芍药苷在皮肤血管炎的抗炎、抗氧化作用及机制研究

Cutaneous vasculitis comprises a wide spectrum of inflammatory diseases directed at vessels, which is characterized by endothelial damage and perivascular leukocyte infiltrates. Our previous work indicated that Peoniflorin (PF) can significantly inhibit chemokine production in TNF-α-induced HMEC-1 cells, ameliorate endothelial damage in H2O2-induced HUVECs. In this study, we observe the therapeutic effects of PF in two mouse models of CV. We focus on the effects of PF on perivascular leukocyte infiltrates and vascular barrier damage in CV mice. Furthermore, we investigate the potential molecular mechanisms underlying therapeutic effects of PF in CV, for example, the release of pro-inflammatory cytokines and chemokines, the expression of adhesion molecules, VE-cadherin disruption and the oxidative stress status. In addition, in our in vitro experiments, we also investigate the effects and mechanisms of PF on adhesion molecules expression and the subsequent leukocyte-endothelial cells adhesion in TNF-α-induced HDMECs, and endothelial damage, endothelial barrier dysfunction and the expression of VE-cadherin in H2O2-treated HDMECs. It would be interesting and important to ascertain the effects and mechanisms of PF in CV through these in vivo as well as in vitro approaches, which may further extend and deepen the understanding of the pharmaceutical functions of PF and provide experimental evidence for the potential clinical use of PF in the treatment of CV.No similar study was reported.

皮肤血管炎(CV) 是一大类病变定位于血管壁的炎症性疾病。血管壁的损伤及炎细胞浸润是其主要特征。我们的前期工作显示芍药苷(PF)能抑制TNF-α诱导的HMEC-1细胞趋化因子产生及H2O2诱导的HUVECs损伤。本项目在此基础上，观察PF对两种CV动物模型的治疗效果（尤其是对炎细胞浸润及屏障功能破坏的效果）及可能的机制，如：促炎症细胞因子、趋化因子释放，粘附分子表达、VE-cadherin破坏及氧化应激状态等。进一步通过体外研究，观察PF对TNF-α诱导的HDMECs粘附分子表达、内皮-白细胞粘附的作用及机制；PF对H2O2诱导的HDMECs氧化损伤、屏障功能破坏及VE-cadherin表达的作用及机制。本项目从分子、细胞到整体来揭示PF对CV的治疗作用及机制，扩展和深化对PF药理作用机制的认识，为进一步开发及应用PF作为CV类疾病的治疗新手段提供理论依据。未见国内外相关研究报道。

项目调查芍药苷(paeoniflorin, PF)对皮肤血管炎 (cutaneous vasculitis, CV)的干预作用及机制，分为体内及体外研究两部分。体外研究发现：PF能抑制肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)诱导的皮肤微血管内皮细胞(human dermal microvascular endothelial cells, HDMECs)细胞E-选择素及细胞间粘附分子(intercellular adhesion molecule-1, ICAM-1)在mRNA及蛋白水平的表达；及人多形核白细胞对HDMECs的粘附。免疫印迹结果显示:PF能抑制TNF–α诱导的抑制性κBα的磷酸化及P38、细胞外信号调节激酶及c-Jun氨基末端激酶途径的活化。免疫荧光显示PF也抑制TNF–α诱导NF–κBp65核转导。PF能明显改善H202诱导人真皮微血管内皮细胞的氧化损伤；PF对H20 2诱导血管内皮细胞屏障功能损伤的修复作用与改善VE-cadherin的表达有关；PF对H2O2诱导血管内皮细胞损伤的抑制作用与抑制MAPKs及PI3K/Akt通路的活化有关。PF明显减轻了免疫复合物(immune complexes, IC)诱导的CV小鼠皮损及组织学改变；减少了局部出血半定量评分；减轻局部FITC-BSA的渗出及MPO活力；及组织标本血管壁E-选择素及ICAM-1表达。PF剂量依赖性的抑制了LPS诱导CV小鼠皮损及组织学改变；减少了局部出血半定量评分；减轻局部FITC-BSA的渗出及MPO活力；抑制了局部组织趋化因子IL-8及促炎症细胞因子TNF–α的mRNA表达。这些结果支持PF具有抗炎及抗氧化作用，能对抗TNF–α诱导的皮肤微血管内皮炎症，减轻H202诱导的皮肤微血管内皮氧化应激。减轻IC及LPS诱导CV小鼠的皮损及组织学改变。PF可能是今后CV治疗的一个新的选择。