Model Of Cell Calcium Overload And Calcium Antagonist Efficacy Evaluation In Traditional Chinese Medicine

Calcium is an essential secondary messenger used to regulate a wide range of cellular processes. Pathological changes and some harmful substances can introduce dysfunction of calcium counterbalance system and disturbance of calcium distribution, as well as result in the abnormal increase of intracellular calcium, which is termed calcium overload. Calcium overload can lead to cellular injury and possibly activate apoptosis. The lesions in vascular smooth muscle and endothelial cells caused by calcium overload can induce angiocardiopathy such as atherosclerosis and hypertension, etc.. Calcium antagonists are important drugs used for treating angiocardiopathy. It is desired to establish an effective calcium overload model for evaluating calcium antagonists.In this project, a new calcium overload-cell model is presented. Extracellular high K⁺ allows the extracellular Ca²+ to enter the cytoplasm through voltage-operated Ca²+ channels (VOCCs); ATP can activate P2 receptors, stimulate the formation of the inositol 1,4,5-trisphosphate (InsP₃), which binds to IP3 receptors and triggers Ca²+ release from intracellular store; Angiotensin II(Ang II) accesses InsP₃Rs Ca²+ store and some receptor-operated Ca²+ channels (ROCCs); and oxidation injury of the H₂O₂ induces calcium overload in cells. The calcium overload in vascular smooth muscle induced by high K⁺, ATP, Ang II and in human umbilical vascular endothelial cells (HUVECs) induced by high K⁺, ATP, and H₂O₂ through different pathways was discussed. The [Ca²+]i changes were determined by laser scanning confocal microtechnique and fluorescent technique. Using this model we evaluated the drug effect of astragaloside IV on calcium overload in vascular smooth muscle cells and the drug effect of puerarin on calcium overload in HUVECs. Results showed that astragaloside IV caused an 84.8%> 47.2% and 34.2% inhibition of the fluorescence intensity increase, stimulated by high K⁺, ATP and Ang II respectively; puerarin inhibits the increasing of fluorescence intensity, caused by high K⁺, ATP and H₂O₂, and their corresponding inhibition ratios are 78.2%, 56.5% and 72.2%.The calcium overload model established in this project has characteristics of celerity, good stabilization, quantitative analysis. This study would be helpful for evaluating drug effects of calcium antagonists and investigating the medicine mechanisms preliminarily.