糖尿病氧化应激环境中α-Klotho对巨噬细胞-血管内皮细胞串扰的影响  

Effect ofα-Klotho on macrophage-vascular endothelial cell crosstalk in diabetic oxidative stress environment

作　　者：李青博 王佩玉 胡立影 李筱荣[1] 邵彦[1,2] Li Qingbo;Wang Peiyu;Hu Liying;Li Xiaorong;Shao Yan(Tianjin Medical University Eye Hospital,School of Optometry&Eye Institute,Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science,Tianjin 300384,China;University of Tibetan Medicine,Lhasa 850000,Tibet Autonomous Region,China)

机构地区：[1]天津医科大学眼科医院,天津医科大学眼视光学院,天津医科大学眼科研究所,天津市视网膜功能与疾病重点实验室,天津市眼科学与视觉科学国际联合研究中心,天津市300384 [2]西藏藏医药大学,西藏自治区拉萨市850000

出　　处：《国际眼科杂志》2024年第7期1020-1026,共7页International Eye Science

基　　金：天津市教委科研计划项目(No.2022KJ258)。

摘　　要：目的:探讨糖尿病氧化应激环境中过表达α-Klotho(KL)的小鼠单核巨噬细胞白血病细胞(RAW264.7)对人脐静脉内皮细胞(HUVECs)增生、迁移、管腔形成以及紧密连接的影响。方法:将RAW264.7细胞分为对照组、4-羟壬二酸酯(4HNE)组、4HNE+KL组,采用免疫荧光实验检测RAW264.7细胞F4/80的表达。制备3组细胞的条件培养基用于培养HUVECs,分为M-NC组、M-4HNE组和M-4HNE+KL组。采用CCK8实验检测血管内皮细胞增生,采用划痕实验和Transwell实验检测迁移,采用管腔形成实验检测管腔形成,采用Western blot实验检测闭合蛋白5(Claudin 5)、咬合蛋白(Occludin)、带状闭合蛋白1(ZO 1)表达水平。结果:免疫荧光实验结果显示,4HNE组RAW264.7细胞F4/80荧光强度较对照组明显增强,而4HNE+KL组F4/80荧光强度较4HNE组明显减弱(均P<0.05)。CCK8实验结果显示,相比于M-NC组,M-4HNE组HUVECs增生显著增加,而M-4HNE+KL组HUVECs增生较M-4HNE组显著下降(均P<0.01)。划痕实验和Transwell实验结果显示,相比于M-NC组,M-4HNE组HUVECs迁移显著增强,而M-4HNE+KL组HUVECs迁移较M-4HNE组显著减弱(均P<0.01)。管腔形成实验结果显示,相比于M-NC组,M-4HNE组HUVECs管腔数显著增加,而M-4HNE+KL组管腔数较M-4HNE组显著下降(均P<0.01)。Western blot实验结果显示,相比于M-NC组,M-4HNE组HUVECs中Claudin 5、Occludin、ZO 1蛋白的相对表达量明显减少,而M-4HNE+KL组Claudin 5、Occludin、ZO 1蛋白的相对表达量较M-4HNE组明显增加(均P<0.01)。结论:KL通过改变糖尿病氧化应激环境中巨噬细胞激活状态抑制了HUVECs的增生、迁移、管腔形成,并增强了HUVECs的紧密连接。AIM:To investigate the effects of overexpressingα-Klotho(KL)in RAW264.7 cells stimulated by oxidative stress on the proliferation,migration,tube-formation and tight junction of human umbilical vein endothelial cells(HUVECs).METHODS:RAW264.7 cells were categorized into control,4-hydroxynonenal(4HNE),and 4HNE+KL groups,with F4/80 expression assessed via immunofluorescence staining.Three groups of conditional media were prepared for HUVECs and culture divided into M-NC,M-4HNE,and M-4HNE+KL groups.Cell proliferation was evaluated using CCK8 assay,while scratch test and Transwell assays were employed to measure cell migration.Additionally,tube-formation assay was conducted to assess cell tubule formation,and Western blot assay was utilized to detect the protein expression levels of Claudin 5,Occludin and ZO 1.RESULTS:The results of immunofluorescence staining showed that the fluorescence intensity of F4/80 of RAW264.7 cells in the 4HNE group was significantly enhanced compared with the control group,while that of F4/80 in the 4HNE+KL group was significantly decreased compared with the 4HNE group(all P<0.05).The CCK8 assay results revealed a significant increase in the proliferation of HUVECs in the M-4HNE group compared with the M-NC group.Conversely,the proliferation of the M-4HNE+KL group exhibited a significant decrease compared with that in the M-4HNE group(all P<0.01).The results of scratch test and Transwell assays demonstrated a significant increase in the migration of HUVECs in the M-4HNE group compared with the M-NC group,while the migration of the M-4HNE+KL group exhibited a significant decrease compared with the M-4HNE group(all P<0.01).In the tube-formation assay,it was observed that the number of tubes formed by HUVECs in the M-4HNE group was significantly increased compared with the M-NC group,while that of tubes formed in the M-4HNE+KL group was significantly decreased compared with the M-4HNE group(all P<0.01).Additionally,the Western blot results revealed a significant decrease in the relative expres

关 键 词：氧化应激 巨噬细胞 血管内皮细胞 增生 迁移 管腔形成 紧密连接 视网膜新生血管 

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