基于核苷酸结合的寡聚结构域1/受体相互作用蛋白2通路探讨N-乙酰-5-羟色胺调控大鼠视网膜缺血再灌注后小胶质细胞极化的机制  

The mechanism of N-acetylserotonin regulating microglial polarization via NOD1/Rip2 pathway in rats after retinal ischemia reperfusion

作　　者：徐莹 刘建亮[1] 赵玉泽 王晨旭 付忻澔 李晓双 王晓莉[2] 赵岩松[1] Xu Ying;Liu Jianliang;Zhao Yuze;Wang Chenxu;Fu Xinhao;Li Xiaoshuang;Wang Xiaoli;Zhao Yansong(Department of Ophthalmology,The Affiliated Hospital of Weifang Medical University,Weifang 261031,China;School of Medical Imaging,Weifang Medical University,Weifang 261053,China;School of Basic Medicine,Weifang Medical University,Weifang 261053,China)

机构地区：[1]潍坊医学院附属医院眼科,潍坊261031 [2]潍坊医学院医学影像学院,潍坊261053 [3]潍坊医学院基础医学院,潍坊261053

出　　处：《中华眼底病杂志》2024年第4期287-295,共9页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(82071888);山东省自然科学基金(ZR2021MH351、ZR2020MH074);山东省医药卫生发展计划项目(2017WS738);潍坊市科学技术计划发展项目(2021GX057、2021YX039)。

摘　　要：目的观察N-乙酰-5-羟色胺(NAS)对大鼠视网膜缺血再灌注损伤(RIRI)后视网膜小胶质细胞极化的影响,并基于核苷酸结合的寡聚结构域1(NOD1)/受体相互作用蛋白2(Rip2)通路初步探讨其机制。方法采用随机数字表法将60只雄性Sprague Dawley大鼠分为假手术组(Sham组)、RIRI组、NAS组,分别为21、21、18只,以右眼为实验眼。RIRI组、NAS组大鼠采用前房高眼压法建立RIRI模型。NAS组大鼠分别于建模前及建模后30 min腹腔注射10 mg/kg NAS。建模后24 h,苏木素-伊红、免疫组织化学染色观察各组大鼠视网膜形态学变化,计数视网膜神经节细胞(RGC);免疫荧光染色观察NAS干预对大鼠视网膜小胶质细胞极化的影响。转录组测序筛选Sham组、RIRI组间差异基因,蛋白质免疫印迹法(Western blot)、实时定量聚合酶链反应(RT-PCR)进行验证。免疫组织化学染色、Western blot、RT-PCR观察NAS对大鼠视网膜组织及小胶质细胞中NOD1、Rip2蛋白、mRNA表达的影响。一般线性回归分析NAS组、RIRI组大鼠视网膜组织中NOD1+细胞数差值与M1、M2型小胶质细胞数差值的相关性。结果Sham组大鼠视网膜可见大量RGC;建模后24 h,与Sham组比较,RIRI组大鼠视网膜内层厚度显著增加、RGC数量显著减少;NAS组大鼠视网膜内层厚度显著变薄,RGC数量显著增多。与Sham组比较,RIRI组大鼠视网膜M1、M2型小胶质细胞数显著增多;与RIRI组比较,NAS组大鼠视网膜M1型小胶质细胞数显著减少,M2型小胶质细胞显著增多。三组大鼠视网膜M1、M2型小胶质细胞数比较,差异均有统计学意义(P<0.05)。转录组测序结果显示,建模后24 h,视网膜NOD1、Rip2为重要差异基因;RIRI组视网膜中NOD1、Rip2 mRNA、蛋白相对表达量显著高于Sham组,差异均有统计学意义(P<0.05)。RIRI组视网膜小胶质细胞中NOD1+、Rip2+细胞数及mRNA、蛋白相对表达量显著高于Sham组,NAS组亦较Sham组显著升高,但低于RIRI组,差异�Objective To investigate the effect of N-acetylserotonin(NAS)on the retinal microglia polarization in retinal ischemia-reperfusion injury(RIRI)rats and explore its mechanism via nucleotide-bound oligomeric domain 1(NOD1)/receptor interacting protein 2(Rip2)pathway.Methods Healthy male Sprague Dawley rats were randomly divided into Sham(n=21),RIRI(n=21)and NAS(injected intraperitoneally 30 min before and after modeling with NAS,10 mg/kg,n=18)groups,using random number table.And the right eye was used experimental eye.The RIRI model of rats in RIRI group and NAS group was established by anterior chamber high intraocular pressure method.Rats in NAS group were intraperitoneally injected with 10 mg/kg NAS before and 30 min after modeling,respectively.The retinal morphology and the number of retinal ganglion cell(RGC)in each group were detected by hematoxylin-eosin staining and immunohistochemical staining.The effect of NAS on polarization of retinal microglia was detected by immunofluorescence staining.Transcriptome sequencing technology was used to screen out the differentially expressed genes between Sham and RIRI groups.Western blot and real-time quantitative polymerase chain reaction(RT-PCR)were used to examine the differentially expressed genes.Immunohistochemical staining,Western blot and RT-PCR were used to investigate the effect of NAS on the expression of NOD1 and Rip2 protein and mRNA in retinal tissue and microglia of rats.General linear regression analysis was performed to determine the correlation between the number difference of NOD1+cells and the number difference of M1 and M2 microglia in retinal tissues of rats in NAS group and RIRI group.Results A large number of RGC were observed in the retina of rats in Sham group.24 h after modeling,compared with Sham group,the inner retinal thickness of rats in RIRI group was significantly increased and the number of RGC was significantly decreased.The thickness of inner retina in NAS group was significantly thinner and the number of RGC was significantly increas

关 键 词：视网膜缺血再灌注损伤 N-乙酰-5-羟色胺 视网膜小胶质细胞 核苷酸结合的寡聚结构域 动物实验 

分 类 号：R774.1[医药卫生—眼科]