探究半乳糖凝集素-3在高糖诱导的视网膜色素上皮细胞氧化损伤和炎症因子表达中的作用  

Effects of galectin-3 on oxidative damage and expression of inflammatory factors in retinal pigment epithelial cells induced by high glucose

作　　者：刘珮 蔡宇 王德成 韩珊珊 LIU Pei;CAI Yu;WANG Decheng;HAN Shanshan(Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,China Three Gorges University,Yichang 443002,Hubei Province,China;College of Basic Medical Science,China Three Gorges University,Yichang 443002,Hubei Province,China)

机构地区：[1]三峡大学肿瘤微环境与免疫治疗湖北省重点实验室,湖北省宜昌市443002 [2]三峡大学基础医学院,湖北省宜昌市443002

出　　处：《眼科新进展》2025年第4期269-275,共7页Recent Advances in Ophthalmology

基　　金：国家自然科学基金青年项目(编号:82000914);肿瘤微环境与免疫治疗湖北省重点实验室开放基金项目(编号:2023KZL017,2023KZL029)。

摘　　要：目的探究半乳糖凝集素-3(Gal3)在高糖诱导的人视网膜色素上皮(hTERT-RPE)细胞内对自噬活动的影响。方法将体外培养的hTERT-RPE细胞随机分为正常组(C组)、高糖组(HG组)、高糖+si-NC组(HG+si-NC组)和高糖+si-Gal3组(HG+si-Gal3组),采用RT-PCR法检测各组hTERT-RPE细胞内Gal3、细胞间黏附分子(ICAM)-1、白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α的mRNA相对表达水平;ELISA检测各组hTERT-RPE细胞上清液中IL-1、IL-6的表达水平;DCFH-DA荧光探针染色、比色法和微板法分别检测各组hTERT-RPE细胞内活性氧(ROS)表达水平、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;Western blot检测各组hTERT-RPE细胞内Gal3、微管相关蛋白1轻链3(LC3)Ⅱ型和Ⅰ型的比值(LC3Ⅱ/LC3Ⅰ)、苄氯素1(Beclin1)、自噬相关(ATG)5、ATG7的蛋白表达水平;在各组hTERT-RPE细胞内分别单独转染双荧光mRFP-GFP-LC3质粒和共同转染RFP-LAMP1、GFP-LC3质粒,激光共聚焦显微镜检测两种方法转染后各组hTERT-RPE细胞内自噬流变化。结果与C组相比,HG组hTERT-RPE细胞内Gal3、IL-1、IL-6、TNF-α、ICAM-1、P62、ROS表达水平以及MDA含量均升高(均为P<0.05),LC3Ⅱ/LC3Ⅰ、Beclin1、ATG7、ATG5表达水平以及SOD活性、双荧光mRFP-GFP-LC3质粒红色荧光斑点(自噬溶酶体)和黄色荧光斑点(自噬小体)数量均降低(均为P<0.05),RFP-LAMP1、GFP-LC3质粒共定位的黄色荧光斑点(自噬溶酶体)数量差异无统计学意义(P>0.05);与HG组相比,HG+si-NC组hTERT-RPE细胞内上述指标的表达水平差异均无统计学意义(均为P>0.05);与HG+si-NC组相比,HG+si-Gal3组hTERT-RPE细胞内Gal3、IL-1、IL-6、TNF-α、ICAM-1、P62、ROS表达水平以及MDA含量均降低(均为P<0.05),LC3Ⅱ/LC3Ⅰ、Beclin1、ATG7、ATG5表达水平以及SOD活性、双荧光mRFP-GFP-LC3质粒红色和黄色荧光斑点数量均升高(均为P<0.05),RFP-LAMP1、GFP-LC3质粒共定位的黄色荧光斑点数量差异无统计学意义(P>0.05)。结论Gal3在Objective To investigate the effects of galectin-3(Gal3)on autophagy in high glucose-induced human telomerase reverse transcriptase-immortalized retinal pigment epithelial(hTERT-RPE)cells.Methods The hTERT-RPE cells cultured in vitro were randomly divided into a normal group(group C),a high glucose group(group HG),a high glucose+si-NC group(group HG+si-NC)and a high glucose+si-Gal3 group(group HG+si-Gal3).Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the relative mRNA expression levels of Gal3,intercellular adhesion molecule(ICAM)-1,interleukin(IL)-6 and tumor necrosis factor(TNF)-αin hTERT-RPE cells of each group.The expression levels of IL-1 and IL-6 in the supernatant of hTERT-RPE cells were measured by enzyme-linked immunosorbent assay(ELISA).The expression level of reactive oxygen species(ROS),the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in hTERT-RPE cells were analyzed by dichlorofluorescin diacetate(DCFH-DA)staining,the colorimetric method and the microplate method,respectively.The protein expression levels of Gal3,the ratio of microtubule associated protein 1 light chain 3(LC3)type II to type I(LC3Ⅱ/LC3Ⅰ),Beclin 1,and autophagy-associated 5(ATG5)and 7(ATG7)in hTERT-RPE cells from each group were detected by Western blot analysis.After the hTERT-RPE cells in each group were transfected with double fluorescent mRFP-GFP-LC3 plasmids alone and with RFP-LAMP1 and GFP-LC3 plasmids jointly,the changes of autophagy flow in hTERT-RPE cells were detected by laser confocal microscopy.Results Compared with the group C,the group HG showed an increase in the expression of Gal3,IL-1,IL-6,TNF-α,ICAM-1,P62,ROS and MDA content(all P<0.05).However,the expression of LC3Ⅱ/LC3Ⅰ,Beclin1,ATG7,ATG5,the SOD activity,and the number of red(autolysosomes)and yellow fluorescence spots(autophagosomes)of the double fluorescent mRFP-GFP-LC3 plasmid were all lower in the group HG than those in the group C(all P<0.05).There was no significant difference in the number

关 键 词：糖尿病视网膜病变 半乳糖凝集素-3 炎症反应 氧化应激 自噬 

分 类 号：R774[医药卫生—眼科]