青光安颗粒剂对高眼压大鼠及谷氨酸诱导损伤的视网膜神经节细胞-5凋亡的影响  

Effects of Qingguangan Granules on apoptosis of retinal ganglion cell-5 in high intraocular pressure rats and glutamate-induced damage

作　　者：时健 王紫艳 刘倩宏 陈立浩 吴凯 姚小磊 SHI Jian;WANG Zi-yan;LIU Qian-hong;CHEN Li-hao;WU Kai;YAO Xiao-lei(Hunan University of Chinese Medicine,Changsha 410208,China;Key Laboratory of Hunan Province for Prevention and Treatment of Eye,Ear,Nose and Throat Diseases with Traditional Chinese Medicine,Changsha 410208,China;Hunan Provincial Engineering and Technological Research Center for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine and Protecting Visual Function,Changsha 410208,China;The First Hospital of Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学,长沙410208 [2]中医药防治眼耳鼻咽喉疾病湖南省重点实验室,长沙410208 [3]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,长沙410208 [4]湖南中医药大学第一附属医院,长沙410208

出　　处：《中华中医药杂志》2022年第5期2507-2512,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金地区科学基金项目(No.81860870);中国博士后科学基金面上资助项目(No.2018M640754);全国中医药创新骨干人才培训项目(No.湘中医药函〔2019〕67号);湖南省高层次卫生人才“225”工程培养项目(No.湘卫函〔2019〕196号);中医药防治眼耳鼻咽喉疾病湖南省重点实验室(No.2017TP1018);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(No.2018TP2008)。

摘　　要：目的:观察青光安颗粒剂对高眼压模型大鼠视网膜神经节细胞(RGCs)的凋亡及其含药血清对谷氨酸诱导损伤的RGC-5凋亡的作用。方法:取SD大鼠60只分为正常组、模型组、青光安组。采用巩膜上静脉烧灼的方法建立慢性高眼压模型,造模后测量各组眼压,3个月后成模,取各组大鼠视网膜行TUNEL法检测RGCs凋亡情况。取RGC-5分为5组:空白血清组(A组)、谷氨酸模型组(B组)、青光安含药血清组(C组)、3-MA阻断剂组(D组)以及青光安含药血清+3-MA组(E组)。通过细胞培养后,除A组以外,其余组均中加入谷氨酸模拟青光眼损伤视神经。采用生物显微镜观察细胞形态,ELISA检测细胞外培养液LDH含量,Western blot检测Cyt C、Caspase-3的蛋白表达情况,以及流式细胞仪检测RGC-5凋亡变化。结果:TUNEL检测显示模型组视网膜各层形态均发生改变,视网膜全层厚度减少,神经节细胞个数明显减少;青光安组少量凋亡细胞存在于RGCs。流式细胞仪检测显示,与A组比较,E组显著抑制细胞凋亡(P<0.05)。Western Blot显示,与A组比较,Cyt C、Caspase-3在其他4组中表达均显著升高(P<0.05);与B组比较,C组和E组中Cyt C、Caspase-3表达水平均显著降低(P<0.05)。ELISA检测显示,与A组比较,C组和E组中LDH含量显著下降(P<0.05)。结论:青光安能够通过减少LDH的释放,减少RGC-5受损程度,并抑制Cyt C的释放从而抑制Caspase-3的激活,逆转了由谷氨酸诱导RGC-5细胞所致的细胞凋亡。Objective:To observe the effects of Qingguangan Granules on the apoptosis of retinal ganglion cells(RGCs)in the retina of high intraocular pressure model and its drug-containing serum on the apoptosis of RGC-5 with glutamate-induced damage.Methods:Sixty SD rats were divided into normal group,model group,and Qingguangan group.The chronic ocular hypertension model was established by the method of suprascleral vein burning.After the modeling,the intraocular pressure of each group was measured.After 3 months,the model was formed,and the optic nerve of each group was taken to determine the apoptosis of RGCs by the TUNEL method.RGC-5 cells were divided into 5 groups:blank serum group(group A),glutamate model group(group B),drug-containing serum group(group C),3-MA blocker group(group D)and drug-containing serum+3-MA group(group E).After cell culture,glutamate was added to other groups except group A,to simulate mitochondrial autophagy in glaucoma damage optic nerve.The cell morphology was observed with a biological microscope,the LDH content of the extracellular culture medium was determined by ELISA,and the protein expression of Cyt C and Caspase-3 was detected by Western blot.Flow cytometer was used to detect the membrane potential changes of RGC-5.Results:TUNEL detection showed that the morphology of all retinal layers was changed in the model group,the whole retinal layer thickness was reduced,and the number of ganglion cells was significantly reduced.A small amount of apoptotic cells in Qingguangan group were present in RGCs.The flow cytometer showed that compared with group A,group E significantly inhibited cell apoptosis(P<0.05).Western blot displayed that compared with group A,Cyt C and Caspase-3 expressions were elevated in the other 4 groups(P<0.05).Compared with group B,the expression level of Cyt C and Caspase-3 in group C and group E was significantly reduced(P<0.05).ELISA detection showed that compared with group A,the LDH content decreased significantly in group C and group E(P<0.05).Conclusion:Qingguang

关 键 词：青光安颗粒剂 视网膜神经节细胞-5 乳酸脱氢酶 细胞色素C 胱天蛋白酶3 青光眼 细胞凋亡 谷氯酸 

分 类 号：R285.5[医药卫生—中药学]