Du Xiaoqiang, Liu Huan, Yue Yonghua, Wu Qingjiang, Jiang Wenli, Qiu Yan, Zeng Ye
Institute of Biomedical Engineering, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, 610041, China.
Chengdu No. 1, Pharmaceutical Co., Ltd, Chengdu, 610031, China.
Cardiovasc Eng Technol. 2021 Feb;12(1):91-100. doi: 10.1007/s13239-020-00486-8. Epub 2020 Sep 15.
Anisodamine hydrobromide (Ani HBr) has been used to improve the microcirculation during cardiovascular disorders and sepsis. Glycocalyx plays an important role in preserving the endothelial cell (EC) barrier permeability and nitric oxide (NO) production. We aimed to test the hypothesis that Ani HBr could protect the EC against permeability and NO production via preventing glycocalyx shedding.
A human cerebral microvascular EC hCMEC/D3 injury model induced by lipopolysaccharide (LPS) was established. Ani HBr was administrated to ECs with the LPS challenge. Cell viability was performed by Cell Counting Kit-8 assay. Cell proliferation and apoptosis were detected by EdU and Hoechst 33342 staining. Apoptosis and cell cycle were also assessed by flow cytometry with annexin V staining and propidium iodide staining, respectively. Then, adherens junction integrity was evaluated basing on the immunofluorescence staining of vascular endothelial cadherin (VE-cadherin). The glycocalyx component heparan sulfate (HS) was stained in ECs. The cell permeability was evaluated by leakage of fluorescein isothiocyanate (FITC)-dextran. Cellular NO production was measured by the method of nitric acid reductase.
Ani HBr at 20 μg/mL significantly increased the viability of ECs with LPS challenge, but significantly inhibited the cell viability at 80 μg/mL, showing a bidirectional regulation of cell viability by Ani HBr. Ani HBr had not significantly change the LPS-induced EC proliferation. Ani HBr significantly reversed the induction of LPS on EC apoptosis. Ani HBr reinstated the LPS-induced glycocalyx and VE-cadherin shedding and adherens junction disruption. Ani HBr significantly alleviated LPS-induced EC layer permeability and NO production.
Ani HBr protects ECs against LPS-induced increase in cell barrier permeability and nitric oxide production via preserving the integrity of glycocalyx. Ani HBr is a promising drug to rescue or protect the glycocalyx.
氢溴酸山莨菪碱（Ani HBr）已被用于改善心血管疾病和脓毒症期间的微循环。糖萼在维持内皮细胞（EC）屏障通透性和一氧化氮（NO）生成方面发挥着重要作用。我们旨在验证Ani HBr可通过防止糖萼脱落来保护内皮细胞免受通透性增加和NO生成影响这一假说。
建立脂多糖（LPS）诱导的人脑微血管内皮细胞hCMEC/D3损伤模型。在LPS刺激内皮细胞时给予Ani HBr。采用细胞计数试剂盒-8法检测细胞活力。通过EdU和Hoechst 33342染色检测细胞增殖和凋亡。分别用膜联蛋白V染色和碘化丙啶染色通过流式细胞术评估凋亡和细胞周期。然后，基于血管内皮钙黏蛋白（VE-钙黏蛋白）的免疫荧光染色评估黏附连接完整性。在内皮细胞中对糖萼成分硫酸乙酰肝素（HS）进行染色。通过异硫氰酸荧光素（FITC）-葡聚糖渗漏评估细胞通透性。采用硝酸还原酶法测定细胞内NO生成量。
20μg/mL的Ani HBr显著提高了受LPS刺激的内皮细胞活力，但在80μg/mL时显著抑制细胞活力，表明Ani HBr对细胞活力具有双向调节作用。Ani HBr对LPS诱导的内皮细胞增殖无显著影响。Ani HBr显著逆转了LPS对内皮细胞凋亡的诱导作用。Ani HBr恢复了LPS诱导的糖萼和VE-钙黏蛋白脱落以及黏附连接破坏。Ani HBr显著减轻了LPS诱导的内皮细胞层通透性增加和NO生成。
Ani HBr通过维持糖萼完整性保护内皮细胞免受LPS诱导的细胞屏障通透性增加和NO生成影响。Ani HBr是一种有望挽救或保护糖萼的药物。
