摘要
目的探讨氧化低密度脂蛋白(ox-LDL)导致的视网膜损伤是否与Toll样受体-4(TLR-4)/MyD88通路激活和活化小胶质细胞使其极化为M1表型相关。方法雄性C57BL/6小鼠分为两组,实验组小鼠视网膜下注射1μL ox-LDL,对照组小鼠注射等体积的磷酸盐缓冲液(PBS);2周后,采用免疫荧光染色检测视网膜小胶质细胞的离子钙结合适配器分子1(Iba-1)、iNOS的表达,TUNEL染色检测光感受器细胞凋亡,OCT检测视网膜外核层(ONL)和内核层(INL)厚度,ERG检测视网膜功能,qPCR检测视网膜Iba-1、TLR-4和MyD88的mRNA和蛋白表达。体外实验使用ARPE-19细胞,ox-LDL组细胞用100 mg·L^(-1) ox-LDL处理24 h,PBS组细胞使用等体积的PBS处理24 h;TUNEL染色检测细胞凋亡,Western blot和qPCR检测细胞TLR-4和MyD88的mRNA和蛋白表达。结果qPCR和Western blot检测结果均显示,与对照组相比,实验组小鼠视网膜中TLR-4、MyD88、Iba-1的mRNA和蛋白表达水平均升高(均为P<0.05);与PBS组相比,ox-LDL组ARPE-19细胞中TLR-4、MyD88的mRNA和蛋白表达水平均升高(均为P<0.01)。免疫荧光染色结果显示,与对照组相比,实验组小鼠视网膜中的Iba-1、iNOS阳性细胞数均显著增加。TUNEL染色结果显示,与对照组相比,实验组小鼠视网膜中TUNEL染色阳性细胞数增加;与PBS组相比,ox-LDL组ARPE-19细胞中TUNEL染色阳性细胞数增加。ERG检测结果显示,实验组小鼠视网膜的a波和b波振幅均较对照组降低(均为P<0.001)。OCT检测结果显示,与对照组相比,实验组小鼠视网膜的ONL/INL比值下降(P<0.01)。结论ox-LDL会引起视网膜损伤,其机制可能与激活TLR-4/MyD88通路,并且使小胶质细胞活化并极化为M1表型有关。
Objective To investigate whether retinal degeneration caused by oxidized low-density lipoprotein(ox-LDL)is associated with the activation of Toll-like receptor-4/myeloid differentiation factor 88(TLR-4/MyD88)pathway and the polarization of microglia to M1 phenotype.Methods Male C57BL/6 mice were subretinally injected with 1μL of ox-LDL in the experimental group and an equal volume of PBS in the control group.Two weeks later,the expression levels of ionized calcium-binding adaptor molecule 1(Iba-1)and inducible nitric oxide synthase(iNOS)in retinal microglia were detected by immunofluorescence.Photoreceptor cell apoptosis was assessed by TUNEL staining.The thickness of the outer nuclear layer(ONL)and inner nuclear layer(INL)was measured by optical coherence tomography(OCT).Retinal function was evaluated by electroretinogram(ERG).The mRNA and protein levels of Iba-1,TLR-4 and MyD88 were detected by quantitative polymerase chain reaction(qPCR).In vitro,ARPE-19 cells were treated with 100 mg·L^(-1) ox-LDL for 24 hours in the ox-LDL group and with 100 mg·L^(-1) PBS for 24 hours in the PBS group.TUNEL staining was used to detect apoptosis,and Western blot and qPCR to detect mRNA and protein levels of TLR-4 and MyD88.Results qPCR and Western blot results showed that compared with the control group,the mRNA and protein levels of TLR-4,MyD88 and Iba-1 in the experimental group were up-regulated(all P<0.05),and compared with the PBS group,the mRNA and protein levels of TLR-4 and MyD88 in the ox-LDL group were also up-regulated(all P<0.01).Immunofluorescence results showed that the number of Iba-1 and iNOS positive cells in the experimental group was significantly increased compared with the control group.TUNEL staining results showed that the number of TUNEL-positive cells in the experimental group was increased compared with the control group,and the number of TUNEL-positive cells in the ox-LDL group was also increased compared with the PBS group.ERG results showed that the amplitudes of a and b waves in the experiment
作者
李雷
杨名珠
周庆儒
邱瑞琪
雷博
LI Lei;YANG Mingzhu;ZHOU Qingru;QIU Ruiqi;LEI Bo(Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Henan Eye Institute,Henan Eye Hospital,People’s Hospital,Henan Provincial People’s Hospital,Zhengzhou 450003,Henan Province,China;Zhengzhou University People’s Hospital,Zhengzhou University,Zhengzhou 450003,Henan Province,China)
出处
《眼科新进展》
CAS
北大核心
2022年第8期597-602,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助项目(编号:81770949,82071008)
河南省卫生健康委员会医学科技计划项目(编号:LHGJ20210072)。
