J Biomed Nanotechnol. 2020 May 1;16(5):603-615. doi: 10.1166/jbn.2020.2923.
The goal of this study was to examine the impact of the mitochondrial-targeted antioxidant peptide, SS31, and its role in promoting autophagy in cone photoreceptor 661W cells that were subjected to oxidative damage. To do so, we examined the viability of 661W cells in the presence of increasing concentrations of H₂O₂ with or without SS31 pre-treatment using the MTT assay and by expression of autophagy and apoptosis-associated proteins LC3-II/I, P62, and caspase-3. Autophagy was evaluated by fluorescence microscopy in cells stained with monodansyl cadaverine (MDC). Autophagy was induced with rapamycin (Rap) and inhibited with bafamycin A1 (bafA1) followed by examination of Reactive oxygen species (ROS) levels in target 661W cells by fluorescence microscopy and flow cytometry. Annexin V/PI staining was used to evaluate the rate of apoptosis and mRNA sequencing (mRNA-seq) analysis (Illumina platform) was performed on H₂O₂-exposed 661W cells treated with SS31. Among our results, we observed a substantial and concentration-dependent decrease in 661W cell viability in response to H₂O₂-exposure; production of ROS, autophagy and apoptosis were induced at 8 h in response to exposure to 100 M of H₂O₂. Pre-treatment with 100 nM SS31 resulted in significant attenuation of H₂O₂-mediated cytotoxicity, together with reduced ROS production and enhanced autophagy observed in response to oxidative stress. Both Rap and bafA1 were used to modulate SS31-mediated autophagy; the impact of Rap was similar to that of SS31. By contrast, administration of bafA1 counteracted autophagy induced by SS31. Furthermore, mRNAseq analysis revealed that SS31 promoted significant alterations in gene expression in 661W cells and suggested that autophagy was induced via the mTORC1-mediated signaling. In conclusion, our results indicate that exposure to H₂O₂ resulted in reduced 661W cell viability via mechanisms associated with oxidative damage, apoptosis, and autophagy. Notably, we demonstrated that pre-treatment with SS31 protects 661W cells from H₂O₂-induced oxidative damage that may result in part from induction of autophagy via mTORC1-mediated signaling pathways.
本研究的目的是探讨靶向线粒体的抗氧化肽 SS31 对氧化损伤的 661W 细胞锥体光感受器中自噬的影响及其作用。为此，我们通过 MTT 测定法和自噬和凋亡相关蛋白 LC3-II/I、P62 和 caspase-3 的表达，检测了存在或不存在 SS31 预处理时，661W 细胞在增加浓度的 H₂O₂中的活力。通过用单丹磺酰尸胺 (MDC) 染色的细胞的荧光显微镜评估自噬。用雷帕霉素 (Rap) 诱导自噬，并用巴弗霉素 A1 (bafA1) 抑制，然后通过荧光显微镜和流式细胞术检测目标 661W 细胞中的活性氧 (ROS) 水平。用 Annexin V/PI 染色评估凋亡率，并对用 SS31 处理的暴露于 H₂O₂的 661W 细胞进行 mRNA 测序 (mRNA-seq) 分析 (Illumina 平台)。在我们的研究结果中，我们观察到 661W 细胞对 H₂O₂暴露的活力显著且浓度依赖性降低；在暴露于 100 μM H₂O₂ 8 小时后，会诱导产生 ROS、自噬和凋亡。用 100 nM SS31 预处理会显著减弱 H₂O₂ 介导的细胞毒性，同时减少 ROS 产生和增强对氧化应激的自噬。Rap 和 bafA1 均用于调节 SS31 介导的自噬；Rap 的作用与 SS31 相似。相比之下，给予 bafA1 会抵消 SS31 诱导的自噬。此外，mRNAseq 分析表明，SS31 促进了 661W 细胞中基因表达的显著变化，并表明自噬是通过 mTORC1 介导的信号转导诱导的。总之，我们的研究结果表明，H₂O₂ 的暴露会通过与氧化损伤、凋亡和自噬相关的机制导致 661W 细胞活力降低。值得注意的是，我们证明了 SS31 的预处理可保护 661W 细胞免受 H₂O₂ 诱导的氧化损伤，这可能部分归因于通过 mTORC1 介导的信号通路诱导自噬。
