丹皮酚通过酸性鞘磷脂酶/神经酰胺通路改善血管紧张素Ⅱ所致动脉内皮功能障碍

Paeonol Ameliorates Angiotensin Ⅱ induced Endothelial Dysfunction through Acid Sphingomyelinase/Ceramide Pathway

目的:探讨丹皮酚(Paeonol,Pae)对血管紧张素II(AngiotensinⅡ,AngⅡ)所致动脉内皮功能障碍的保护作用及其机制。方法:采用实时定量聚合酶链式反应(RT-PCR)和Westernblot检测酸性鞘磷脂酶(acidsphingomyelinase,ASM)基因及蛋白表达;采用免疫组织荧光检测血管环神经酰胺(ceramide,Cer)含量变化;采用血管环张力描记技术检测主动脉血管环的舒张功能;采用二氢乙啶荧光探针(dihydroethidium,DHE)检测血管环超氧阴离子(superoxideanion,O2-·)水平。结果:与对照组相比,AngⅡ孵育后主动脉对乙酰胆碱(acetylcholine,Ach)的舒张反应显著降低,Pae与AngⅡ共孵育则无明显改变;AngⅡ及Pae孵育后,动脉对硝普钠(sodiumnitroprusside,SNP)的舒张反应均无明显变化;AngⅡ孵育可增高动脉环O2-·水平,这一反应被Pae明显抑制;与对照组相比,AngⅡ孵育动脉ASMmRNA水平无明显改变,但蛋白表达显著增加,Cer含量显著增多,Pae与AngⅡ共孵育动脉ASM蛋白及Cer水平与对照组相比均无明显改变;采用神经酰胺酶抑制剂N-oleoylethanolamine(OEA)增加动脉Cer含量后,Pae降低动脉O2-·水平以及增强Ach舒张反应的效应被显著抑制。结论:Pae通过ASM/Cer通路改善AngⅡ所致动脉氧化应激增强和内皮功能障碍。

Objective： Angiotensin Ⅱ（Ang Ⅱ） is a crucial mediator inducing dysfunction of endothelial cell contributing to the development of hypertension and atherosclerosis. Paeonol（Pae） owns the ability to enhance activity of endothelial nitric oxide synthase and lessen oxidative stress in multiple cells. Acid sphingomyelinase（ASM）/ceramide（Cer） regulates the activity of NADPH oxidase and mitochondrial function influencing the level of reactive oxygen species upon a host pathological stimulus. We sought to investigate the protective effect of Pae on Ang Ⅱ induced arterial endothelial dysfunction and elucidate the role of ASM/Cer pathway in the situation. Methods：Reverse transcriptase polymerase chain reaction（RT-PCR） and Western blot were used to detect the gene and protein abundance of ASM.Immunohistochemical fluorescence technique was carried out to show the content of Cer. Isometric force recording system was done to detect the vasodilatation response. Dihydroethidium fluorescent probes（DHE） was applied to investigate the superoxide anion（O_2^-·） level of vascular rings. Results： As compared to control level, the vasodilation of aorta to acetylcholine（Ach） was decreased significantly by Ang Ⅱ（100 nmol/L）（P〈0.05） which could be reversed by Pae. The vasodilation to sodium nitroprusside（SNP） did not change with treatment of either Ang Ⅱ or Pae. The O_2^-·level of aorta ring was increased after Ang Ⅱ incubation which could also be reverted by Pae. The m RNA abundance of ASM was not changed by Ang Ⅱ whereas the protein level of it was upregulated significantly（P〈0.05）. At the same time, the Cer content was increased by Ang Ⅱ too（P〈0.05）. Similarly, the change of both the ASM protein expression and Cer content were prevented by Pae treatment. N-oleoylethanolamine（OEA）, an acid ceramidase inhibitor was used to increase the Cer level of aorta which was found blocking the effect of Pae reducing O_2^-·level and promoting vasodilation response of aorta to Ach. Conclusion： Paeonol ameliorates angiotensin Ⅱ elicited oxidative stress and endothelial dysfunction through ASM/ Cer pathway.