College of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, People's Republic of China.
College of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, People's Republic of China; Maoming Maternal and Child Health Hospital, Maoming, Guangdong, 525000, People's Republic of China.
J Ethnopharmacol. 2024 Dec 5;335:118636. doi: 10.1016/j.jep.2024.118636. Epub 2024 Jul 30.
Ischemic stroke is an acute central nervous system disease that poses a threat to human health. It induces a series of severe pathological mechanisms, ultimately leading to neuronal cell death in the brain due to local ischemia and hypoxia. Buyang Huanwu decoction (BYHWD), as a representative formula for treating ischemic stroke, has shown good therapeutic effects in stroke patients.
This study aimed to explore the mechanism of BYHWD in promoting neural remodeling after ischemic stroke from the perspective of neuronal synaptic plasticity, based on the cAMP/PKA/CREB signaling pathway.
A modified suture technique was employed to establish a rat model of MCAO. The rats were divided into sham, model, and BYHWD (20 g/kg) groups. After the corresponding intervention, rat brains from each group were collected. TMT quantitative proteomics technology was employed for the research. Following proteomics studies, we investigated the mechanism of BYHWD in the intervention of ischemic stroke through animal experiments and cell experiments. The experimental animals were divided into sham, model, and BYHWD (5 g/kg, 10 g/kg, and 20 g/kg) groups. Infarct volume and severity of brain injury were measured by TTC staining. HE staining was utilized to evaluate alterations in tissue morphology. The Golgi staining was used to observe changes in cell body, dendrites, and dendritic spines. Transmission electron microscopy was used to observe the ultrastructure of synapses in the cortex and hippocampus. TUNEL staining was conducted to identify apoptotic neurons. Meanwhile, a stable and reliable (OGD/R) SH-SY5Y cell model was established. The effect of BYHWD-containing serum on SH-SY5Y cell viability was measured by CCK-8 kit. The apoptosis situation of SH-SY5Y cells was determined by Annexin V-FITC/PI. Immunofluorescence was employed to measure the fluorescence intensity of synaptic-related factors Syt1, Psd95, and Syn1. Synaptic plasticity pathways were assessed by using RT-qPCR and Western blot to determine the expression levels of cAMP, Psd95, Prkacb, Creb1/p-Creb1, BDNF, Shank2, Syn1, Syt1, Bcl-2, Bcl-2/Bax mRNA and proteins.
After treatment with BYHWD, notable alterations were detected in the signaling pathways linked to synaptic plasticity and the cAMP signaling pathway-related targets among the intervention targets. This trend of change was also reflected in other bioinformatics analyses, indicating the important role of synaptic plasticity changes before and after modeling and drug intervention. The results of vivo and vitro experiments showed that BYHWD improved local pathological changes, and reduced cerebral infarct volume, and neurological function scores in MCAO rats. It increased dendritic spine density, improved synaptic structural plasticity, and had a certain neuroprotective effect. BYHWD increased the postsynaptic membrane thickness, synaptic interface curvature, and synaptic quantity. 10% BYHWD-containing serum was determined as the optimal concentration for treatment. 10% BYHWD-containing serum significantly reduced the overall apoptotic rate of (OGD/R) SH-SY5Y cells. Immunofluorescence experiments demonstrated that 10% BYHWD-containing serum could improve synaptic plasticity and increase the relative expression levels of synaptic-related proteins Syt1, Psd95, and Syn1. BYHWD and decoction-containing serum upregulated the mRNA and protein expression levels in (OGD/R) SH-SY5Y cells and MCAO rats, suggesting its ability to improve damaged neuronal synaptic plasticity and enhance transmission efficiency, which might be achieved through the regulation of the cAMP/PKA/CREB pathway.
This study may provide a basis for clinical medication by elucidating the underlying experimental evidence for the promotion of neural plasticity after ischemic stroke by BYHWD.
缺血性中风是一种严重威胁人类健康的急性中枢神经系统疾病。它会引起一系列严重的病理机制，最终导致由于局部缺血和缺氧而导致大脑神经元细胞死亡。补阳还五汤（BYHWD）作为治疗缺血性中风的代表性方剂，在中风患者中显示出良好的治疗效果。
本研究旨在从神经元突触可塑性的角度，基于 cAMP/PKA/CREB 信号通路，探讨 BYHWD 促进缺血性中风后神经重塑的机制。
采用改良缝线技术建立 MCAO 大鼠模型。将大鼠分为假手术组、模型组和 BYHWD（20 g/kg）组。在相应干预后，从每组大鼠中收集脑组织。采用 TMT 定量蛋白质组学技术进行研究。在蛋白质组学研究之后，我们通过动物实验和细胞实验研究了 BYHWD 干预缺血性中风的机制。实验动物分为假手术组、模型组和 BYHWD（5 g/kg、10 g/kg 和 20 g/kg）组。通过 TTC 染色测量梗死体积和脑损伤严重程度。HE 染色用于评估组织形态的变化。高尔基染色用于观察细胞体、树突和树突棘的变化。透射电子显微镜用于观察皮质和海马突触的超微结构。TUNEL 染色用于鉴定凋亡神经元。同时，建立了稳定可靠的（OGD/R）SH-SY5Y 细胞模型。通过 CCK-8 试剂盒测量 BYHWD 含血清对 SH-SY5Y 细胞活力的影响。通过 Annexin V-FITC/PI 测定 SH-SY5Y 细胞的凋亡情况。免疫荧光法测量突触相关因子 Syt1、Psd95 和 Syn1 的荧光强度。通过 RT-qPCR 和 Western blot 评估突触可塑性途径，以确定 cAMP、Psd95、Prkacb、Creb1/p-Creb1、BDNF、Shank2、Syn1、Syt1、Bcl-2、Bcl-2/Bax mRNA 和蛋白的表达水平。
经 BYHWD 治疗后，干预靶点中的突触可塑性相关信号通路和 cAMP 信号通路相关靶标发生了明显变化。这种变化趋势也反映在其他生物信息学分析中，表明建模前后和药物干预时突触可塑性变化的重要作用。体内和体外实验结果表明，BYHWD 改善了 MCAO 大鼠的局部病理变化，减少了脑梗死体积和神经功能评分，增加了树突棘密度，改善了突触结构可塑性，具有一定的神经保护作用。BYHWD 增加了突触后膜厚度、突触界面曲率和突触数量。10% BYHWD 含血清被确定为最佳治疗浓度。10% BYHWD 含血清显著降低了（OGD/R）SH-SY5Y 细胞的总凋亡率。免疫荧光实验表明，10% BYHWD 含血清可改善突触可塑性，增加突触相关蛋白 Syt1、Psd95 和 Syn1 的相对表达水平。BYHWD 和含汤血清上调了（OGD/R）SH-SY5Y 细胞和 MCAO 大鼠的 mRNA 和蛋白表达水平，表明其通过调节 cAMP/PKA/CREB 通路改善受损神经元突触可塑性和增强传递效率的能力。
本研究可能为临床用药提供依据，阐明了 BYHWD 促进缺血性中风后神经可塑性的潜在实验证据。
