通过PINK1 / Parkin途径介导的线粒体自噬 保护 H9c2心肌细胞免受藏红花素氧化应激
Summary
本研究在体外实验的基础上揭示了藏红花素通过影响线粒 体 自噬修复心肌细胞氧化应激损伤的机制，其中PINK1/Parkin信号通路起重要作用。
Abstract
本研究旨在通过体外实验探讨藏红花素对H2O2介导的H9c2心肌细胞的氧化应激保护作用，并进一步探讨其机制是否与线粒体自噬的影响有关。本研究还旨在证明红花酸对心肌细胞氧化应激的治疗作用，并探讨其机制是否与线粒体自噬的作用有关。本文构建了基于H2O2的氧化应激模型，通过检测乳酸脱氢酶（LDH）、肌酸激酶（CK）、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH Px）的水平，评估了心肌细胞的氧化应激损伤程度。采用活性氧（ROS）检测荧光染料DCFH-DA、JC-1染料和TUNEL染料评估线粒体损伤和凋亡。通过转染Ad-mCherry-GFP-LC3B腺病毒测量自噬通量。然后通过蛋白质印迹和免疫荧光检测线粒体自噬相关蛋白。然而，藏红花素（0.1-10μM）可以显着提高细胞活力，减少由H2O2引起的细胞凋亡和氧化应激损伤。在自噬过度激活的细胞中，藏红花素还可以减少自噬流量和线粒体自噬相关蛋白PINK1和Parkin的表达，并逆转Parkin向线粒体的转移。藏红花素可减少H2O2介导的氧化应激损伤和H9c2细胞凋亡，其机制与线粒体自噬密切相关。
Introduction
急性心肌梗死（AMI）是一种危及生命的心肌坏死，由冠状动脉严重和持续的缺血和缺氧引起1,2。经皮冠状动脉介入治疗 （PCI） 是 AMI 的一线治疗策略之一，通常可保护心肌细胞免受缺血性损伤 3,4。如果AMI后不及时有效治疗，远端心肌将缺乏血液和氧气供应，从而导致缺血性坏死和进一步的心血管并发症5,6。在错过PCI手术机会后，促进心肌细胞恢复并尽量减少不可逆的心肌损伤一直是研究热点。AMI后心肌细胞处于缺血缺氧状态，导致线粒体氧化磷酸化受到抑制，NAD+还原为NADPH，单电子还原增加7。结果，氧气的不完全还原反应产生过量的活性氧（ROS），并最终导致心肌细胞的氧化应激损伤8。ROS的过度积累会引发脂质过氧化，进一步破坏线粒体膜的结构和功能。结果是线粒体通透性过渡孔的持续打开和线粒体膜电位的降低，诱导细胞凋亡和坏死。
血管紧张素转换酶 （ACE） 抑制剂、血管紧张素受体阻滞剂 （ARB）、β肾上腺素受体抑制剂、醛固酮拮抗剂和其他 AMI 标准药物有助于增强心肌梗死后的心脏功能，预防恶性事件的发生，如心律失常和左心室重塑9.然而，梗死后的生存率和预后受梗死大小的影响很大，降低心肌细胞凋亡尚未取得满意的结果10,11。因此，开发促进心肌梗死后心肌细胞恢复的药物已成为一个紧迫的问题。
多年来，传统医学一直是现代药物研究的灵感来源12,13,14,15。中医（TCM）在AMI的治疗方面有着悠久的历史，近年来的一系列随机对照试验证实，中医确实可以改善患者的预后16,17。根据中医理论，AMI是由血瘀引起的18,19，因此促进血液循环的药物通常用于急性期20的AMI的治疗。其中，藏红花被认为对血液活化和瘀滞有强大的作用，常用于AMI的急性治疗。藏红花素是藏红花的主要成分，可能在保护心肌细胞方面发挥关键作用21。
本研究以H2O2 诱导H9c2心肌细胞模拟AMI心肌细胞损伤的心肌缺血/再灌注，以藏红花素为干预措施，研究其对氧化应激诱发心肌损伤的保护作用。通过线粒体自噬进一步探索藏红花素保护心肌细胞的机制。更重要的是，本文为线粒体自噬研究的技术方法提供了参考，并详细描述了整个实验过程。
Protocol
实验在中国北京中医药大学生理学实验室进行。所有研究方法均按照北京大学的相关指导方针和规定进行。 1. 细胞培养 在Dulbecco的改良Eagle培养基（DMEM）碱性培养基（含4.5 g/L D-葡萄糖、4.g.g/L L-谷氨酰胺和110 mg/L丙酮酸钠;见 材料表）中加入10%胎牛血清和1%青霉素/链霉素，制备DMEM完全培养基。 在37°C的温水中解冻液氮冷冻的H9c2心肌细胞…
Representative Results
藏红花素对细胞活力的影响0.1 μM、0.5 μM、1 μM、5 μM、10 μM、50 μM 和 100 μM 的藏红花素对细胞有显着的增殖作用，而浓度高于 200 μM 的藏红花素显着抑制 H9c2 细胞的增殖（图 1A）。用400μM H 2 O2处理4小时后，细胞活力大大降低，藏红花素可以在一定程度上逆转这种变化（图1B）。由于在H2O2诱导的H9c2细胞?…
Discussion
通过先进技术从天然药物的复杂化合物中探索有效成分一直是中医药研究的热点29，验证后可为未来的药物开发提供实验室证据。红花是治疗“促进血液循环，减少血瘀”的代表药物，广泛用于治疗心肌梗塞30,31。藏红花被认为具有与红花相似的功效，其促进血液循环和祛血瘀的效果明显优于红花31,32<sup class="…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了北京自然科学基金（第7202119号）和国家自然科学基金（第82274380号）的支持。
Materials
| 0.25% trypsin | Gibco | 2323363 | |
| 1% Penicillin-streptomycin | Sigma | V900929 | |
| 5x protein loading buffer | Beijing Pulilai Gene Technology | B1030-5 | |
| Ad-mCherry GFP-LC3B adenovirus | Beyotime | C3011 | |
| Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) | Zhongshan Golden Bridge Biotechnology Co., Ltd. | ZF-0514 | |
| Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L) | Zhongshan Golden Bridge Biotechnology Co., Ltd. | ZF-0513 | |
| Animal-free blocking solution | CST | 15019s | |
| Anti-Parkin antibody | Santa Cruz | sc-32282 | |
| Anti-PINK1 antibody | ABclonal | A11435 | |
| Anti-TOM20 antibody | ABclonal | A19403 | |
| Anti-β-actin antibody | ABclonal | AC026 | |
| BCA protein assay kit | KeyGEN Biotech | KGP902 | |
| Blood cell counting plate | Servicebio | WG607 | |
| CAT assay kits | Nanjing Jiancheng Bioengineering Institute | A007-1-1 | |
| Chemiluminescence detection system | Shanghai Qinxiang Scientific Instrument Factory | ChemiScope 6100 | |
| CK assay kits | Nanjing Jiancheng Bioengineering Institute | A032-1-1 | |
| Coenzyme Q10 (CoQ 10) | Macklin | C6129 | |
| Crocetin | Chengdu Ruifensi Biotechnology Co., Ltd. | RFS-Z01802006012 | |
| DAPI-containing antifluorescence quenching tablets | Zhongshan Golden Bridge Biotechnology Co., Ltd. | ZLI-9557 | |
| DCFH-DA | Beyotime | S0033S | |
| DMSO | Solarbio | D8371 | |
| Dulbecco's modified eagle medium (DMEM) | Gibco | 8122091 | |
| Enhanced Chemiluminescence (ECL) solution | NCM Biotech | P10100 | |
| Fetal bovine serum (FBS) | Corning-Cellgro | 35-081-CV | |
| GraphPad Prism 7.0 | https://www.graphpad.com/ | ||
| GSH-Px assay kits | Nanjing Jiancheng Bioengineering Institute | A005-1-2 | |
| H9c2 myocardial cells | Beijing Dingguochangsheng Biotech Co., Ltd. | CS0062 | |
| Horseradish peroxidase (HRP)-conjugated goat anti-goat IgG (H+L) | Zhongshan Golden Bridge Biotechnology Co., Ltd. | ZB-2305 | |
| Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) | Zhongshan Golden Bridge Biotechnology Co., Ltd. | ZB-2301 | |
| JC-1 mitochondrial membrane potential assay kit | LABLEAD | J22202 | |
| LDH assay kits | Nanjing Jiancheng Bioengineering Institute | A020-2-2 | |
| MDA assay kits | Nanjing Jiancheng Bioengineering Institute | A003-2-2 | |
| Methanol | Aladdin | A2114057 | |
| MTS assay | Promega | G3581 | |
| Perhydrol | G-clone | CS7730 | |
| Phosphatase inhibitor | CWBIO | CW2383 | |
| Polybrene | Beyotime | C0351 | |
| Polyvinylidene difluoride (PVDF) membranes | Millipore | ISEQ00010 | |
| Radioimmunoprecipitation assay (RIPA) lysis buffer | Solarbio | R0010 | |
| SDS-PAGE gels | Shanghai Epizyme Biomedical Technology | PG112 | |
| SDS-PAGE running buffer powder | Servicebio | G2018-1L | |
| SDS-PAGE transfer buffer powder | Servicebio | G2017-1L | |
| SOD assay kits | Nanjing Jiancheng Bioengineering Institute | A001-2-2 | |
| Tris-buffered saline powder | Servicebio | G0001-2L | |
| Triton X-100 | Sigma | SLCC9172 | |
| TUNEL apoptosis assay kit | Beyotime | C1086 | |
| Tween-20 | Solarbio | T8220 |
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Chen, J., Li, Y., Zhang, Y., Du, T., Lu, Y., Li, X., Guo, S. Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy. J. Vis. Exp. (195), e65105, doi:10.3791/65105 (2023).