Eye Center, The Second Hospital of Jilin University, Changchun, Jilin 130000, P.R. China.
Int J Mol Med. 2021 Apr;47(4). doi: 10.3892/ijmm.2021.4892. Epub 2021 Feb 19.
Cataracts have a high incidence and prevalence rate worldwide, and they are the leading cause of blindness. Lens epithelial cell (LEC) apoptosis is often analysed in cataract research since it is the pathological basis of cataracts, except for congenital cataract. Chloride channels are present in ocular tissues, such as in trabecular cells, LECs and other cells. They serve an important role in apoptosis and participate in endoplasmic reticulum (ER) stress and oxidative stress. However, their role in the apoptosis of LECs has not been discussed. The present study examined the effects of the chloride channel blocker 5‑nitro‑2‑(3‑phenylpropylamino) benzoic acid (NPPB) in human LECs (HLECs) to elucidate the role of NPPB in HLECs and investigate the role and mechanism of chloride channels in cataract formation. HLECs were exposed to NPPB. Cell survival rate was evaluated using Cell Counting Kit‑8 assays. Oxidative stress was detected as reactive oxygen species (ROS) in cells by using a ROS assay kit. Apoptosis was examined by assessing mitochondrial membrane potential and using a JC‑1 assay kit, and western blot analysis was performed to measure the expression levels of mitochondrial‑dependent apoptosis pathway‑associated proteins. ER stress was evaluated by determining the intracellular calcium ion fluorescence intensity, and western blot analysis was performed to measure ER stress‑associated protein expression. The results revealed that NPPB treatment decreased the viability of HLECs and increased apoptosis. Additionally, NPPB increased intracellular ROS levels, as well as the number of JC‑1 monomers and the protein expression levels of B‑cell lymphoma‑2 (Bcl‑2)‑associated X and cleaved caspase‑3, and decreased Bcl‑2 protein expression. NPPB increased intracellular calcium ions, the protein expression levels of activating transcription factor 6, JNK, C/EBP homologous protein and caspase‑12, and the phosphorylation of protein kinase R‑like endoplasmic reticulum kinase. N‑acetylcysteine and 4‑phenylbutyric acid inhibited NPPB‑induced oxidative stress, ER stress and apoptosis. Therefore, NPPB treatment decreased cell viability and promoted apoptosis of HLECs via the promotion of oxidative and ER stress.
白内障在全球范围内具有较高的发病率和患病率，是导致失明的主要原因。除先天性白内障外，晶状体上皮细胞（LEC）凋亡通常被用于白内障研究，因为它是白内障的病理基础。氯通道存在于眼部组织中，如小梁细胞、LEC 和其他细胞中。它们在凋亡中发挥重要作用，并参与内质网（ER）应激和氧化应激。然而，它们在 LEC 凋亡中的作用尚未得到讨论。本研究通过研究氯通道阻滞剂 5-硝基-2-（3-苯丙氨基）苯甲酸（NPPB）对人晶状体上皮细胞（HLECs）的影响，来阐明 NPPB 在 HLECs 中的作用，并探讨氯通道在白内障形成中的作用和机制。用 NPPB 处理 HLECs。通过使用细胞计数试剂盒-8 评估细胞存活率。通过使用 ROS 测定试剂盒检测细胞中的活性氧物种（ROS）来检测氧化应激。通过评估线粒体膜电位和使用 JC-1 测定试剂盒检测凋亡，以及通过蛋白质印迹分析测定线粒体依赖性凋亡途径相关蛋白的表达水平来检测凋亡。通过确定细胞内钙离子荧光强度来评估 ER 应激，并通过蛋白质印迹分析测定 ER 应激相关蛋白的表达水平。结果表明，NPPB 处理降低了 HLEC 的活力并增加了细胞凋亡。此外，NPPB 增加了细胞内 ROS 水平，以及 JC-1 单体的数量和 B 细胞淋巴瘤-2（Bcl-2）相关 X 和 cleaved caspase-3 的蛋白表达水平，并降低了 Bcl-2 蛋白表达。NPPB 增加了细胞内钙离子、激活转录因子 6、JNK、C/EBP 同源蛋白和 caspase-12 的蛋白表达水平以及蛋白激酶 R 样内质网激酶的磷酸化。N-乙酰半胱氨酸和 4-苯基丁酸抑制了 NPPB 诱导的氧化应激、ER 应激和细胞凋亡。因此，NPPB 处理通过促进氧化应激和 ER 应激降低了 HLEC 的细胞活力并促进了细胞凋亡。
