丹皮酚通过调控动脉粥样硬化中的miR-152-3p/ASPH轴抑制氧化低密度脂蛋白诱发的小鼠血管内皮细胞凋亡、炎症反应和氧化应激  

Paeonol inhibits ox-LDL-induced apoptosis,inflammation,and oxidative stress of mouse vascular endothelial cells by regulating the miR-152-3p/ASPH axis in atherosclerosis

作　　者：刘岩 张磊 安硕 史玉娟 秦素霞[3] LIU Yan;ZHANG Lei;AN Shuo;SHI Yujuan;QIN Suxia(Henan Medical College,Zhengzhou 451191,China;Henan Provincial Veteran Cadres Rehabilitation Hospital,Zhengzhou 450003,China;Qiannan Medical College for Nationalities,Duyun 558000,China)

机构地区：[1]河南医学高等专科学校,河南郑州451191 [2]河南省老干部康复医院,河南郑州450003 [3]黔南民族医学高等专科学校,贵州都匀558000

出　　处：《沈阳药科大学学报》2024年第9期1244-1253,共10页Journal of Shenyang Pharmaceutical University

基　　金：河南省科技厅科技发展计划项目(212400410181);河南省职业教育教学改革研究与实践项目(豫教[2024]05846)。

摘　　要：目的揭示丹皮酚通过调控miR-152-3p/ASPH轴在ox-LDL诱导的血管内皮细胞(vascular endothelial cells,VECs)进展中的作用。方法首先,用不同浓度的氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)(0、25、50、100μg·mL^(-1))处理小鼠VECs,选取100μg·mL^(-1)ox-LDL进行实验。然后,用不同浓度的丹皮酚(30、60、120μmol·L^(-1))或辛伐他汀(30μmol·L^(-1))处理VECs。接着,使用细胞计数试剂盒-8和流式细胞术分别用于测定细胞活力和凋亡。此外,使用酶联免疫吸附法分析了IL-1β和IL-6的水平,并使用相关试剂盒检测了活性氧、乳酸脱氢酶和丙二醛的含量。此外,通过qRT-PCR或Western blot检测miR-152-3p和天冬氨酰(天冬酰胺)β-羟化酶(aspartate-β-hydroxylase,ASPH)的表达。miR-152-3p和ASPH之间的相互作用由starBase预测,然后通过双荧光素酶报告基因实验和RNA结合蛋白免疫沉淀实验证实。结果100μg·mL^(-1)ox-LDL显著抑制VECs活力,并促进细胞凋亡、炎症反应和氧化损伤。而丹皮酚的处理以剂量依赖性的方式增加细胞活力,抑制ox-LDL诱导的细胞凋亡。丹皮酚以剂量依赖性的方式降低ox-LDL诱导的IL-1β和IL-6的水平,以及以剂量依赖性的方式减弱ox-LDL对ROS、MDA和LDH含量的促进作用。另外,ox-LDL对miR-152-3p表达的抑制作用和对ASPH表达的促进作用也被丹皮酚逆转,且120μmol·L^(-1)丹皮酚效果最好。120μmol·L^(-1)丹皮酚能够上调miR-152-3p或下调ASPH,进一步促进丹皮酚对VECs生长的保护作用,miR-152-3p靶向负调控ASPH表达。结论丹皮酚通过调节miR-152-3p/ASPH轴减弱ox-LDL对小鼠VECs生长的影响,为AS的治疗提供理论基础。Objective The study was to uncover the action of paeonol in the progression of ox-LDL-stimulated vascular endothelial cells(VECs)via modulating microRNA(miR)-152-3p/aspartate-β-hydroxylase(ASPH)axis.Methods First,mouse VECs were treated with different concentrations of ox-LDL(0,25,50,100μg·mL^(-1)),and 100μg·mL^(-1)was selected for further experiments.Then,VECs were treated with different doses of paeonol(30,60,120μmol·L^(-1))or simvastatin(30μmol·L^(-1)).Then,determination of cell viability and apoptosis was via adopting cell counting kit-8(CCK-8)and flow cytometry.Additionally,analysis of interleukin(IL)-1βand IL-6 was conducted,and test of reactive oxygen species(ROS),lactate dehydrogenase(LDH)and malondialdehyde(MDA)contents was via adopting relevant kits.Furthermore,examination of miR-152-3p and ASPH was performed.Forecast of the interaction of miR-152-3p with ASPH was via starBase with verification.Results Ox-LDL(100μgmL^(-1))significantly inhibited the viability of VECs and promoted apoptosis,inflammatory response and oxidative damage.Paeonol treatment increased cell viability and inhibited ox-LDL-induced apoptosis in a dose-dependent manner.Moreover,paeonol reduced the levels of IL^(-1)βand IL-6 induced by ox-LDL in a dose-dependent manner,and attenuated the promoting effects of ox-LDL on ROS,MDA and LDH content.In addition,the inhibitory effect of ox-LDL on miR-152-3p expression and the promotion effect on ASPH expression were also reversed by paeonol,and paeonol(120μmol·L^(-1))had the best effect.Therefore,paeonol(120μmol·L^(-1))was selected for subsequent transfection experiments.Up-regulation of miR-152-3p or down-regulation of ASPH could further promote the protective effect of paeonol on the growth of VECs,and miR-152-3p targeted and negatively regulated ASPH expression.Conclusion Paeonol attenuates the impact of ox-LDL on the growth of VECs in mice via controlling miR-152-3p/ASPH axis,offering a theoretical basis for AS cure.

关 键 词：丹皮酚 氧化低密度脂蛋白 miR-152-3p 天冬氨酰(天冬酰胺)β-羟化酶 血管内皮细胞 动脉粥样硬化 

分 类 号：R96[医药卫生—药理学]