丹芪化瘀方对光诱导的人视网膜色素上皮细胞氧化应激的影响  

Effects of Danqi Huayu For⁃mula on Oxidative Stress in Light induced Human Retinal Pigment Epithelium Cells

作　　者：周媛 喻京生[2] 颜家朝[2] 秦裕辉 朱定耀 于奕 李一枝 柳葵 ZHOU Yuan;YAN Jiazhao;QIN Yuhui;YU Jingsheng;ZHU Dingyao;YU Yi;LI Yizhi;LIU Kui(The First Clinical College of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学第一中医临床学院,长沙410208 [2]湖南中医药大学第一附属医院,长沙410007

出　　处：《中国中医眼科杂志》2025年第2期108-113,126,共7页China Journal of Chinese Ophthalmology

基　　金：国家自然科学基金项目(82274583);国家基本中医药循证能力建设-中医眼科专病循证能力提升建设项目(2019XZZX-YK007);湖南省自然科学基金项目(2022JJ30460);湖南省中医药科研计划项目(D2022014);湖南省教育厅科学研究项目(21C0251);湖南省眼科疾病(中医)临床医学研究中心项目(2023SK4038)。

摘　　要：目的观察丹芪化瘀方对光诱导的人视网膜色素上皮(RPE)细胞氧化应激的影响,探讨其保护视网膜的作用机制。方法2组大鼠分别以31.05 g/kg丹芪化瘀方中药颗粒水溶液和等体积蒸馏水灌胃,制备含药血清和空白血清。常规培养的RPE细胞使用白色发光二极管(LED)冷光灯于(7,500±200)LUX光照强度的下直落式照射12 h,建立RPE细胞光损伤模型,随机分为模型组(MG)、低剂量丹芪化瘀方组(L-DQHY)、中剂量丹芪化瘀方组(M-DQHY)、高剂量丹芪化瘀方组(H-DQHY),另将采用锡纸遮光的对照细胞设为对照组(CG)。CG组细胞不加任何试剂,MG组细胞加入大鼠空白血清,L-DQHY组、M-DQHY组、HDQHY组细胞分别加入4%、8%、16%浓度的丹芪化瘀方大鼠血清,培养24 h。细胞计数试剂-8(CCK-8)法检测细胞活性,流式细胞仪检测活性氧(ROS)表达,实时荧光定量PCR(RTqPCR)法检测还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)mRNA表达,酶联免疫吸附法(ELISA)检测超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)表达水平。结果(1)细胞活性:MG组细胞活性低于CG组(t=13.592,P=0.000),M-DQHY组、H-DQHY组细胞活性高于MG组(tM-DQHY=3.910,P=0.001;tH-DQHY=9.028,P=0.000),差异均有统计学意义。(2)ROS:MG组ROS表达水平高于CG组(t=13.457,P=0.000),L-DQHY组、MDQHY组、H-DQHY组ROS表达水平低于MG组,(tL-DQHY=7.697、tM-DQHY=8.325、tH-DQHY=13.219,均P=0.000),差异均有统计学意义。(3)NOX4 mRNA:MG组NOX4 mRNA表达水平高于CG组(t=42.469,P=0.000),L-DQHY组、M-DQHY组、H-DQHY组NOX4 mRNA表达水平低于MG组(tL-DQHY=17.238、tM-DQHY=28.764、tH-DQHY=35.210,均P=0.000),差异均有统计学意义。(4)SOD、GSH-Px、MDA:MG组MDA表达水平高于CG组(t=50.375,P=0.000),SOD、GSH-Px表达水平低于CG组(tSOD=39.847、tGSH-Px=45.431,均P=0.000);L-DQHY组、M-DQHY组、H-DQHY组MDA表达水平低于MG组(tL-DQHY=13.821、tM-DQHY=20.223、tH-DQHY=40.067,均P=0.000),SOD、GSH-Px表达OBJECTIVE To observe the effects of Danqi Huayu Formula on oxidative stress in light-induced human retinal pigment epithelium(RPE)cells and explore its mechanism in retinal protection.METHODS The rats of two groups were intragastrically administered with either 31.05 g/kg of the Danqi Huayu Formula herbal granule solution or an equal volume of distilled water to prepare medicine-containing serum and blank serum,respectively.Cultured RPE cells were exposed to white light-emitting diode(LED)light at an intensity of(7,500±200)LUX for 12 h to establish an RPE cell photodamage model.The cells were randomly divided into five groups:model group(MG),low-dose Danqi Huayu Formula group(L-DQHY),medium-dose Danqi Huayu Formula group(M-DQHY),high-dose Danqi Huayu Formula group(H-DQHY),and a control group(CG)shielded from light using tin foil.No reagents were added to the CG,blank rat serum was added to the MG,and 4%,8%,and 16%concentrations of Danqi Huayu Formulacontaining rat serum were added to the L-DQHY,M-DQHY,and H-DQHY,respectively,followed by 24 h of incubation.Cell viability was detected using the Cell Counting Kit-8(CCK-8)assay,reactive oxygen species(ROS)expression was assessed by flow cytometry,nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)mRNA expression was analyzed by real-time quantitative PCR(RT-qPCR),and the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)were measured by enzyme-linked immunosorbent assay(ELISA).RESULTS(1)Cell Viability:Cell viability in the MG was lower than that in the CG(t=13.592,P=0.000),while the M-DQHY and H-DQHY exhibited higher viability compared to the MG(tM-DQHY=3.910,P=0.001;tH-DQHY=9.028,P=0.000),with significant differences.(2)ROS:ROS levels in the MG were higher than those in the CG(t=13.457,P=0.000).ROS levels in the LDQHY,M-DQHY,and H-DQHY were lower than those in the MG(tL-DQHY=7.697,tM-DQHY=8.325,tH-DQHY=13.219,all P=0.000),with significant differences.(3)NOX4 mRNA:NOX4 mRNA levels in the MG were higher than those in t

关 键 词：视网膜光损伤 丹芪化瘀方 氧化应激 视网膜色素上皮细胞 NOX4 ROS 

分 类 号：R774.1[医药卫生—眼科] R285.5[医药卫生—临床医学]