Cytotoxic Effects of Mesaconitine, the Aconitum carmichaelii Debx Bioactive Compound, on HBEC-5i Human Brain Microvascular Endothelial Cells: Role of Ca2+ Signaling-Mediated Pathway

Abstract

Mesaconitine, one of Aconitum carmichaelii Debx bioactive compounds, was shown to evoke Ca2+ homeostasis and its related physiological effects in endothelial cell types. However, the effect of mesaconitine on Ca2+ signaling and cell viability in human brain microvascular endothelial cells is unclear. This study focused on exploring whether mesaconitine changed cytosolic Ca2+ concentrations ([Ca2+]i), affected cell viability, and established the relationship between Ca2+ signaling and viability in HBEC-5i human brain microvascular endothelial cells. In HBEC-5i cells, cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. Mesaconitine (10–100 μM) concentration dependently induced [Ca2+]i rises. Ca2+ removal reduced the signal by approximately 25%. Mesaconitine (40–100 μM) caused cytotoxicity in HBEC-5i cells. This cytotoxic response was significantly reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-containing medium, mesaconitine-induced Ca2+ entry was inhibited by 25% by modulators of store-operated Ca2+ channels and protein kinase C (PKC). Furthermore, mesaconitine also induced Mn2+ influx suggesting of Ca2+ entry. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished mesaconitine-evoked [Ca2+]i rises. Conversely, treatment with mesaconitine abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished mesaconitine-induced [Ca2+]i rises. In sum, mesaconitine caused cytotoxicity that was triggered by preceding [Ca2+]i rises. Furthermore, mesaconitine induced [Ca2+]i rises by evoking Ca2+ entry via PKC-sensitive store-operated Ca2+ channels and PLC-dependent Ca2+ release from the endoplasmic reticulum. It suggests that Ca2+ signaling have a potential cytotoxic effect on mesaconitine-treated human brain microvascular endothelial cells.

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Acknowledgments

This work was supported by the Department of Pharmacy and Master Program, College of Pharmacy and Health Care, Tajen University, Pingtung County 90741, Taiwan.

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Correspondence to Wei-Zhe Liang.

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Hsu, S., Liang, W. Cytotoxic Effects of Mesaconitine, the Aconitum carmichaelii Debx Bioactive Compound, on HBEC-5i Human Brain Microvascular Endothelial Cells: Role of Ca2+ Signaling-Mediated Pathway. Neurotox Res (2020). https://doi.org/10.1007/s12640-020-00249-2

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Keywords

  • Mesaconitine
  • Ca2+ signaling
  • Cytotoxicity
  • Human brain microvascular endothelial cells
  • Store-operated Ca2+ channel
  • Endoplasmic reticulum