Xiao Yan-Ling, Wang Xiao-Rui, Ye Kejing, Chen Wan-Zhu, Zheng Bing-Ru, Huang Yi-Hong, Wu Yu-Yu
Department of Ophthalmology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian Province, China.
J Ophthalmol. 2022 Sep 10;2022:6026464. doi: 10.1155/2022/6026464. eCollection 2022.
To investigate the effect of 3-methyladenine (3-MA) and starvation on the expression of matrix metalloproteinase (MMP-2) in patients with primary open-angle glaucoma.
Primary TM cells were cultured and divided into three groups. The control group was treated with a normal medium, the 3-MA group was stimulated with 3-MA, and the starvation group received nutrient depletion by replacing the normal media with Earle's balanced salt solution. Cellular mRNA and protein were measured at different 3-MA concentrations and starvation time periods. The level of autophagy was accessed by monodansylcadaverine fluorescent staining and expression of specific autophagy-related genes, light chain 3 (LC3), and Beclin1. The effects of 3-MA and starvation on cell proliferation were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay kit. The mRNA and protein expression of LC3-II, Beclin1, and MMP-2 were measured by reverse transcription-polymerase chain reaction and western blot, respectively.
Compared to the control group, starvation significantly upregulated LC3-II and Beclin1 in TM cells after 3 h of stimulation, which peaked at 6 h and 9 h, respectively. Increased MDC-labeled cells were also observed. Starvation downregulated the expression of MMP-2. On the contrary, 3-MA suppressed the activation of autophagy, as shown by the marked downregulation of LC3-II and Beclin1. The expressions of MMP-2 were higher in the 3-MA group compared to the control group, reaching a peak at a concentration of 5 mM.
Autophagy may be involved in the pathogenesis of POAG via regulating the expression of MMP-2 and, subsequently, the deposition of the extracellular matrix.
探讨3-甲基腺嘌呤（3-MA）和饥饿对原发性开角型青光眼患者基质金属蛋白酶（MMP-2）表达的影响。
培养原代小梁网细胞并分为三组。对照组用正常培养基处理，3-MA组用3-MA刺激，饥饿组用Earle平衡盐溶液替换正常培养基以造成营养缺乏。在不同的3-MA浓度和饥饿时间段测量细胞的mRNA和蛋白质。通过单丹磺酰尸胺荧光染色以及特异性自噬相关基因轻链3（LC3）和Beclin1的表达来评估自噬水平。用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐检测试剂盒测定3-MA和饥饿对细胞增殖的影响。分别通过逆转录-聚合酶链反应和蛋白质免疫印迹法测量LC3-II、Beclin1和MMP-2的mRNA和蛋白质表达。
与对照组相比，饥饿刺激3小时后小梁网细胞中LC3-II和Beclin1显著上调，分别在6小时和9小时达到峰值。还观察到单丹磺酰尸胺标记的细胞增加。饥饿下调了MMP-2的表达。相反，3-MA抑制了自噬的激活，表现为LC3-II和Beclin1明显下调。3-MA组中MMP-2的表达高于对照组，在浓度为5 mM时达到峰值。
自噬可能通过调节MMP-2的表达，进而参与原发性开角型青光眼的发病机制以及细胞外基质的沉积。
