Lv Bochang, Chen Tao, Xu Zhiguo, Huo Fuquan, Wei Yanyan, Yang Xinguang
Shaanxi Ophthalmic Medical Center, Xi'an No. 4 Hospital, Affiliated Guangren Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China.
Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
Int J Mol Med. 2016 Jan;37(1):225-32. doi: 10.3892/ijmm.2015.2418. Epub 2015 Nov 23.
Glaucoma is a degenerative nerve disorder that results in irreversible blindness. It has been reported that the apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. Oxidative stress is one of the major factors that cause apoptosis of RGCs. Crocin has many beneficial effects, including antioxidant and anti-apoptotic actions. However, the mechanism by which crocin protects against oxidative stress‑induced damage to RGCs remains unclear. The present study aimed to investigate the mechanism by which crocin protects RGC-5 cells against H2O2-induced damage. H2O2 was used to establish a model of oxidative stress injury in RGC-5 cells to mimic the development of glaucoma in vitro. Different concentrations (0.1 and 1 µM) of crocin were added to test whether crocin was capable of protecting RGCs from H2O2-induced damage. WST-1, lactic dehydrogenase (LDH) release and Annexin V/FITC assays were then performed. Levels of reactive oxygen species (ROS) were detected using a ROS assay kit, mitochondrial membrane potential (ΔΨm) was analyzed by JC-1 staining, caspase-3 activity was examined using a Caspase-3 assay kit, and the protein levels of Bax, Bcl-1 and cytochrome c were measured using western blot analysis. In addition, the protein level of phosphorylated nuclear factor-κB (p-NF-κB) p65 was also evaluated using western blot analysis. The results showed that crocin protected RGC-5 cells from apoptosis, decreased LDH release and enhanced cell viability. Additional experiments demonstrated that crocin decreased ROS levels, increased ΔΨm, downregulated the protein expression of Bax and cytochrome c, promoted Bcl-2 protein expression and activated NF-κB. Taken together, the findings of this study indicate that crocin prevented H2O2‑induced damage to RGCs through the mitochondrial pathway and activation of NF-κB.
青光眼是一种导致不可逆失明的退行性神经疾病。据报道，视网膜神经节细胞（RGCs）的凋亡是青光眼的一个标志。氧化应激是导致RGCs凋亡的主要因素之一。藏红花素具有许多有益作用，包括抗氧化和抗凋亡作用。然而，藏红花素保护RGCs免受氧化应激诱导损伤的机制仍不清楚。本研究旨在探讨藏红花素保护RGC-5细胞免受H2O2诱导损伤的机制。使用H2O2建立RGC-5细胞氧化应激损伤模型，以模拟体外青光眼的发展。添加不同浓度（0.1和1µM）的藏红花素，以测试其是否能够保护RGCs免受H2O2诱导的损伤。然后进行WST-1、乳酸脱氢酶（LDH）释放和膜联蛋白V/FITC检测。使用活性氧（ROS）检测试剂盒检测ROS水平，通过JC-1染色分析线粒体膜电位（ΔΨm），使用Caspase-3检测试剂盒检测Caspase-3活性，并使用蛋白质印迹分析测量Bax、Bcl-1和细胞色素c的蛋白质水平。此外，还使用蛋白质印迹分析评估磷酸化核因子-κB（p-NF-κB）p65的蛋白质水平。结果表明，藏红花素保护RGC-5细胞免于凋亡，减少LDH释放并增强细胞活力。进一步的实验表明，藏红花素降低了ROS水平，增加了ΔΨm,下调了Bax和细胞色素c的蛋白质表达，促进了Bcl-2蛋白质表达并激活了NF-κB。综上所述，本研究结果表明，藏红花素通过线粒体途径和NF-κB的激活预防了H2O2诱导的RGCs损伤。
