大黄?虫丸对ox-LDL诱导血管内皮细胞泛素蛋白酶体系统的影响

Effect of Dahuang Zhechong Pill on Ubiquitin-proteasome System of Vascular Endothelial Cells Induced by ox-LDL

目的探讨氧化低密度脂蛋白(ox-LDL)损伤人脐静脉血管内皮细胞(HUVEC)泛素蛋白酶体系统(UPS)的变化以及大黄[庶虫]虫丸对泛素(Ub)、泛素激活酶(UbE1)、核转录因子(NF-κB)的影响。方法体外培养HUVEC,加入ox-LDL共同孵育制成细胞损伤模型。将药物血清作用于细胞,实验分为正常对照组、ox-LDL组、大黄[庶虫]虫丸(1 400 mg·kg^-1、700 mg·kg^-1)干预组、辛伐他汀阳性对照组。观察各组细胞形态,ELISA法检测ICAM-1含量,免疫组化法检测Ub表达,RT-PCR法测定UbE1、NF-κB、IκB mRNA表达,Western Blot法检测UbE1、NF-κB、I-κB的蛋白表达。结果大黄[庶虫]虫丸干预后细胞形态随浓度变化趋于正常;ICAM-1含量降低(P<0.05);Ub呈弱阳性;UbE1、NF-κB mRNA表达减少(P<0.05),蛋白表达量下降;IκB mRNA上调(P<0.01),蛋白表达量增加。结论大黄[庶虫]虫丸可通过阻抑泛素蛋白酶体系统活化NF-κB干预基因的表达,从而减轻动脉粥样硬化(AS)的炎症反应。

Objective The change of ubiquitin-proteasome system(UPS)of oxidized low density lipoprotein(oxLDL)stimulated human umbilical vein endothelial cells(HUVEC)and the effects of Dahuang Zhechong pill(DHZCP)on ubiquitin(Ub),ubiquitin-activating enzymes(UbE1),nuclear transcription factor-kappa B(NF-κB)were discussed.Methods HUVECs cultivated in vitro were incubated with addition of ox-LDL to induce vascular endothelial injury model.Drug containing serum were added to cells.Cells were randomly divided into normal control group,ox-LDL group,Dahuang Zhechong pill intervention group(1 400 mg·kg^-1,700 mg·kg^-1),and positive control(simvastatin)group.Morphology of cells was observed.ELISA was used to detect content of intercellular adhesion molecules-1(ICAM-1).Immunohistochemistry test was used to detect expression of Ub.RT-PCR was used to detect mRNA levels of UbE1,NF-κB and the inhibitor of nuclear factor-kappa B(IκB).Western Blot was used to visualize the protein contents of UbE1,NF-κB and IκB.Results The cell morphology tended to be normal with the increase of concentration of Dahuang Zhechong pill drug-containing serum intervention.Content of ICAM-1 decreased(P<0.05).Weak positive expression of Ub was observed.The mRNA levels of UbE1,NF-κB were reduced(P<0.05),along with decreased expression of proteins.IκB mRNA raised(P<0.01),and the protein expression increased.Conclusion Through the inhibition of activating NF-κB intervention gene expression by ubiquitin proteasome system,DHZCP reduced the inflammation in atherosclerosis(AS).