Notch1和自噬对高糖诱导下的人视网膜色素上皮细胞的作用  

Role of Notch1 and autophagy on high glucose-induced human retinal pigment epithelial cells

作　　者：秦婷婷 王苏涵 张乐颖 王娇娇 宋宗明 QIN Tingting;WANG Suhan;ZHANG Leying;WANG Jiaojiao;SONG Zongming(Graduate School of Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Department of Ophthalmology,Henan Provincial People’s Hospital,Henan Eye Hospital,Zhengzhou 450003,Henan Province,China;Department of Ophthalmology,Henan University People’s Hospital,Zhengzhou 450003,Henan Province,China)

机构地区：[1]新乡医学院研究生院,河南省新乡市453003 [2]河南省人民医院眼科,河南省立眼科医院,河南省郑州市450003 [3]河南大学人民医院眼科,河南省郑州市450003

出　　处：《眼科新进展》2024年第10期780-785,共6页Recent Advances in Ophthalmology

基　　金：中原科技领军人才项目(编号:224200510013);河南省科技攻关联合共建项目(编号:LHGJ20190817);河南省立眼科医院基础研究专项(编号:20JCZD001);河南省重大科技专项(221100310200)。

摘　　要：目的观察高糖条件下Notch1与自噬之间的关系,并探讨Notch1抑制剂DAPT和自噬抑制剂3-MA对高糖培养的人视网膜色素上皮细胞的影响。方法预实验选定25 mmol·L^(-1)葡萄糖作为ARPE-19细胞的高糖培养液,5 mmol·L^(-1)3-MA作为自噬抑制剂浓度。将体外培养的ARPE-19细胞随机分为对照组(添加5 mmol·L^(-1)葡萄糖培养48 h)、高糖组(添加25 mmol·L^(-1)葡萄糖培养48 h)、高糖+DAPT组(添加25 mmol·L^(-1)葡萄糖+40μmol·L^(-1)DAPT,40μmol·L^(-1)DAPT先处理2 h,然后更换25 mmol·L^(-1)葡萄糖处理48 h)及高糖+3-MA组(添加25 mmol·L^(-1)葡萄糖+5 mmol·L^(-1)3-MA,5 mmol·L^(-1)3-MA处理2 h,然后更换25 mmol·L^(-1)葡萄糖处理48 h)。采用透射电镜观察各组细胞超微结构;CCK-8和划痕实验检测各组细胞增殖和迁移情况;Western blot检测各组细胞Notch1和自噬相关蛋白LC3、Beclin1的蛋白表达情况;RT-PCR法检测各组细胞中Notch1、LC3、Beclin1的mRNA相对表达量。结果透射电子显微镜下对照组细胞结构正常,核呈圆形或卵圆形,自噬小体少量可见;高糖组细胞损伤略微明显,胞质不均且可见较多自噬溶酶体。与对照组相比,高糖组细胞增殖、迁移能力均上升,Notch1、LC3、Beclin1的mRNA和蛋白表达水平均增高(均为P<0.05);与高糖组相比,高糖+DAPT组细胞增殖、迁移能力均下降,Notch1、LC3、Beclin1的mRNA和蛋白表达水平均降低(均为P<0.05);与高糖组相比,高糖+3-MA组细胞增殖、迁移能力均下降,LC3、Beclin1的mRNA和蛋白表达水平均降低(均为P<0.05)。结论高糖可激活Notch1和自噬过程并促进ARPE-19细胞的增生,而Notch1抑制剂DAPT和自噬抑制剂3-MA在一定程度上可抑制自噬的进程,发挥抑制细胞增生的作用。Objective To investigate the relationship between Notch1 and autophagy under high glucose conditions and to explore the effects of Notch1 inhibitor DAPT and autophagy inhibitor 3-MA on human retinal pigment epithelial cells cultured in high glucose conditions.Methods Via preliminary experiment,25 mmol·L^(-1)glucose was used as the high glucose culture medium of adult retinal pigment epithelial(ARPE)-19 cells,and 5 mmol·L^(-1)3-MA was adopted as the autophagy inhibitor.ARPE-19 cells cultured in vitro were randomly divided into four groups:control group(treated with 5 mmol·L^(-1)glucose for 48 h),high glucose group(treated with 25 mmol·L^(-1)glucose for 48 h),high glucose+DAPT group(treated with 40μmol·L^(-1)DAPT for 2 h and then 25 mmol·L^(-1)glucose for 48 h),and high glucose+3-MA group(treated with 5 mmol·L^(-1)3-MA for 2 h and then 25 mmol·L^(-1)glucose for 48 h).A transmission electron microscope was used to observe the ultrastructure of cells in each group.Cell proliferation and migration were observed using Cell Counting Kit-8 and scratch assays.Western blot was used to detect the protein expression levels of Notch1 and autophagy-related proteins LC3 and Beclin1.Reverse transcription-polymerase chain reaction was used to measure the relative messenger ribonucleic acid(mRNA)expression levels of Notch1,LC3 and Beclin1 of cells in each group.Results Transmission electron microscope showed that cells in the control group had normal structures,with round or oval nuclei and a few autophagosomes.In the high glucose group,cells exhibited slightly obvious injury,with uneven cytoplasm and numerous autolysosomes.Compared to the control group,ARPE-19 cells in the high glucose group had increased proliferation and migration abilities,and higher mRNA and protein expression levels of Notch1,LC3 and Beclin1(all P<0.05).Compared to the high glucose group,ARPE-19 cells in the high glucose+DAPT group showed decreased proliferation and migration abilities,and lower mRNA and protein expression levels of Notch1,LC3 and B

关 键 词：自噬 NOTCH信号通路 人视网膜色素上皮细胞 高糖 

分 类 号：R774.1[医药卫生—眼科]