丹皮酚对大鼠糖尿病视网膜病变的改善作用及其调节miR-802-5p表达的作用机制  

Improvement effect of paeonol on diabetic retinopathy in rats and its mechanism of regulating miR-802-5p expression

作　　者：孙俊波[1] 高达[2] 赵逸菲 许华[2] 邱帆 赵璐[2] SUN Junbo;GAO Da;ZHAO Yifei;XU Hua;QIU Fan;ZHAO Lu(Department of Internal Medicine,Second Affiliated Hospital,Henan University of Traditional Chinese Medicine,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Department of Endocrinology,Third Affiliated Hospital,Henan University of Traditional Chinese Medicine,Zhengzhou 450008,China)

机构地区：[1]河南中医药大学第二附属医院河南省中医院内科,河南郑州450002 [2]河南中医药大学第三附属医院内分泌科,河南郑州450008

出　　处：《吉林大学学报（医学版）》2022年第1期82-93,共12页Journal of Jilin University：Medicine Edition

基　　金：河南省中医药管理局资助项目(2021ZY2225,豫中医科教[2018]35号)。

摘　　要：目的:探讨丹皮酚对大鼠糖尿病视网膜病变(DR)的改善作用,并阐明其可能的作用机制。方法:40只SPF级SD大鼠随机分为对照组、DR模型组、15 mg·kg^(-1)丹皮酚组和30 mg·kg^(-1)丹皮酚组,每组10只,除对照组外其余3组大鼠腹腔注射55 mg·kg^(-1)链脲佐菌素(STZ)构建DR模型。建模成功后,对照组和DR模型组大鼠皮下注射8 mL·kg^(-1)生理盐水,15和30 mg·kg^(-1)丹皮酚组大鼠皮下注射15和30 mg·kg^(-1)丹皮酚磺酸钠注射液,每日1次,共2周。取大鼠视网膜组织用于实时荧光定量PCR(RT-qPCR)、Western blotting和酶联免疫吸附测定(ELISA)检测,HE染色观察各组大鼠视网膜组织病理形态表现。采用高糖(HG,33 mmol·L^(-1)葡萄糖)培养基培养人视网膜微血管内皮细胞(HRMECs)。采用不同剂量(0、5、25、50、70和100 mg·L^(-1))丹皮酚分别干预HRMECs和HG处理24 h后的HRMECs,采用CCK-8法和流式细胞术检测各组细胞活性和凋亡率,以确定后续实验中丹皮酚最适作用剂量。HRMECs进行转染后分为对照组、HG组、HG+NC inhibitor组、HG+miR-802-5p inhibitor组、HG+丹皮酚(25 mg·L^(-1))组、HG+丹皮酚+NC mimic组和HG+丹皮酚+miR-802-5p mimic组。采用RT-qPCR法检测各组大鼠视网膜组织和各组细胞中miR-802-5p表达水平,荧光素酶报告基因和RNA免疫共沉淀法检测HRMECs中miR-802-5p与SIRT6的结合关系,Western blotting法检测各组大鼠视网膜组织和各组细胞中沉默调节蛋白6(SIRT6)、色素上皮衍生因子(PEDF)和血管内皮生长因子(VEGF)蛋白表达水平,ELISA法检测各组大鼠视网膜组织和各组细胞中活性氧(ROS)水平及过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性。结果:与对照组比较,DR模型组大鼠视网膜组织中miR-802-5p表达水平和VEGF蛋白表达水平明显升高(P<0.01),SIRT6和PEDF蛋白表达水平明显降低(P<0.01),ROS水平明显升高(P<0.01),CAT和SOD活性明显降低(P<0.01);与DR模型组比较,不同剂量丹皮酚组大Objective:To investigate the improvement effect of paeonol on diabetic retinopathy(DR)in the rats,and to elucidate its possible mechanism.Methods:A total of 40 SPF grade SD rats were randomly divided into control group,DR model group,15 mg·kg^(-1) paeonol group and 30 mg·kg^(-1) paeonol group;there were 10 rats in each group.Except control group,the rats in other groups were intraperitoneally injected with 55 mg·kg^(-1) streptozotocin(STZ)to establish the DR medels.After successeful modeling,the rats in control group and DR model group were subcutaneously injected with normal saline,and the rats in 15 and 30 mg·kg^(-1) paeonol groups were subcutaneously injected with 15 and 30 mg·kg^(-1) paeonol sodium sulfonate.The rat retina tissue was harvested for real-time fluorescence quantitative PCR(RT-qPCR),Western blotting,and ELISA assays and HE staining was performed to observe the pathomorphology of retina tissue of the rats in various groups.The human retinal microvascular endothelial cells(HRMECs)were cultured in high glucose(HG,33 mmol·L^(-1) glucose medium).Different doses(0,5,25,50,70 and 100 mg·L^(-1))of paeonol were used to intervene the HRMECs and the HRMECs treated with HG for 24 h,respectively;the cell viabilities and apoptotic rates of cells in various groups were detected by CCK-8 assay and flow cytometry,respectively.The optimum dose used in the follow-up experiment was confirmed.The transfected HRMECs were divided into control group,HG group,HG+NC inhibitor group,HG+miR-802-5p inhibitor group,HG+paeonol(25 mg·L^(-1))group,HG+paeonol+NC mimic group,and HG+paeonol+miR-802-5p mimic group.The expression levels of miR-802-5p in the retina tissue and the cells in various groups were determined by RT-qPCR method,and the association between miR-802-5p and SIRT6 in the HRMECs was determined by luciferase reporter gene assay and RNA co-immunoprecipitation assay;the protein expression levels of silent information regulator 6(SIRT6),pigment epithelium derived factor(PEDF)and vascular endothelial growth fact

关 键 词：糖尿病视网膜病变 丹皮酚 微小RNA-802-5p 沉默调节蛋白6 高糖 

分 类 号：R587.1[医药卫生—内分泌]