滋阴明目方调控GRP78/IRE1/ATF6通路改善视网膜色素变性的体外研究  

Ziyin Mingmu Formula improve retinitis pigmentosa through the GRP78/IRE1/ATF6 pathway in vitro study

作　　者：欧晨 宋厚盼[2] 谢薇 彭俊[1,2] 曾梅艳 彭清华[1,2] OU Chen;SONG Houpan;XIE Wei;PENG Jun;ZENG Meiyan;PENG Qinghua(The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,长沙410007 [2]湖南中医药大学

出　　处：《北京中医药大学学报》2024年第6期773-781,共9页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.82074196,No.82004427);湖南省自然科学基金项目(No.2023JJ40474);湖南省教育厅科学研究项目(No.23B0347);湖南省重点实验室建设项目(No.2017TP1018)。

摘　　要：目的探讨滋阴明目方改善视网膜色素变性的作用机制。方法将20只SD大鼠按随机数字表法分为空白血清组和滋阴明目方含药血清组,每组10只。滋阴明目方含药血清组大鼠灌胃滋阴明目方(46.875 g/kg),空白血清组大鼠灌胃蒸馏水。制备滋阴明目方含药血清及空白血清,采用液质联用技术分析滋阴明目方含药血清的化学成分。采用衣霉素干预人视网膜色素上皮(ARPE-19)细胞构建内质网应激损伤模型。CCK-8法筛选滋阴明目方含药血清最优体积分数。将ARPE-19细胞分为空白组、模型组、空白血清组、滋阴明目方含药血清组、牛磺熊去氧胆酸组,分别进行干预。干预24 h后,采用多时间动态细胞功能分析系统对细胞进行形态观察,CCK-8法检测各组细胞存活率,Annexin V-FITC/PI法检测各组细胞凋亡率,蛋白质印迹法和微滴式数字PCR法检测各组细胞葡萄糖调节蛋白78(GRP78)、1型内质网转膜蛋白激酶(IRE1)、活化转录因子6(ATF6)的蛋白及mRNA表达。结果液质联用分析得到滋阴明目方含药血清中包含熊果苷、水杨酸、木犀草素、丹酚酸A、刺桐碱、牛磺酸、檞皮素、麦芽酚、黄芩苷、丹参素等主要化学成分。与模型组比较,滋阴明目方含药血清组细胞数量增多,生长较均匀,漂浮的死亡ARPE-19细胞及碎片减少;滋阴明目方含药血清组和牛磺熊去氧胆酸组的细胞存活率均升高,细胞凋亡率均下降(均P<0.05);滋阴明目方含药血清组GRP78、IRE1、ATF6蛋白表达降低,牛磺熊去氧胆酸组IRE1、ATF6蛋白表达降低(均P<0.05);滋阴明目方含药血清组和牛磺熊去氧胆酸组GRP78、IRE1、ATF6 mRNA表达降低(均P<0.05)。结论滋阴明目方可以减少ARPE-19细胞内质网应激损伤模型的细胞凋亡,其分子机制可能与下调GRP78、IRE1、ATF6的表达,抑制细胞内质网应激反应有关。Objective We aimed to explore the mechanism of Ziyin Mingmu Formula in improving retinitis pigmentosa.Methods Twenty SD rats were randomly divided into the blank serum group and the Ziyin Mingmu Formula containing serum group using a random number table method,with 10 rats in each group.The rats in the Ziyin Mingmu Formula containing serum group were given Ziyin Mingmu Formula(46.875 g/kg),while the rats in the blank serum group were given distilled water.The Ziyin Mingmu Formula containing serum and blank serum were prepared.Analysis of chemical components in Ziyin Mingmu Formula containing serum using liquid chromatography-mass spectrometry technology.Adult retinal pigment epithelial cell line-19(ARPE-19)cells were treated with tunicamycin to induce endoplasmic reticulum stress injury model.The optimal volume fraction of Ziyin Mingmu Formula containing serum was screened by CCK-8.ARPE-19 cells were divided into the blank group,the model group,the blank serum group,the Ziyin Mingmu Formula containing serum group and the tauroursodeoxycholic acid group,each group is intervened separately.After 24 hours of intervention,the morphological observation of cells was performed using a multi-time dynamic cell function analysis system,cell survival rate was detected by CCK-8,cell apoptosis rate was detected by Annexin V-FITC/PI,and protein and mRNA expressions of 78 kDa glucose regulated protein(GRP78),inositolrequiring enzyme 1(IRE1)and activating transcription factor 6(ATF6)were detected by Western blotting and ddPCR.Results The main chemical components in Ziyin Mingmu Formula containing serum were obtained by liquid chromatography-mass spectrometry analysis,such as arbutin,salicylic acid,luteolin,Salvianolic acid A,erysovine,taurine,quercetin,maltol,baicalin,and danshensu.Compared with the model group,the number of cells in Ziyin Mingmu Formula containing serum group increased,the growth was more uniform,and the floating dead ARPE-19 cells and fragments decreased.The cell survival rate of Ziyin Mingmu Formula containin

关 键 词：滋阴明目方 人视网膜色素上皮细胞系-19 内质网应激 葡萄糖调节蛋白78/1型内质网转膜蛋白激酶/活化转录因子6通路 凋亡 

分 类 号：R285.5[医药卫生—中药学]