信号素蛋白3A通过Notch1信号通路参与糖尿病大鼠视网膜神经节细胞凋亡  

Semaphorin 3A affects apoptosis of retinal ganglion cells in diabetic rats via Notch1 signaling pathway

作　　者：徐家窈[1] 郑海生[1] 陈海燕 冼文光[2] XU Jiayao;ZHENG Haisheng;CHEN Haiyan;XIAN Wenguang(Department of Corneal Diseases,Hainan Eye Hospital,Zhongshan Ophthalmic Center,Key Laboratory of Ophthalmology of Hainan Province,Sun Yat-sen University,Haikou 571300,Hainan,China;Department of Fundus Diseases,Hainan Eye Hospital,Zhongshan Ophthalmic Center,Key Laboratory of Ophthalmology of Hainan Province,Sun Yat-sen University,Haikou 571300,Hainan,China)

机构地区：[1]中山大学中山眼科中心海南眼科医院(海南省眼科医院)角膜病科,海南省眼科学重点实验室,海口571300 [2]中山大学中山眼科中心海南眼科医院(海南省眼科医院)眼底病科,海南省眼科学重点实验室,海口571300

出　　处：《海军军医大学学报》2023年第11期1297-1302,共6页Academic Journal of Naval Medical University

基　　金：海南省临床医学中心资助项目,海南省自然科学基金面上项目(819MS133)。

摘　　要：目的 探讨轴索导向分子信号素蛋白3A(SEMA3A)对糖尿病大鼠视网膜神经节细胞(RGC)凋亡的影响及其可能机制。方法 40只成年雄性SD大鼠随机分为对照组(正常喂养)、模型组[腹腔注射链脲佐菌素建立糖尿病视网膜病变(DR)模型]、si-SEMA3A组(建立DR模型后,双眼玻璃体内注射病毒滴度为1×10^(9)IU/mL的SEMA3A-si RNA慢病毒10μL)、si-SEMA3A+DAPT组[建立DR模型后,双眼玻璃体内注射病毒滴度为1×10^(9)IU/mL的SEMA3A-siRNA慢病毒10μL和浓度为10μmol/L的Notch1通路抑制剂(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)10μL],每组10只。各组处理12周后取样,采用SEMA3A和RGC标志物POU结构域转录因子3a(Brn3a)双标记免疫荧光染色检测大鼠视网膜组织中SEMA3A蛋白的表达定位;H-E染色检测大鼠RGC密度,TUNEL染色检测大鼠RGC凋亡情况,ELISA法检测大鼠视网膜组织中炎症因子IL-6和TNF-α的表达水平,蛋白质印迹法检测大鼠视网膜组织中SEMA3A、Notch1及caspase 3蛋白的表达水平。结果 双标记免疫荧光染色确定SEMA3A在大鼠RGC中特异性表达。与对照组比较,模型组大鼠的RGC密度较低(P<0.05);si-SEMA3A组大鼠的RGC密度大于模型组和si-SEMA3A+DAPT组(P均<0.05)。对照组大鼠视网膜组织中未检测到TUNEL阳性RGC,模型组大鼠视网膜组织中TUNEL阳性RGC较多,RGC凋亡率为(49.55±7.82)%;与模型组比较,si-SEMA3A组大鼠RGC凋亡率较低(P<0.05),而si-SEMA3A+DAPT组大鼠RGC凋亡率高于si-SEMA3A组(P<0.05)。模型组大鼠视网膜组织中IL-6、TNF-α、SEMA3A和caspase 3的表达水平均高于对照组,Notch1表达水平低于对照组(P均<0.05);与模型组比较,si-SEMA3A组大鼠视网膜组织中IL-6、TNF-α、SEMA3A和caspase 3表达水平均降低,Notch1表达水平升高(P均<0.05);si-SEMA3A+DAPT组大鼠视网膜组织中IL-6、TNF-α、SEMA3A和caspase 3表达水平均高于si-SEMA3A组,Notch1表达水平低于si-SEMA3A组(P均<0.05)。结论 SEMA3A可Objective To investigate the effect and mechanism of axon guidance molecule semaphorin 3A(SEMA3A)on the apoptosis of retinal ganglion cells(RGCs)in diabetic rats.Methods Forty adult male SD rats were randomly divided into control group(normal feeding),model group(establishing diabetic retinopathy[DR]model by intraperitoneal injection of streptozotocin),si-SEMA3A group(intravitreal injection of 10μL SEMA3A-siRNA lentivirus[titer 1×10^(9) IU/mL]after establishing DR model),and si-SEMA3A+DAPT group(intravitreal injection of 10μL SEMA3A-9 siRNA lentivirus[titer 1×10^(9) IU/mL]and 10μL Notch1 pathway inhibitor N-(N-((3,5-difluorophenyl)acetyl)-L-alanyl-L-2-phenyl)glycine-1,1-dimethylethyl ester[DAPT,10μmol/L]after establishing DR model),with 10 rats in each group.Samples were collected after 12 weeks of treatment in each group;immunofluorescence staining by double labeling of SEMA3A and POU domain transcription factor 3a(Brn3a,a RGC marker)was used to detect the expression and localization of retinal SEMA3A protein;hematoxylin-eosin staining was used to detect the density of RGCs;terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)method was used to detect the apoptosis of RGCs;enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of retinal inflammatory factors interleukin 6(IL-6)and tumor necrosis factorα(TNF-α);and Western blotting was used to detect the expression levels of SEMA3A,Notch1,and cysteine aspartic acid specific protease 3(caspase 3)in retinal tissues.Results The specific expression of SEMA3A in RGCs was confirmed by double-labeled immunofluorescence staining.Compared with the control group,the density of RGCs in the model group was significantly lower(P<0.05).The density of RGCs in the si-SEMA3A group was significantly higher than that in the model group and si-SEMA3A+DAPT group(both P<0.05).No TUNEL-positive RGCs were detected in the retinal tissue of control rats.In the model group,there were more TUNEL-positive RGCs in the retinal tissue

关 键 词：信号素蛋白3A 糖尿病视网膜病变 神经节细胞 细胞凋亡 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]