反应氧族促进神经保护的量效关系及其机制研究

doi:10.5246/jcps.2024.01.005

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (1): 46-56.DOI: 10.5246/jcps.2024.01.005

反应氧族促进神经保护的量效关系及其机制研究

黄荣¹, 胡小娇², 张润芳², 李晓晖¹^,^*(), 岑娟¹^,²^,^*()

^1. 河南大学第一附属医院神经内科, 河南开封 475000
^2. 河南大学药学院天然药物与免疫工程重点实验室, 河南开封 475004

收稿日期:2023-05-25 修回日期:2023-07-11 接受日期:2023-08-20 出版日期:2024-01-31 发布日期:2024-01-31
通讯作者: 李晓晖, 岑娟

Study on the dose-effect relationship and mechanism of reactive oxygen species promoted neuroprotection

Rong Huang¹, Xiaojiao Hu², Runfang Zhang², Xiaohui Li¹^,^*(), Juan Cen¹^,²^,^*()

¹ Department of Neurology, The First Affiliated Hospital of Henan University, Kaifeng 475000, Henan, China
² The Key Laboratory of Natural Medicine and Immune-Engineering, School of Pharmacy, Henan University, Kaifeng 475004, Henan, China

Received:2023-05-25 Revised:2023-07-11 Accepted:2023-08-20 Online:2024-01-31 Published:2024-01-31
Contact: Xiaohui Li, Juan Cen
Supported by:
Henan Medical Science and Technology Research Program (Joint Construction Project) Funded Project (Grant No. 2018020317), Project of Kaifeng Science and Technology Bureau (Grant No. 2003033).

摘要/Abstract

摘要：

反应氧族(ROS)的大量产生是缺血性中风损伤的关键因素。尽管大量研究表明抗氧化剂具有神经保护作用, 然而其长期临床疗效始终差强人意。事实上, ROS在特定浓度下具有促进细胞增殖、促进保护性自噬、激活自身抗氧化能力等积极的生理作用, 在抗氧化干预时尽量保留ROS的优势作用或是解决问题的关键。本研究探索了不同剂量下H₂O₂对PC12和SH-SY5Y神经细胞增殖、自噬、凋亡等生理病理功能的影响, 检测了相应细胞内及线粒体内ROS的变化规律, 绘制了ROS发挥优势活性的剂量区间。进一步使用自噬抑制剂及信号通路抑制剂等进行研究, 结果表明100 μM的H₂O₂可通过AKT/m-TOR介导的HIF-1α和TFEB信号激活保护性自噬, 从而实现神经细胞的保护作用。本研究揭示了ROS发挥优势作用的浓度范围, 并阐明了其神经保护机制, 为全面优化脑缺血后抗氧化剂的使用提供了重要参考。

关键词: 反应氧族, 脑缺血, 保护性自噬, HIF-1α, TFEB

Abstract:

The overproduction of reactive oxygen species (ROS) is widely acknowledged as a pivotal factor in the occurrence of ischemic stroke injuries. While numerous experimental studies have demonstrated the significant neuroprotective effects of antioxidants, their long-term clinical efficacy has consistently fallen short of expectations. Interestingly, ROS can exert positive physiological effects, such as promoting cell proliferation, activating protective autophagy, and enhancing antioxidant capacity, at certain concentrations. Consequently, preserving the advantageous aspects of ROS is crucial for addressing the limitations of antioxidant therapy. This study investigated the impact of ROS at varying concentrations on physiological and pathological functions, including neural cell proliferation, autophagy, and apoptosis. We also assessed the fluctuations in intracellular and mitochondrial ROS levels and delineated the dose range within which these advantageous functions occur. Additionally, we conducted further research using autophagy inhibitors and signaling pathway inhibitors. The findings demonstrated that 100 μM H₂O₂ could induce protective autophagy through HIF-1α and TFEB signaling pathways mediated by AKT/m-TOR, thereby conferring a protective effect on nerve cells. This study elucidated the concentration range at which ROS exerted a protective role and unraveled its neuroprotective mechanism, offering a crucial reference for optimizing the comprehensive application of antioxidants following cerebral ischemia.

Key words: Reactive oxygen species, Cerebral ischemia, Protective autophagy, HIF-1α, TFEB

Supporting:

黄荣, 胡小娇, 张润芳, 李晓晖, 岑娟. 反应氧族促进神经保护的量效关系及其机制研究[J]. 中国药学(英文版), 2024, 33(1): 46-56.

Rong Huang, Xiaojiao Hu, Runfang Zhang, Xiaohui Li, Juan Cen. Study on the dose-effect relationship and mechanism of reactive oxygen species promoted neuroprotection[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(1): 46-56.

图/表 8

Figure 1. The viability of neuron cells was influenced by different levels of ROS in a concentration-dependent manner. (A) Incubation with H2O2 demonstrated a concentration- and time-dependent increase, with no toxic effects observed on the cell viability of PC12 cells; (B) Microscopy observations revealed morphological changes in PC12 cells at various H2O2 concentrations (200× magnification); (C) Similarly, concentration-dependent incubation with H2O2 showed no toxicity to toxic effects on the cell viability of SH-SY5Y cells; (D) Microscopy observations depicted morphological changes in SH-SY5Y cells at different H2O2 concentrations (200× magnification). The data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. the control group.

Figure 2. Treatment with H2O2 at various concentrations resulted in changes in intracellular ROS levels in neuron cells. Cells were treated with the specified concentrations of H2O2 for 6 h and then stained with DCFH-DA according to the instructions. Flow cytometry was utilized to analyze the intracellular ROS content. The data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001 vs. the control group.

Figure 3. Treatment with H2O2 at different concentrations led to an increase in mitochondrial ROS content in a concentration-dependent manner. Cells were treated with the specified concentrations of H2O2 for 6 h and then stained with MitoSOX according to the instructions. Flow cytometry was utilized to analyze the mitochondrial ROS content. The data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. the control group.

Figure 4. The proliferation ability of cells displayed a specific concentration-dependent response to H2O2. Cells were treated with various concentrations of H2O2 for 6 h, followed by staining with Ki67 antibody according to the provided instructions. Flow cytometry was utilized to analyze the percentage of Ki67+ cells. The data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. the control group.

Figure 5. H2O2 induced a concentration-dependent increase in mitophagy in PC12 and SH-SY5Y cells. Cells were treated with different concentrations of H2O2 for 6 h, followed by staining with PINK1 antibody according to the provided instructions. Flow cytometry was utilized to analyze the percentage of PINK1+ cells. The data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. the control group.

Figure 6. H2O2 induced a concentration-dependent increase in the apoptosis rate of PC12 and SH-SY5Y cells. Cells were treated with different concentrations of H2O2 for 6 h, followed by staining with Annexin V/PI double stain agents according to the provided instructions. Flow cytometry was used to analyze the apoptosis rate. The data are presented as mean ± SD (n = 3). **P < 0.01 vs. the control group.

Figure 7. Moderate levels of ROS activated mitophagy and provided neuroprotection in PC12 and SH-SY5Y cells. However, when autophagy inhibitor 3-MA or mitophagy inhibitor Mdivi-1 was applied, cell injury was induced even after treatment with a subtoxic concentration of H2O2 at 100 μM. Cells were pretreated with or without 5 mM 3-MA or 20 μM Mdivi-1 for 2 h, followed by incubation with the indicated concentration of H2O2 for an additional 6 h. Afterward, the cells were collected and subjected to the MTT assay. The data are expressed as mean ± SD (n = 3). Statistical analysis showed that ***P < 0.001 as vs. the control group, indicating significant differences between the groups.

Figure 8. The AKT/mTOR-mediated HIF-1α and TFEB signaling pathways were found to be involved in H2O2-induced mitophagy in PC12 and SH-SY5Y cells. (A) Changes in protein levels were observed in whole cells for p-Akt, Akt, p-mTOR, mTOR, HIF-1α, TFEB, and P-gp, as well as in the nucleus for HIF-1α and TFEB; (B) Inhibitors targeting HIF-1α and TFEB led to a decrease in cell viability in SH-SY5Y cells; (C) Treatment with inhibitors of HIF-1α and TFEB resulted in a reduction in H2O2-induced mitophagy activation. SH-SY5Y cells were pretreated with 100 μM YC-1, 10 μM CCI-779, 50 μM LY294002, and 400 nM Rapamycin for 2 h, followed by treatment with or without 50, 100, or 200 μM H2O2 for an additional 6 h. Western blotting analysis and MTT assay were performed using the collected cells. The data are presented as mean ± SD (n = 3). Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. 100 μM H2O2 treatment group.

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反应氧族促进神经保护的量效关系及其机制研究

Study on the dose-effect relationship and mechanism of reactive oxygen species promoted neuroprotection

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