Purpose

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.

Methods

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.

Results

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.

Conclusion

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.

中文翻译：

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山莨菪碱氢溴酸盐保护糖萼并防止脂多糖诱导的微血管内皮层渗透性和一氧化氮产生增加。

目的

山莨菪碱氢溴酸盐 (Ani HBr) 已被用于改善心血管疾病和败血症期间的微循环。糖萼在保持内皮细胞 (EC) 屏障通透性和一氧化氮 (NO) 产生方面起着重要作用。我们旨在检验 Ani HBr 可以通过防止糖萼脱落保护 EC 防止渗透性和 NO 产生的假设。

方法

建立脂多糖(LPS)诱导的人脑微血管EC h​​CMEC/D3损伤模型。Ani HBr 被给予具有 LPS 挑战的 EC。细胞活力通过细胞计数试剂盒-8 测定进行。通过EdU和Hoechst 33342染色检测细胞增殖和凋亡。细胞凋亡和细胞周期也分别通过流式细胞术与膜联蛋白 V 染色和碘化丙啶染色进行评估。然后，根据血管内皮钙粘蛋白（VE-钙粘蛋白）的免疫荧光染色评估粘附连接的完整性。糖萼组分硫酸乙酰肝素 (HS) 在 ECs 中染色。通过荧光素异硫氰酸酯 (FITC)-葡聚糖的渗漏来评估细胞渗透性。用硝酸还原酶法测定细胞NO产生量。

结果

20 μ g/mL 的Ani HBr 在LPS 攻击下显着增加 ECs 的活力，但在 80 μ g/mL 时显着抑制细胞活力，显示 Ani HBr 对细胞活力的双向调节。Ani HBr 没有显着改变 LPS 诱导的 EC 增殖。Ani HBr 显着逆转了 LPS 对 EC 细胞凋亡的诱导。Ani HBr 恢复了 LPS 诱导的糖萼和 VE-钙粘蛋白脱落和粘附连接中断。Ani HBr 显着减轻了 LPS 诱导的 EC 层渗透性和 NO 产生。

结论

Ani HBr通过保持糖萼的完整性来保护 ECs 免受 LPS 诱导的细胞屏障通透性和一氧化氮产生的增加。Ani HBr 是一种很有前途的药物，可以挽救或保护糖萼。