青光安Ⅱ号方有效组分对慢性高眼压DBA/2J小鼠视网膜神经节细胞的保护作用及机制  

Protective effects and mechanism of active components of Qingguang AnⅡFormula on retinal ganglion cells in DBA/2J mice with chronic intraocular hypertension

作　　者：曾志成[1,2] 李银鑫 秦惠钰 蒋鹏飞 彭俊[1,3] 彭清华 ZENG Zhicheng;LI Yinxin;QIN Huiyu;JIANG Pengfei;PENG Jun;PENG Qinghua(Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The First People's Hospital of Guiyang County,Chenzhou,Hunan 424400,China;The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南桂阳县第一人民医院,湖南郴州424400 [3]湖南中医药大学第一附属医院,湖南长沙410007

出　　处：《湖南中医药大学学报》2022年第12期1973-1981,共9页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金项目(81874492,81904260);湖南省自然科学基金项目(2018JJ3389,2020JJ5436);湖南省研究生科研创新项目(CX2018B470,CX20190584);中医药防治眼耳鼻咽喉疾病湖南省重点实验室(2017TP1018);湖南中医药大学中医学国内一流建设学科资助项目;中央财政支持地方高校重点学科和中医眼科创新团队建设项目;国家中医药管理局重点学科中医眼科学建设项目;湖南省重点学科中医五官科学建设项目;湖南省中医药防治眼耳鼻咽喉疾病与视功能保护工程技术研究中心。

摘　　要：目的观察青光安Ⅱ号方及其低、中、高浓度有效组分对青光眼模型小鼠视网膜神经节细胞的保护作用机制。方法将8只雌性C57BL/6小鼠设为正常对照组(A组)并灌胃蒸馏水,48只38周龄雌性DBA/2J小鼠随机分成6组:模型组(B组)、益脉康分散片组(C组)、青光安Ⅱ号汤剂组(D组)、青光安Ⅱ号有效组分低浓度组(E组)、青光安Ⅱ号有效组分中浓度组(F组)、青光安Ⅱ号有效组分高浓度组(G组),分别灌胃蒸馏水、益脉康分散片混悬液、青光安Ⅱ号方汤剂、青光安Ⅱ号方有效组分低/中/高浓度汤剂。干预4周后,HE染色观察小鼠视网膜组织结构,TUNEL染色观察视网膜神经节细胞凋亡,免疫组化染色观察视网膜组织神经节细胞层糖原合酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、β-连环蛋白(β-Catenin)及同源盒基因-6(paired box gene-6,Pax6)表达情况,Western blot法测定视网膜组织GSK-3β、β-Catenin及Pax6蛋白相对表达量。结果眼压测量发现,DBA/2J小鼠眼压在26周及以后明显上升,从第14周开始DBA/2J小鼠眼压明显高于C57BL/6J组(P<0.05或P<0.01);A组小鼠视网膜各层组织结构清晰、完整、染色均匀、清晰可见3个核层,神经节细胞呈单层排列,比较连续无间断,细胞呈圆形或椭圆形;B组小鼠视网膜以神经节细胞层(ganglion cell layer,GCL)损伤最为明显,神经节细胞明显减少,连续性中断,胞内空泡样变性,核固缩;C组、D组、E组、F组、G组神经节细胞数目增多,胞内空泡样变性改变均有所改善。与A组比较,B组凋亡指数明显升高(P<0.01);与B组比较,C组、D组、E组、F组、G组凋亡指数均明显降低(P<0.01);与C组、D组比较,E组凋亡指数升高(P<0.05)。与A组比较,B组视网膜GSK-3β表达量明显升高(P<0.01),β-Catenin和Pax6表达量明显降低(P<0.01);与B组比较,C组、D组、E组、F组、G组GSK-3β表达量均明显降低(P<0.01),C组、D组、G组β-Catenin表达量均有升高(P<0.Objective To observe the protective mechanism of Qingguang An ⅡFormula and its active components with low,medium and high concentration on retinal ganglion cells in glaucoma model mice.Methods Eight female C57BL/6 mice were set as normal control group(group A)with intragastric gavage of distilled water.Forty-eight 38-week-old female DBA/2J mice were randomly divided into 6 groups:Model group(group B),Yimai Kang Dispersing Tablet group(group C),decoction group of Qingguang An ⅡFormula(group D),low concentration group of active component in Qingguang An ⅡFormula(group E),medium concentration group of active component in Qingguang An ⅡFormula(group F),and high concentration group of active component in Qingguang An ⅡFormula(group G),and the distilled water,suspension of Yimai Kang Dispersible Tablet,decoction of Qingguang An ⅡFormula and low/medium/high concentration decoction of effective component in Qingguang An ⅡFormula were administered by intragastric gavage respectively.After 4 weeks of intervention,HE staining was used to observe the structure of retinal tissue and TUNEL staining was applied to observe the apoptosis of retinal ganglion cells.In addition,immunohistochemical staining was used to observe the expressions of glycogen synthase kinase-3β(GSK-3β),β-Catenin and paired box gene-6(Pax6)in the ganglion cell layer of retinal tissue.The relative expressions of GSK-3β,β-Catenin and Pax6 in retinal tissue were measured by Western blot.Results The intraocular pressure(IOP)of DBA/2J mice increased significantly after 26 weeks,and the IOP of DBA/2J mice was significantly higher than that of C57BL/6J group from week 14(P<0.05 or P<0.01).In group A,the tissue structure of each layer of the retina of mice was clear,complete,uniformly stained,and three nuclear layers were clearly visible.Ganglion cells were arranged in a single layer,which was continuous without interruption,and the cells were round or oval.In group B,ganglion cell layer(GCL)was the most obvious layer of retinal damage.Ganglion

关 键 词：青光安Ⅱ号方 青光眼 DBA/2J小鼠 Wnt/β-Catenin/Pax6信号通路 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]