Effects of Qingguang’an Granules on mitochondrial autophagy of retinal ganglion cells in rats with chronic ocular hypertension  

青光安颗粒剂对慢性高眼压大鼠模型视网膜神经节细胞线粒体自噬的影响

作　　者：TANG Yu ZHU Bingyao SHI Jian LIU Qianhong CHEN Lihao PENG Qinghua PENG Jun YAO Xiaolei 汤钰;朱冰瑶;时健;刘倩宏;陈立浩;彭清华;彭俊;姚小磊(湖南中医药大学第一附属医院眼科,湖南长沙410007;湖南中医药大学研究生院,湖南长沙410208;中医药防治眼耳鼻咽喉疾病湖南省重点实验室,湖南长沙410208;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,湖南长沙410208)

机构地区：[1]Department of Ophthalmology,The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China [2]Graduate School,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China [3]Hunan Provincial Key Laboratory for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine,Changsha,Hunan 410208,China [4]Hunan Provincial Engineering and Technological Research Center for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine and Protecting Visual Function,Changsha,Hunan 410208,China

出　　处：《Digital Chinese Medicine》2022年第3期295-304,共10页数字中医药（英文）

基　　金：Regional Fund Project of National Natural Science Foundation of China(81860870);China Postdoctoral Science Foundation(2018M640754);Hunan Natural Science Foundation Project(2020JJ5436);Program of Chinese Medicine Innovative-Backbone Talents of China(Xiang CM[2019]67);Hunan Province“225”Program for Cultivation of High-level Health Talents(Xiang CM[2019]196);Open Fund Project of Hunan Provincial Engineering Technology Research Center for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases and Visual Function Protection with Chinese Medicine(2018YZD02).

摘　　要：Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage目的探讨青光安颗粒剂(QGAG)对慢性高眼压大鼠模型视网膜神经节细胞(RGCs)线粒体自噬的影响及其作用机制。方法将60只SD大鼠,雌雄各半,随机分为对照组、模型组和QGAG组(2.5 g/kg),每组20只。模型组和QGAG组大鼠采用巩膜上静脉烧灼的方法建立慢性高眼压模型。造模成功后,QGAG组大鼠予以QGAG灌胃,对照组和模型组予以等剂量的生理盐水灌胃。灌胃3个月后,检测各组大鼠眼压。免疫荧光共定位检测线粒体自噬发生情况;JC-1法检测线粒体膜电位;透射电镜观察RGCs中线粒体自噬的形态学改变;荧光金法标记大鼠RGCs,计算各组RGCs密度的平均值;原位末端转移酶标记技术(TUNEL)观察RGCs凋亡情况。Western blot检测Parkin、optineurin、微管相关蛋白1A/1B-轻链3-Ⅱ/微管相关蛋白1A/1B-轻链3-Ⅰ-Ⅰ(LC3-Ⅱ/LC3-Ⅰ)、溶酶体相关膜蛋白(LAMP1)、B淋巴细胞瘤-2基因(Bcl-2)在视网膜神经节细胞中的表达情况,并辅以实时荧光定量聚合酶链反应法(qRT-PCR)检测其相应mRNA。结果QGAG能降低慢性高眼压模型大鼠的眼压(P<0.05)。QGAG能抑制RGCs线粒体自噬,并抑制RGCs凋亡(P<0.05)。QGAG能上调慢性高眼压模型大鼠视神经节细胞中Parkin和Bcl-2的表达水平(P<0.05),抑制optineurin、LAMP1和LC3-Ⅱ/LC3-Ⅰ的表达(P<0.05)。结论QGAG对慢性高眼压大鼠模型RGCs线粒体自噬具有抑制作用,对青光眼导致的视神经损伤具有一定的保护作用,其作用可能是通过线粒体自噬泛素化Parkin/PINK1相关通路进行调节。

关 键 词：Glaucoma Qingguang’an Granules(青光安颗粒剂 QGAG) Retinal ganglion cells(RGCs) Mitochondrial autophagy Parkin/PINK1 pathway 

分 类 号：R285.5[医药卫生—中药学]