基于HSP72探讨通络明目方对青光眼模型大鼠RGC和视神经的保护作用  

Protective Effect of Tongluo Mingmu Formula on Retinal Ganglion Cell and Optic Nerve in Rat Model of Glaucoma Based on HSP72 Regulation

作　　者：赵颖 张珊 刘子豪[1] 孙雨辰 段琪 邓桂江[1] 赵静如[1] ZHAO Ying;ZHANG Shan;LIU Zihao;SUN Yuchen;DUAN Qi;DENG Guijiang;ZHAO Jingru(Dong-zhimen Hospital,Beijing University of Chinese Medicine,Beijing 101121,China)

机构地区：[1]北京中医药大学东直门医院,北京101121

出　　处：《中国中医眼科杂志》2024年第5期401-407,共7页China Journal of Chinese Ophthalmology

基　　金：北京中医药大学基本科研业务费项目(2021-JYB-XJSJJ-054)。

摘　　要：目的探讨通络明目方调控热休克蛋白72(HSP72)对青光眼模型大鼠视网膜神经节细胞(RGC)和视神经的保护作用。方法采用Shareef-Sharma法将40只大鼠建立青光眼模型,随机分为模型组(MG)和通络明目方组(TM),各20只,另将进行假手术的24只大鼠设为对照组(CG)。TM组大鼠每日通络明目方8.61 g/kg灌胃,CG、MG组大鼠每日等体积0.9%氯化钠注射液灌胃,连续给药21 d。造模后第1、7、14、21天时,使用眼压计测量大鼠眼压,采集大鼠造模对侧眼眼眶血0.5 mL,酶联免疫吸附法(ELISA)检测大鼠血清HSP72水平。给药完成后,苏木精-伊红(HE)染色观察大鼠视网膜形态结构,原位末端转移酶标记技术(TUNEL)检测大鼠RGC细胞数量及凋亡率,测定视网膜厚度;免疫荧光和免疫组织化学法检测RGC细胞HSP72表达。结果(1)眼压:MG组大鼠造模后1、7、14、21 d眼压均高于CG组(t_(1 d)=7.758、t_(7 d)=10.913、t_(14 d)=17.353、t_(21 d)=7.955,均P=0.000),TM组大鼠造模后7、14、21 d眼压均低于MG组(t_(7 d)=-2.604,P=0.021;t_(14 d)=-8.815,P=0.000;t_(21 d)=-4.163,P=0.001),差异均有统计学意义。其他时间比较,差异均无统计学意义(P>0.05)。(2)血清HSP72:MG组大鼠造模后1、7、14、21 d血清HSP72水平均高于CG组(t_(1 d)=16.450、t_(7 d)=23.576、t_(14 d)=13.206、t_(21 d)=8.934,均P=0.000),TM组大鼠造模后21 d血清HSP72水平高于MG组(t=5.936,P=0.000),差异均有统计学意义;其他时间比较,差异均无统计学意义(P>0.05)。(3)视网膜形态和厚度:MG组大鼠RGC出现数目减少和空泡化现象,内核层细胞开始排列紊乱、疏松,神经纤维层水肿,内核层疏松,厚度较CG组薄;而TM组大鼠视网膜RGC数目轻度减少、空泡化、内核层细胞状态与MG相比均较轻,厚度较MG组厚。(4)RGC数量和凋亡率:MG组大鼠造模后14、21 d视网膜RGC数量低于CG组(t_(14 d)=-5.886,P=0.000;t_(21 d)=-4.424,P=0.001),1、7、14、21 d RGC凋亡率高于CG组(t_(1 d)=22.165、t_(7 d)=36.OBJECTIVE To investigate the protective effect of Tongluo Mingmu Formula on retinal ganglion cell(RGC)and optic nerve in rat models of glaucoma by regulating heat shock protein 72(HSP72).METHODS A total of 40 rats were used to establish the glaucoma model using the Shareef-Sharma method and were randomly divided into the model group(MG)and the Tongluo Mingmu Formula group(TM),with 20 rats in each group.An additional 24 rats underwent sham surgery and served as the control group(CG).Rats in the TM group were orally administered Tongluo Mingmu Formula at a dose of 8.61 g/kg daily,while rats in CG and MG were orally administered an equivalent volume of 0.9%sodium chloride injection daily for 21 days.On the first,seventh,14th,and 21st day after modeling,intraocular pressure was measured,and 0.5 mL of blood was collected from the retro-orbital sinus of the opposite eye of the rats.The enzyme-linked immunosorbent assay(ELISA)was used to detect serum HSP72 levels.After completion of treatment,hematoxylin-eosin(HE)staining was performed to observe the morphological structure of the rat retina,and TdT-mediated dUTP nick-end labeling(TUNEL)assay was used to detect the number and apoptosis rate of RGC in rats.Retinal thickness was also measured.Immunofluorescence and immunohistochemical methods were used to detect the expression of HSP72 in RGC.RESULTS(1)Intraocular pressure:In the MG group,intraocular pressure was higher than that in the CG group at first,seventh,14th,and 21st day after modeling(t_(1 d)=7.758,t_(7 d)=10.913,t_(14 d)=17.353,t_(21 d)=7.955,all P=0.000).In the TM group,intraocular pressure was lower than that in the MG group at first,seventh,14th,and 21st dayafter modeling(t_(7 d)=-2.604,P=0.021;t_(14 d)=-8.815,P=0.000;t_(21 d)=-4.163,P=0.001),with statistically significant differences.There were no statistically significant differences at other time points(P>0.05).(2)Serum HSP72:Serum HSP72 levels in the MG group were higher than those in the CG group at first,seventh,14th,and 21st day after modeling(t_(1 d)

关 键 词：热休克蛋白72 青光眼 通络明目方 视神经 眼压 

分 类 号：R285.5[医药卫生—中药学]