刺五加对颈性眩晕患者多巴胺D2受体基因CpG岛甲基化及体外小胶质细胞的M1/M2极性的影响  

Effects of Acanthopanax senticosus on CpG island methylation of dopamine D2 receptor gene and M1/M2 polarity of microglia in vitro in patients with cervical vertigo

作　　者：郝军[1] 常虎飞 盛旻[1] 杨春艳 邢娟丽[2] HAO Jun;CHANG Hu-fei;SHENG Min;YANG Chun-yan;XING Juan-li(Department of Neurology,Yulin Second Hospital,Yulin 719000,China;Department of Otorhinolaryngology Head and Neck Surgery,the First Affiliated Hospital of Xi′an Jiaotong University School of Medicine,Xi′an 710061,China)

机构地区：[1]榆林市第二医院神经内科,陕西榆林719000 [2]西安交通大学医学院第一附属医院耳鼻咽喉头颈外科,陕西西安710061

出　　处：《实用药物与临床》2022年第1期26-31,共6页Practical Pharmacy and Clinical Remedies

基　　金：陕西省自然科学基础研究计划项目(2019JM-575)。

摘　　要：目的研究刺五加对颈性眩晕(CV)患者多巴胺D2受体(DRD2)基因CpG岛甲基化及体外小胶质细胞hmc-3的M1/M2极性的影响。方法收集30例未经治疗的CV患者、30例经刺五加治疗的CV患者、30例健康者的血液,样本分别分为CV组、治疗组、健康组。培养hmc-3小胶质细胞,分别应用20μg、40μg、80μg刺五加处理hmc-3细胞,分为对照组和刺五加组。提取血液全基因组DNA,提取血液或小胶质细胞中的总RNA。RT-qPCR检测DRD2、M1表型标志物CD16、CD32、CD86和M2表型标志物Arginase 1、CD206的mRNA水平。应用亚硫酸氢盐修饰后测序法检测CV组、治疗组、健康组、对照组及刺五加组的DRD2甲基化。CCK-8法检测hmc-3细胞的增殖率变化。蛋白免疫印迹法检测DRD2、CD16、CD32、CD86、Arginase 1、CD206的蛋白水平。细胞免疫荧光法检测hmc-3细胞中的DRD2表达。结果与健康组比较,CV组DRD2的mRNA表达下调且甲基化率上调(P<0.05),但与CV组比较,治疗组的mRNA表达上调且甲基化率下调(P<0.05)。40μg、80μg的刺五加上调小胶质细胞hmc-3的增殖率和DRD2表达(P<0.05)。与对照组比较,刺五加下调M1表型CD16、CD32、CD86的mRNA和蛋白水平(P<0.05),但上调M2表型Arginase 1、CD206的表达(P<0.05)。结论刺五加抑制CV患者DRD2基因CpG岛的甲基化率,促进体外小胶质细胞的M2极性表型。Objective To study the effect of Acanthopanax senticosus on the CpG island methylation of dopamine D2 receptor(DRD2)gene and the M1/M2 polarity of microglia hmc-3 in vitro in patients with cervical vertigo(CV).Methods The blood of 30 untreated CV patients,30 CV patients treated with Acanthopanax senticosus,and 30 healthy individuals was collected.The samples were divided into CV group,treatment group,and healthy group.Hmc-3 microglia were cultured and treated with 20μg,40μg,and 80μg Acanthopanax senticosus respectively.The cells were divided into control group and acanthopanax group.The blood whole genome DNA and total RNA in blood or microglia were extracted.The mRNA levels of DRD2,M1 phenotype markers CD16,CD32,CD86 and M2 phenotype marker Arginase 1 and CD206 were detected by RT-qPCR.The DRD2 methylation of CV group,treatment group,healthy group,control group and Acanthopanax senticosus group was detected by sequencing method after modification with bisulfite.CCK-8 method was used to detect the change in the proliferation rate of hmc-3 cells.Western blot was used to detect the protein levels of DRD2,CD16,CD32,CD86,Arginase 1,and CD206.Cellular immunofluorescence method was used to detect the expression of DRD2 in hmc-3 cells.Results Compared with the healthy group,the mRNA expression of DRD2 in CV group was down-regulated,while the methylation rate was up-regulated(P<0.05);compared with CV group,the mRNA expression of treatment group was up-regulated,while the methylation rate was down-regulated(P<0.05).Acanthopanax senticosus 40μg and 80μg up-regulated the proliferation rate and DRD2 expression of microglia hmc-3(P<0.05).Compared with control group,Acanthopanax senticosus down-regulated the mRNA and protein levels of M1 phenotype CD16,CD32 and CD86(P<0.05),while it up-regulated the expression of M2 phenotype Arginase 1 and CD206(P<0.05).Conclusion Acanthopanax senticosus inhibits the methylation rate of CpG island of DRD2 gene in CV patients and promotes the M2 polar phenotype of microglia in vitro.

关 键 词：刺五加 颈性眩晕 多巴胺D2受体 CPG岛甲基化 小胶质细胞 M1/M2极性 

分 类 号：R274.9[医药卫生—中西医结合]