Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia (K.W.C., Y.S.L., D.M., M.R.M.); and State Key Laboratory of Pharmaceutical Biotechnology, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, People's Republic of China (P.M.V.).
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia (K.W.C., Y.S.L., D.M., M.R.M.); and State Key Laboratory of Pharmaceutical Biotechnology, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, People's Republic of China (P.M.V.)
J Pharmacol Exp Ther. 2018 Mar;364(3):420-432. doi: 10.1124/jpet.117.245217. Epub 2017 Dec 19.
Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.
内皮细胞的炎症损伤导致细胞凋亡和内皮功能障碍。本研究探讨了丹皮酚在脂多糖（LPS）诱导的炎症损伤、内皮细胞凋亡和内皮功能障碍中的作用及机制。采用Western blot、流式细胞术和活性氧（ROS）测定法检测丹皮酚对人脐静脉内皮细胞（HUVEC）和 C57BL/6J 小鼠 LPS 诱导的炎症损伤的影响。利用血管张力测定仪检测分离的小鼠主动脉的血管反应性。LPS 暴露于 HUVEC 可增加 Toll 样受体 4（TLR4）、骨形态发生蛋白 4（BMP4）、BMP 受体 1A、烟酰胺腺嘌呤二核苷酸磷酸氧化酶亚基 2、丝裂原活化蛋白激酶（MAPK）、诱导型一氧化氮合酶（iNOS）和裂解的 caspase 3 的蛋白表达，同时降低磷酸化内皮型一氧化氮合酶的表达；丹皮酚处理可预防这些作用。同样，丹皮酚也可逆转 BMP4 诱导的 HUVEC 凋亡。LPS 暴露后，小鼠主动脉对内皮依赖性血管舒张剂乙酰胆碱的舒张反应受损；丹皮酚、noggin（BMP4 抑制剂）、TAK242（TLR4 拮抗剂）、apocynin（ROS 清除剂）、MAPK 抑制剂和 AG（iNOS 抑制剂）联合处理可逆转这种内皮功能障碍。BMP4 小干扰 RNA（siRNA）可消除 LPS 诱导的 BMP4 和裂解的 caspase 3 蛋白的上调，但对 TLR4 siRNA 处理的细胞无效，反之亦然。TLR4 和 BMP4 的沉默消除了丹皮酚对 LPS 诱导的 cleaved caspase 3 激活的抑制作用。本研究结果表明，丹皮酚通过独立抑制 TLR4 和 BMP4 信号通路，减轻 LPS 诱导的内皮功能障碍和凋亡。
