养血活血解毒方及其拆方药物血清对佛波酯诱导的血管内皮细胞ERK/NF-κB通路的影响

Effects of Yangxue Huoxue Jiedu Fang(养血活血解毒方) and Its Separated Formula Containing Serum on ERK/NF-κB Pathway of Phorbol Ester Induced in Vascular Endothelial Cells

目的探讨养血活血解毒方治疗银屑病的可能作用机制。方法将养血活血解毒方拆分为养血活血类药物和解毒类药物。80只Wistar大鼠随机分为空白组、全方组、养血活血方组、解毒方组,每组20只。全方组、养血活血方组、解毒方组分别给予相应药物10.18、6.17、4.01 g/(kg·d)剂量灌胃,空白组予4 ml的蒸馏水灌胃,连续4天,制备含药血清。人真皮微血管内皮细胞(HDMEC)分为空白组、模型组、全方组、养血活血组、解毒组。除空白组外,其余各组采用佛波酯诱导细胞活化。同时加入相应药物的10%含药血清作用24 h。检测各组内皮细胞增殖情况、迁移情况、管腔形成情况,以及血管内皮细胞生长因子(VEGF)、血管内皮细胞生长因子受体(VEGFR)、细胞外调节蛋白激酶1(ERK1)、细胞外调节蛋白激酶2(ERK2)mRNA表达,检测VEGF/VEGFR通路及细胞外调节蛋白激酶/核因子κB(ERK/NF-κB)通路蛋白表达。结果与空白组比较,模型组细胞增殖,迁移细胞数量,管腔形成数量,VEGF、VEGFR、ERK2 mRNA及VEGF/VEGFR通路、EKR/NF-κB通路相关蛋白表达升高(P<0.01);与模型组比较,全方组和解毒组细胞增殖,迁移细胞数量,VEGF、VEGFR mRNA表达和p-ERK1、p-EKR2、p-NF-κB p65蛋白表达水平均降低,全方组VEGF、p-VEGFR蛋白表达降低,且全方组细胞增殖、ERK2 mRNA表达低于解毒组(P<0.05或P<0.01)。结论养血活血解毒方可通过干预ERK/NF-κB通路,调控促血管新生因子VEGF及其受体的表达,从而对银屑病血管新生的病理环节发挥治疗作用,且全方药效最佳,其养血活血组分未对内皮细胞增生环节有影响。

Objective To investigate the possible action mechanism of Yangxue Huoxue Jiedu Fang（养血活血解毒方,YXHXJDF） in the treatment of psoriasis. Methods YXHXJDF was divided into herbs of nourishing blood and activating blood（YXHXF）,and herbs of detoxification（JDF）. Eighty Wistar rats were randomly divided into the blank group,YXHXJDF group,YXHXF group,and JDF group,with 20 rats in each group. The YXHXJDF group,YXHXF group,and JDF group were given the corresponding drugs 10. 18,6. 17,4. 01 g/（kg·d） for intragastric administration,and the blank group was given 4 ml distilled water for 4 days. The drug-containing serum was prepared. Human dermal microvascular endothelial cells（HDMEC） were divided into blank group,model group,YXHXJDF group,YXHXF group,and JDF group. Except the blank group,the other groups used phorbol ester to induce cell activation. At the same time,10% drug-containing serum of the corresponding drug was added for 24 hours. The endothelial cell proliferation,migration,lumen formation,and the expressions of vascular endothelial growth factor（VEGF）,vascular endothelial growth factor receptor（VEGFR）,extracellular regulated protein kinase1（ERK1）,extracellular regulation protein kinase 2（ERK2） mRNA expression were detected. The expression of VEGF/VEGFR pathway and extracellular regulated protein kinases/nuclear factor-kappa（ERK/NF-κB） pathway protein were detected. Results Compared with the blank group,the cell proliferation,migration cell number,lumen formation number,VEGF,VEGFR,ERK2 mRNA and VEGF/p-VEGFR pathway,ERK/NF-κB pathway related protein expressions in the model group were increased（P〈0. 01）. Compared with the model group,cell proliferation,migration cell number,VEGF,VEGFR mRNA expression and p-ERK1,p-EKR2,p-NF-κB p65 protein expressions in the YXHXJDF group and JDF group were decreased,and the expression of VEGF and p-VEGFR protein in the YXHXJDF group was decreased. The cell proliferation and ERK2 mRNA expression in the YXHXJDF group were lower than those in the JDF group（P〈0. 05 or P〈0. 01）. Conclusion YXHXJDF can regulate the expression of angiogenic factor VEGF and its receptor by interfering with ERK/NF-κB pathway,so as to play a therapeutic role in the pathological process of angiogenesis of psoriasis,and the YXHXJDF has the best effects. Its YXHXF components have no effect on the proliferation of endothelial cells.