Hebei University of Chinese Medicine, Hebei Key Laboratory of Chinese Medicine Research on Cardio-Cerebrovascular Disease, No. 3 Xingyuan Road, Luquan District, Shijiazhuang 050200, Hebei Province, China.
J Ethnopharmacol. 2025 Jan 30;337(Pt 3):118982. doi: 10.1016/j.jep.2024.118982. Epub 2024 Oct 23.
Cerebral ischemia-reperfusion (I/R) injury is a common complication of ischemic stroke, with autophagy and pyroptosis playing key roles. Huangqi and Danggui (HQDG) are a commonly used drug pair of Chinese traditional medicine for clinical treatment of ischemic stroke.
The study aims to investigate the interaction between autophagy and pyroptosis regulated by HQDG through the AMPK/mTOR signaling pathway during cerebral I/R injury.
Model of middle-cerebral artery occlusion/reperfusion (MCAO/R) in SD rats was established using the Longa suture method. The components of traditional Chinese medicine were detected by liquid chromatography coupled to quadrupole orbitrap high resolution mass spectrometry (LC/MS). Neurological deficits were evaluated by neurological function score. Changes of cerebral blood flow were detected by a laser speckle blood flow imaging instrument. The volume of cerebral infarction was observed by 2,3,5-Chlorotriphenyltetrazolium (TTC) staining. The permeability of the blood-brain barrier was measured by Evans blue test. Neurovascular unit and autophagosomes in brain tissue were assessed by transmission electron microscopy. Neuronal pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/Caspase-1 staining. The expression of autophagy related proteins, pyroptosis related proteins, and AMPK/mTOR pathway related proteins were detected by Western blot.
After cerebral I/R injury, autophagy and pyroptosis, were characterized by increased number of autophagosomes and pyroptosis cells, upregulated expression of Beclin 1, LC3-II/LC3-I, NLRP3, cleaved Caspase-1, IL-1beta, IL-18 proteins, and downregulated expression of P62 proteins. HQDG significantly improved neurological function, reduced the volume of cerebral infarction, increased cerebral blood flow, improved blood-brain barrier permeability and the function of neurovascular units. Autophagy was further activated and pyroptosis was significantly inhibited by HQDG, which promoted increased number of autophagosomes, enhanced expression of Beclin 1, LC3-II/LC3-I proteins, reduced expression of P62, NLRP3, cleaved Caspase-1, IL-1beta, and IL-18 proteins, and downregulated the number of pyroptosis cells. On the other hand, after administering 3-Methyladenine (3-MA) to inhibit autophagy, the above effects of HQDG were significantly inhibited. Besides, HQDG promoted AMPK phosphorylation, and weakened mTOR phosphorylation. However, after the administration of AMPK inhibitor Compound C, HQDG caused increase in Beclin 1 and LC3-II/LC3-I, reduced P62 and NLRP3, and cleaved Caspase-1 protein expression, whereas cerebral blood flow decreased.
HQDG alleviated pyroptosis by promoting autophagy via AMPK/mTOR signaling pathway after middle-cerebral artery occlusion/reperfusion in rats, showing its potential for treatment of cerebral I/R injury in humans.
脑缺血再灌注（I/R）损伤是缺血性中风的常见并发症，自噬和细胞焦亡起着关键作用。黄芪和当归（HQDG）是一种常用于治疗缺血性中风的中药药对。
本研究旨在通过 AMPK/mTOR 信号通路研究 HQDG 对脑 I/R 损伤中自噬和细胞焦亡的调节作用。
采用 Longa 缝线法建立 SD 大鼠大脑中动脉闭塞/再灌注（MCAO/R）模型。采用液相色谱-四极杆轨道阱高分辨质谱联用（LC/MS）检测中药成分。通过神经功能评分评估神经功能缺损。采用激光散斑血流成像仪检测脑血流变化。采用 2,3,5-氯化三苯基四氮唑（TTC）染色观察脑梗死体积。采用 Evans 蓝试验检测血脑屏障通透性。通过透射电子显微镜评估神经血管单元和脑组织中的自噬体。通过末端脱氧核苷酸转移酶（TdT）dUTP 缺口末端标记（TUNEL）/Caspase-1 染色检测神经元细胞焦亡。采用 Western blot 检测自噬相关蛋白、细胞焦亡相关蛋白和 AMPK/mTOR 通路相关蛋白的表达。
脑 I/R 损伤后，自噬和细胞焦亡表现为自噬体数量增加和焦亡细胞增多，自噬相关蛋白 Beclin 1、LC3-II/LC3-I 表达上调，自噬相关蛋白 P62 表达下调，细胞焦亡相关蛋白 NLRP3、cleaved Caspase-1、IL-1beta、IL-18 表达上调。HQDG 可显著改善神经功能，减少脑梗死体积，增加脑血流量，改善血脑屏障通透性和神经血管单元功能。HQDG 进一步激活自噬，显著抑制细胞焦亡，增加自噬体数量，增强 Beclin 1、LC3-II/LC3-I 蛋白表达，降低 P62、NLRP3、cleaved Caspase-1、IL-1beta 和 IL-18 蛋白表达，减少焦亡细胞数量。另一方面，用 3-甲基腺嘌呤（3-MA）抑制自噬后，HQDG 的上述作用明显受到抑制。此外，HQDG 促进 AMPK 磷酸化，减弱 mTOR 磷酸化。然而，在用 AMPK 抑制剂 Compound C 处理后，HQDG 引起 Beclin 1 和 LC3-II/LC3-I 增加，P62 和 NLRP3 减少，cleaved Caspase-1 蛋白表达减少，而脑血流量减少。
HQDG 通过 AMPK/mTOR 信号通路促进自噬，减轻大鼠大脑中动脉闭塞/再灌注后的细胞焦亡，为治疗人类脑 I/R 损伤提供了潜在的治疗方法。
