Evaluation of anisodamine-mediated amelioration of hypoxic injury in brain microvascular endothelial cells

Abstract

Purpose: To investigate the therapeutic effect of anisodamine on hypoxic injury of brain microvascular endothelial cells, and the underlying mechanism of action. Methods: A hypoxic injury model of rat primary brain microvascular endothelial cells was established. Cell viability, proliferation, percent survival and apoptosis were assessed using MTT, EdU staining, trypan blue staining and TUNEL staining assays, respectively. The activities of lactate dehydrogenase (LDH) and matrix metalloproteinase-9 (MMP-9) were measured using colorimetry and enzyme-linked immunosorbent assay (ELISA), respectively. Expression levels of key proteins in the PI3K/Akt signal route were determined by Western blotting. Results: Anisodamine treatment significantly reduced apoptosis and LDH leakage in the rat cerebral vascular endothelial cell hypoxia injury model group (p < 0.001). At doses of 3 and 10 mM, anisodamine markedly reduced the cellular level of MMP-9 in rat cerebral vascular endothelial cell hypoxia injury model group (p < 0.001). Treatment of rat brain microvascular endothelial cells with anisodamine significantly reduced levels of caspase-3 and Bax, but up-regulated the expression levels of Bcl2, p-Akt and PI3K (p < 0.001). Conclusion: Anisodamine exerts significant protective effect against hypoxic injury in rat brain microvascular endothelial cells by modulating PI3K/Akt signaling pathway. This finding provides a scientific basis for the clinical application of anisodamine in the treatment and prevention of ischemic cerebrovascular disease.