Zelenak Christine, Pasham Venkanna, Jilani Kashif, Tripodi Paola M, Rosaclerio Luisa, Pathare Ganesh, Lupescu Adrian, Faggio Caterina, Qadri Syed M, Lang Florian
Department of Physiology, University of Tuebingen, Tuebingen, Germany.
Cell Physiol Biochem. 2012;30(1):282-94. doi: 10.1159/000339064. Epub 2012 Jun 19.
Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca(2+)-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin-luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca(2+). Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca(2+) entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.
丹参酮IIA是一种从丹参根茎中提取的具有抗菌、抗氧化、抗过敏、抗纤维化、血管舒张、抗动脉粥样硬化、器官保护和抗肿瘤作用的成分，已知其可引发肿瘤细胞凋亡。丹参酮IIA部分通过线粒体去极化和基因表达改变发挥作用。红细胞缺乏线粒体和细胞核，但可能会发生红细胞凋亡，这是一种类似凋亡的自杀性细胞死亡，其特征是细胞收缩和细胞膜磷脂酰丝氨酸外翻。红细胞凋亡由胞质Ca(2+)活性增加、ATP耗竭和神经酰胺形成引发。本研究探讨了丹参酮IIA是否会引发红细胞凋亡。通过Fluo3荧光测定胞质Ca(2+)浓度，通过前向散射测定细胞体积，通过荧光膜联蛋白V结合测定磷脂酰丝氨酸外翻，通过上清液中血红蛋白浓度测定溶血，利用荧光素 - 荧光素酶测定ATP浓度，利用荧光抗神经酰胺抗体测定神经酰胺形成。通过CFSE标记估计循环红细胞的清除情况。暴露于丹参酮IIA（≥10 µM）48小时可显著增加胞质Ca(2+)浓度，降低ATP浓度（25 µM），增加乳酸浓度（25 µM），增加神经酰胺形成（25 µM），降低前向散射，增加膜联蛋白V结合，并增加（尽管程度小得多）溶血。在名义上无Ca(2+)的情况下，25 µM丹参酮IIA对膜联蛋白V结合的作用部分逆转。与未处理的红细胞相比，标记的经丹参酮IIA处理的红细胞从循环血液中清除得更快。本研究结果揭示了丹参酮IIA一种全新的作用，即引发红细胞Ca(2+)内流、ATP耗竭和神经酰胺形成，这些事件最终导致红细胞凋亡，伴有细胞收缩和细胞膜磷脂酰丝氨酸外翻。
