Anisodamine Hydrobromide Protects Glycocalyx and Against the Lipopolysaccharide-Induced Increases in Microvascular Endothelial Layer Permeability and Nitric Oxide Production.

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.