Institute of Clinical Pharmacology, Central South University, Changsha 410078, China.
J Ethnopharmacol. 2013 Mar 27;146(2):543-51. doi: 10.1016/j.jep.2013.01.019. Epub 2013 Jan 26.
Paeonol is an active compound isolated from traditional Chinese medicine, and has been shown to have anti-atherosclerosis, anti-inflammatory, antioxidant effects. The present investigation was undertaken to determine the suppression effects of paeonol on oxidized low-density lipoprotein (ox-LDL) induced endothelial cell line HUVEC apoptosis and to uncover some of the underlying mechanisms of these effects. Cell viability and lactate dehydrogenase (LDH) were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33342 staining and flow cytometry. Intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Real-time PCR was used to confirm the expression of LOX-1 mRNA. Western blotting was used to evaluate the protein expression of LOX-1 and Bcl-2, as well as caspase-3 cleavage, p38-mitogen-activated protein kinase (p38MAPK) phosphorylation. NF-κB nuclear translocation was detected by Western blotting and immunofluorescence. Caspase-3 activity was measured using a colorimetric protease assay kit. The results showed that ox-LDL significantly decreased cell viability and increased the LDH release, as well as the apoptotic rate (P<0.01). Pre-treatment of paeonol resulted in remarkable increase of cell viability, decrease of LDH release and cell apoptosis in a concentration-dependent manner. Besides, ox-LDL caused the up-regulation of LOX-1, the down-regulation of Bcl-2, the phosphorylation of p38MAPK, the translocation of NF-κB and the activation of caspase-3. Paeonol pre-treatment reversed these effects introduced by ox-LDL. Moreover, paeonol also showed its inhibition effects on ox-LDL induced ROS overproduction. These results indicate the preventive effects of paeonol on ox-LDL induced endothelial cell apoptosis. The effects might, at least partly, be obtained via inhibition of LOX-1-ROS- p38MAPK-NF-κB signaling pathway.
丹皮酚是一种从中药中分离出来的活性化合物，具有抗动脉粥样硬化、抗炎、抗氧化作用。本研究旨在探讨丹皮酚对氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞系（HUVEC）凋亡的抑制作用，并揭示其部分作用机制。通过测定细胞活力和乳酸脱氢酶（LDH）来评估细胞损伤。通过 Hoechst 33342 染色和流式细胞术评估细胞凋亡。通过 2'，7'-二氯荧光素二乙酸酯（DCFH-DA）检测细胞内活性氧（ROS）的产生。实时 PCR 用于验证 LOX-1 mRNA 的表达。Western blot 用于评估 LOX-1 和 Bcl-2 的蛋白表达，以及 caspase-3 裂解、p38 丝裂原激活蛋白激酶（p38MAPK）磷酸化。通过 Western blot 和免疫荧光检测 NF-κB 核转位。通过比色蛋白酶测定试剂盒测定 caspase-3 活性。结果表明，ox-LDL 显著降低细胞活力，增加 LDH 释放和细胞凋亡率（P<0.01）。丹皮酚预处理呈浓度依赖性显著增加细胞活力，降低 LDH 释放和细胞凋亡率。此外，ox-LDL 导致 LOX-1 上调、Bcl-2 下调、p38MAPK 磷酸化、NF-κB 核转位和 caspase-3 激活。丹皮酚预处理逆转了 ox-LDL 引起的这些效应。此外，丹皮酚还显示出对 ox-LDL 诱导的 ROS 过度产生的抑制作用。这些结果表明丹皮酚对 ox-LDL 诱导的内皮细胞凋亡具有预防作用。这些作用至少部分可能是通过抑制 LOX-1-ROS-p38MAPK-NF-κB 信号通路获得的。
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