阿魏酸对糖尿病小鼠视网膜和高糖诱导的人RPE细胞损伤的抑制作用及其机制  

Inhibitory effect of ferulic acid on the retina of diabetic mice and high glucose-induced human retinal pigmentepithelium cell injury and the mechanism

作　　者：朱德军[1] 邹文青[1] 曹相枚 王潇飞 陆钊罡[4] Zhu Dejun;Zou Wenqing;Cao Xiangmei;Wang Xiaofei;Lu Zhaogang(Ningxia Eye Hospital,People's Hospital of Ningxia Hui Autonomous Region,Yinchuan 750002,China;Department of Pathology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750002,China;Department of Pathology,North China University of Science and Technology Affiliated Hospital,Tangshan 063000,China;Department of Pharmacy,People's Hospital of Ningxia Hui Autonomous Region,Yinchuan 750002,China)

机构地区：[1]宁夏回族自治区人民医院宁夏眼科医院,银川750002 [2]宁夏医科大学基础医学院病理学系,银川750002 [3]华北理工大学附属医院病理科,唐山063000 [4]宁夏回族自治区人民医院药学部,银川750002

出　　处：《中华实验眼科杂志》2024年第8期705-715,共11页Chinese Journal Of Experimental Ophthalmology

基　　金：宁夏回族自治区重点研发计划(2021BEG03110)。

摘　　要：目的探讨阿魏酸对糖尿病小鼠视网膜和高糖诱导的人视网膜色素上皮(RPE)细胞损伤的抑制作用及其机制。方法选取SPF级雄性8周龄2型糖尿病db/db小鼠30只,采用随机数字表法将小鼠分为模型组和阿魏酸组,每组15只,另选取15只同周龄db/m小鼠作为对照组。模型组和对照组每日采用生理盐水灌胃(5 ml/kg),阿魏酸组采用阿魏酸溶液灌胃(0.05 g/kg),治疗后2个月处死各组小鼠并摘除眼球。采用苏木精-伊红染色观察小鼠视网膜组织形态学变化;采用免疫荧光染色法和Western blot法检测各组小鼠视网膜组织线粒体钙离子单向转运蛋白(MCU)、p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)蛋白荧光强度和表达水平。取人RPE细胞,将其分为对照组、二甲基亚砜(DMSO)组、高糖组和高糖+阿魏酸组,其中对照组不做任何处理,其余各组用相应试剂培养24 h。采用活性氧簇(ROS)检测试剂盒检测各组RPE细胞ROS水平;采用线粒体膜电位检测试剂盒(JC-1)检测各组RPE细胞线粒体膜电位水平;采用微丝绿色荧光探针检测各组RPE细胞MCU和微丝荧光强度;通过慢病毒转染技术沉默和过表达MCU蛋白水平探讨MCU与p38 MAPK、p-p38MAPK间的调控关系;采用免疫荧光染色法和Western blot法检测各组RPE细胞MCU、p38 MAPK、p-p38MAPK蛋白荧光强度和表达水平。结果与对照组比较,模型组小鼠视网膜组织外核层、内核层和神经节细胞层细胞间隙增大、排列紊乱,阿魏酸组小鼠视网膜组织明显改善。与对照组比较,模型组、阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著升高,差异均有统计学意义(均P<0.05);与模型组比较,阿魏酸组小鼠视网膜组织MCU、p-p38 MAPK和MCU+p-p38 MAPK蛋白荧光强度显著降低,差异均有统计学意义(均P<0.05)。与对照组比较,模型组小鼠视网膜组织MCU、p38 MAPK和p-p38 MAPK蛋白相�Objective To investigate the inhibitory effect of ferulic acid on the retina of diabetic mice and high glucose-induced human retinal pigment epithelium(RPE)cell injury and the mechanism.Methods Thirty 8-weekold SPF male type 2 diabetic db/db mice were selected and divided into a model group and a ferulic acid group by the random number table method,with 15 mice in each group.Another 15 db/m mice of the same age were selected as a control group.The model and control groups received normal saline(5 ml/kg)by gavage daily,and the ferulic acid group received ferulic acid solution(0.05 g/kg)by gavage daily.After two months of treatment,the mice were sacrificed and the eyeballs were removed.The morphological changes of mouse retinal tissues were observed by hematoxylin-eosin staining.The fluorescence intensity and expression levels of mitochondrial calcium uniporter(MCU),p38 mitogen-activated protein kinase(p38 MAPK)and phosphorylated p38 MAPK(p-p38 MAPK)in mouse retinal tissues were detected by immunofluorescence staining and Western blot.Human RPE cells were divided into control group,dimethyl sulfoxide(DMSO)group,high glucose group and high glucose+ferulic acid group.The control group received no treatment,and the other cell groups were cultured with the corresponding reagents for 24 hours.The reactive oxygen(ROS)level of RPE cells in each group was detected with the ROS detection kit.The mitochondrial membrane potential level of RPE cells was detected with the a mitochondrial membrane potential detection kit(JC-1).The MCU and microfilament fluorescence intensity of RPE cells were detected with the a microfilament green fluorescent probe.To explore the regulatory relationship between MCU,p38 MAPK and p-p38 MAPK,the MCU protein level was silenced and overexpressed by lentivirus transfection technology.The fluorescence intensity and expression levels of MCU,p38 MAPK and p-p38 MAPK proteins in RPE cells were detected by immunofluorescence staining and Western blot.The use and feeding of experimental animals followed the

关 键 词：阿魏酸 糖尿病 视网膜 视网膜色素上皮细胞 线粒体钙离子单向转运蛋白 氧化应激 线粒体功能障碍 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]