School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, 010070, PR China; Inner Mongolia Key Laboratory of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010059, PR China.
Inner Mongolia Key Laboratory of Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010059, PR China.
J Ethnopharmacol. 2021 Jun 28;274:114020. doi: 10.1016/j.jep.2021.114020. Epub 2021 Mar 11.
The Zhenbao pill (ZBP) is composed of 29 traditional Chinese medicines and has been proven to exhibit a valid therapeutic effect in nervous system diseases, such as stroke and hemiplegia sequelae.
Whether ZBP has a protective effect on vascular endothelial cells remains unknown. In this study, we established hydrogen peroxide (HO)-induced oxidative injury in human umbilical vein endothelial cells (HUVECs) as an in vitro model to investigate the pharmacological effects of ZBP.
Following the intragastric administration of ZBP (0.25, 0.5, and 1 g/kg for seven days) in rats, drug-containing serum was obtained and cultivated with HUVECs before HO treatment. The viability of HUVECs in the presence of HO was measured by Cell Counting Kit-8 assay, lactate dehydrogenase assay, and flow cytometry. Furthermore, we estimated the effects of ZBP on the production of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Autophagic puncta were detected using a fluorescence microscope. Western blotting and real-time polymerase chain reaction were used to detect the expression levels of several genes associated with apoptosis and autophagy.
Drug-containing serum separated from rats at 1 h after intragastric administration of ZBP (0.5 g/kg) significantly offered a protective effect to HUVECs and reduced cell apoptosis rates. Meanwhile, ZBP-containing serum also repressed ROS production induced by HO exposure and maintained MMP. Further investigation revealed that ZBP-containing serum effectively reduced the accumulation of autophagic puncta. ZBP-mediated inhibition on cell autophagy was found to contribute to ameliorating cell apoptosis. Western blotting also confirmed that ZBP maintained AKT and mTOR phosphorylation and antagonized the imbalance of BCL2/BAX, thereby protecting cells from apoptosis.
Taken together, our data indicate that ZBP inhibits ROS production, mitochondrial damage, cell autophagy, and cell apoptosis. ZBP can offer protection to vascular endothelial cells against oxidative injury through the antagonism of apoptosis and autophagy. Thus, this study enhances the understanding of the therapeutic effects and mechanisms of ZBP in the process of recovery from myocardial and cerebral ischemic stroke.
镇脑宝丸（ZBP）由 29 种中药组成，已被证明对神经系统疾病（如中风和偏瘫后遗症）具有有效的治疗作用。
ZBP 是否对血管内皮细胞具有保护作用尚不清楚。在这项研究中，我们建立了过氧化氢（HO）诱导的人脐静脉内皮细胞（HUVEC）氧化损伤的体外模型，以研究 ZBP 的药理作用。
大鼠灌胃 ZBP（0.25、0.5 和 1 g/kg，连续 7 天）后，获得含药血清，并用 HO 处理前培养 HUVEC。通过细胞计数试剂盒-8 测定法、乳酸脱氢酶测定法和流式细胞术测定 HO 存在时 HUVEC 的活力。此外，我们评估了 ZBP 对活性氧（ROS）和线粒体膜电位（MMP）产生的影响。用荧光显微镜检测自噬斑点。Western blot 和实时聚合酶链反应用于检测与细胞凋亡和自噬相关的几个基因的表达水平。
ZBP（0.5 g/kg）灌胃后 1 小时大鼠分离的含药血清对 HUVEC 具有显著的保护作用，降低细胞凋亡率。同时，ZBP 含药血清还抑制了 HO 暴露引起的 ROS 产生，并维持了 MMP。进一步研究表明，ZBP 含药血清有效减少了自噬斑点的积累。ZBP 介导的细胞自噬抑制有助于改善细胞凋亡。Western blot 也证实，ZBP 维持了 AKT 和 mTOR 的磷酸化，并拮抗了 BCL2/BAX 的失衡，从而保护细胞免受凋亡。
综上所述，我们的数据表明，ZBP 抑制 ROS 产生、线粒体损伤、细胞自噬和细胞凋亡。ZBP 通过拮抗细胞凋亡和自噬，为血管内皮细胞提供氧化损伤保护。因此，本研究增强了对 ZBP 在心肌和脑缺血性中风恢复过程中治疗作用和机制的理解。
