Li Yi, Chen Yan-Ming, Sun Ming-Ming, Guo Xiao-Dan, Wang Ya-Chen, Zhang Zhong-Zhi
Department of Ophthalmology, First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, China.
Chin Med J (Engl). 2016 Apr 20;129(8):976-83. doi: 10.4103/0366-6999.179785.
Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival.
RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis.
Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group.
The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy.
青光眼是一种进行性视神经病变，其特征是由于视网膜神经节细胞（RGCs）丢失导致神经元变性。高眼压（HIOP）是主要危险因素，可导致视神经损伤。然而，HIOP诱导RGC死亡的确切机制尚未完全阐明。本研究旨在确定升高的静水压诱导的RGC-5细胞凋亡，探讨层粘连蛋白是否与压力下的细胞凋亡相关，层粘连蛋白是否能保护RGCs免于凋亡，并明确调节RGCs存活过程的机制。
将RGC-5细胞分别置于压力培养箱中，暴露于0、20、40和60 mmHg压力下6、12和24小时。通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色、3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐检测以及裂解的半胱天冬酶-3蛋白的蛋白质印迹法测定升高的静水压对RGC-５细胞的影响。通过免疫荧光检测层粘连蛋白的定位和表达。采用实时聚合酶链反应和蛋白质印迹分析研究β1整合素的表达、粘着斑激酶（FAK）和蛋白激酶B（PKB，或AKT）的磷酸化情况。
升高的静水压诱导培养的RGC-5细胞凋亡。40 mmHg压力作用24小时诱导的细胞凋亡最多。RGC-5细胞在40 mmHg压力下暴露24小时后，层粘连蛋白减少。用层粘连蛋白预处理后，RGC-5细胞在高压下存活。此外，与40 mmHg组相比，β1整合素以及FAK和AKT的磷酸化增加。
数据显示RGC-5细胞在静水压升高时有凋亡倾向。层粘连蛋白可通过β1整合素/FAK/AKT信号通路保护RGC-5细胞免受高压影响。这些结果表明，RGC-5细胞中层粘连蛋白减少可能是静水压升高诱导细胞凋亡的原因，层粘连蛋白或激活β1整合素/FAK/AKT通路可能是预防青光眼性视神经病变中RGC丢失的潜在治疗方法。
