Abstract

Age-related macular degeneration (AMD) is a complex multifactorial disease associated with the dysfunction of retinal pigment epithelium (RPE). Aloperine is a quinolizidine alkaloid that has been proven to possess broad pharmacological activities. However, the effects of aloperine on AMD remain unclear. In the present study, we used hydrogen peroxide (H₂O₂) to induce oxidative injury in human RPE cells (ARPE-19 cells). ARPE-19 cells were pretreated with different concentrations of aloperine for 2 h, followed by H₂O₂ exposure. Cell cytotoxicity was determined using lactate dehydrogenase (LDH) release assay. Cell viability was measured using Cell Counting Kit-8 (CCK-8) assay. The reactive oxygen species (ROS) generation, malondialdehyde (MDA) level, superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-PX) activity were detected to reflect oxidative status. Western blot was performed to detect the expressions of bcl-2, bax, nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1). The activity of caspase-3 was also assessed to indicate cell apoptosis. In addition, ARPE-19 cells were transfected with siNrf2 to knock down Nrf2. Our results showed that pretreatment with aloperine elevated the reduced cell viability of H₂O₂-induced ARPE-19 cells in a dose-dependent manner. Aloperine greatly decreased the production of ROS and MDA, and increased the activities of SOD and GSH-PX in H₂O₂-stimulated ARPE-19 cells. H₂O₂-caused a decrease in bcl-2 expression and increases in bax expression and caspase-3 activity were mitigated by aloperine. Moreover, aloperine treatment enhanced the expression levels of Nrf2 in nuclear fraction and the HO-1 expression in lysates. Knockdown of Nrf2 reversed the protective effects of aloperine on H₂O₂-induced ARPE-19 cells. In conclusion, these findings demonstrated that aloperine protected ARPE-19 cells from H₂O₂-induced oxidative stress and apoptosis in part via activating the Nrf2/HO-1 signaling pathway. The findings suggested a therapeutic potential of aloperine for the treatment of ADM.

摘要

年龄相关性黄斑变性 (AMD) 是一种与视网膜色素上皮 (RPE) 功能障碍相关的复杂多因素疾病。Aloperine 是一种喹啉类生物碱，已被证明具有广泛的药理活性。然而，阿洛普林对 AMD 的影响仍不清楚。在本研究中，我们使用过氧化氢 (H ₂ O ₂ ) 在人 RPE 细胞 (ARPE-19 细胞) 中诱导氧化损伤。ARPE-19细胞用不同浓度的阿洛普林预处理2小时，然后用H ₂ O ₂接触。使用乳酸脱氢酶 (LDH) 释放测定法测定细胞细胞毒性。使用 Cell Counting Kit-8 (CCK-8) 测定法测量细胞活力。检测活性氧 (ROS) 生成、丙二醛 (MDA) 水平、超氧化物歧化酶 (SOD) 活性和谷胱甘肽过氧化物酶 (GSH-PX) 活性以反映氧化状态。Western blot检测bcl-2、bax、核因子-红细胞2相关因子2（Nrf2）和血红素加氧酶-1（HO-1）的表达。还评估了 caspase-3 的活性以指示细胞凋亡。此外，用 siNrf2 转染 ARPE-19 细胞以敲低 Nrf2。我们的结果表明，用 aloperine 预处理提高了 H ₂ O _{2的细胞活力降低}以剂量依赖性方式诱导的 ARPE-19 细胞。Aloperine大大降低了ROS和MDA的产生，并增加了H ₂ O ₂刺激的ARPE-19细胞中SOD和GSH-PX的活性。H ₂ O ₂ -导致 bcl-2 表达降低，并且 bax 表达和 caspase-3 活性的增加被 aloperine 减轻。此外，aloperine 处理增强了核部分中 Nrf2 的表达水平和裂解物中的 HO-1 表达。Nrf2 的敲低逆转了 aloperine 对 H ₂ O ₂诱导的 ARPE-19 细胞的保护作用。总之，这些发现表明，aloperine 保护 ARPE-19 细胞免受 H ₂ O ₂- 部分通过激活 Nrf2/HO-1 信号通路诱导氧化应激和细胞凋亡。研究结果表明，aloperine 具有治疗 ADM 的治疗潜力。