese Medicinal Formula, Anhui University of Chinese Medicine, Hefei 230012, PR China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, PR China; Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Traditional Chinese Medicine, Hefei 230012, PR China.
Phytomedicine. 2022 Nov;106:154437. doi: 10.1016/j.phymed.2022.154437. Epub 2022 Sep 7.
Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear.
Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury.
Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively.
HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax.
By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.
活血化瘀是中风治疗的关键环节，通窍活血汤（TQHXD）方常用于此。然而，TQHXD 对脑缺血再灌注（I/R）损伤的保护作用机制尚不清楚。
鉴定 TQHXD 中负责其保护作用的成分，并确定其对脑 I/R 损伤的作用模式。
采用气相色谱（GC）和高效液相色谱（HPLC）法测定 TQHXD 的活性成分。在 TCMSP 和 HERB 数据库中查找 TQHXD 的活性成分和靶点；在 Genecards、OMIM、TTD 和 DrugBank 数据库中查找与脑梗死相关的靶点，并获取交集靶点。利用 Cytoscape 3.7.1 和 STRING 网站构建药物-成分-靶点-疾病网络和 PPI 网络。使用 Autodock VINA 对 HPLC 和 GC 检测到的 TQHXD 核心靶点 ASK1 与活性成分进行分子对接。成功建立大脑中动脉闭塞（MCAO）大鼠模型后，采用三苯基四氮唑（TUNEL）-NeuN 染色和透射电镜（TEM）定量海马区细胞凋亡。RT-qPCR 和 Western blot 分别用于检测蛋白和 mRNA 的表达。
HPLC 和 GC 共鉴定出 6 种活性成分。网络药理学分析共检测到 66 个交集靶点，包括 ASK1、MKK4 和 JNK。在分子对接研究中，阿魏酸、HPLC、藁本内酯、芍药苷和麝香均与 ASK1 显示出高结合亲和力。TQHXD 在 I/R 大鼠实验模型中具有神经保护作用。与模型组相比，TQHXD 降低了细胞凋亡率，降低了 p-ASK1、caspase 3、p-MKK4、CytC、p-c-Jun、Bax/Bcl-2 和 p-JNK 的蛋白水平，同时显著增加了 Bcl-2 的 mRNA 水平，降低了 Bax 的 mRNA 水平。
TQHXD 通过调控 ASK1/MKK4/JNK 通路，保护神经元免受 I/R 损伤，防止细胞凋亡。因此，TQHXD 可能对缺血性中风的治疗有效。本研究为 TQHXD 的这些治疗作用机制提供了依据。
