Name: protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy
Uploaded: 2023-05-26T15:00:00
Description: Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

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。

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
<ol><li>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。</li>
	<li>在37°C的温水中解冻液氮冷冻的H9c2心肌细胞...

<ol>
	<li>Anderson, J. L., Morrow, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction.">Acute myocardial infarction.</a> The New England Journal of Medicine. 376 (21), 2053-2064 (2017).</li><li>Samsky, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiogenic+shock+after+acute+myocardial+infarction:+a+review.">Cardiogenic shock after acute myocardial infarction: a review.</a> JAMA. 326 (18), 1840-1850 (2021).</li><li>Abbate, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+cardiac+remodeling+benefits+in+patients+undergoing+late+percutaneous+coronary+intervention+of+the+infarct-related+artery:+evidence+from+a+meta-analysis+of+randomized+controlled+trials.">Survival and cardiac remodeling benefits in patients undergoing late percutaneous coronary intervention of the infarct-related artery: evidence from a meta-analysis of randomized controlled trials.</a> Journal of the American College of Cardiology. 51 (9), 956-964 (2008).</li><li>Santoro, G. M., Carrabba, N., Migliorini, A., Parodi, G., Valenti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+heart+failure+in+patients+with+acute+myocardial+infarction+treated+with+primary+percutaneous+coronary+intervention.">Acute heart failure in patients with acute myocardial infarction treated with primary percutaneous coronary intervention.</a> European Journal of Heart Failure. 10 (8), 780-785 (2008).</li><li>Dhruva, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+use+of+an+intravascular+microaxial+left+ventricular+assist+device+vs+intra-aortic+balloon+pump+with+in-hospital+mortality+and+major+bleeding+among+patients+with+acute+myocardial+infarction+complicated+by+cardiogenic+shock.">Association of use of an intravascular microaxial left ventricular assist device vs intra-aortic balloon pump with in-hospital mortality and major bleeding among patients with acute myocardial infarction complicated by cardiogenic shock.</a> JAMA. 323 (8), 734-745 (2020).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+associated+with+major+cardiovascular+events+1+year+after+acute+myocardial+infarction.">Risk factors associated with major cardiovascular events 1 year after acute myocardial infarction.</a> JAMA Network Open. 1 (4), e181079 (2018).</li><li>Jou, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+protects+against+common+deletion+of+mitochondrial+DNA-augmented+mitochondrial+oxidative+stress+and+apoptosis.">Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis.</a> Journal of Pineal Research. 43 (4), 389-403 (2007).</li><li>La Piana, G., Fransvea, E., Marzulli, D., Lofrumento, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+supported+by+exogenous+cytochrome+c+oxidation+mimics+the+early+stages+of+apoptosis.">Mitochondrial membrane potential supported by exogenous cytochrome c oxidation mimics the early stages of apoptosis.</a> Biochemical and Biophysical Research Communications. 246 (2), 556-561 (1998).</li><li>De Filippo, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+secondary+prevention+medical+therapies+on+outcomes+of+patients+suffering+from+Myocardial+Infarction+with+NonObstructive+Coronary+Artery+disease+(MINOCA):+A+meta-analysis.">Impact of secondary prevention medical therapies on outcomes of patients suffering from Myocardial Infarction with NonObstructive Coronary Artery disease (MINOCA): A meta-analysis.</a> International Journal of Cardiology. 368, 1-9 (2022).</li><li>Davidson, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multitarget+strategies+to+reduce+myocardial+ischemia/reperfusion+injury:+JACC+review+topic+of+the+week.">Multitarget strategies to reduce myocardial ischemia/reperfusion injury: JACC review topic of the week.</a> Journal of the American College of Cardiology. 73 (1), 89-99 (2019).</li><li>Caricati-Neto, A., Errante, P. R., Menezes-Rodrigues, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+pharmacological+and+non-pharmacological+strategies+of+cardioprotection.">Recent advances in pharmacological and non-pharmacological strategies of cardioprotection.</a> International Journal of Molecular Sciences. 20 (16), 4002 (2019).</li><li>Chen, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+pharmacology+analysis+and+experimental+validation+to+investigate+the+mechanism+of+total+flavonoids+of+rhizoma+drynariae+in+treating+rheumatoid+arthritis.">Network pharmacology analysis and experimental validation to investigate the mechanism of total flavonoids of rhizoma drynariae in treating rheumatoid arthritis.</a> Drug Design, Development, and Therapy. 16, 1743-1766 (2022).</li><li>Wei, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+has+great+potential+as+candidate+drugs+for+lung+cancer:+A+review.">Traditional Chinese medicine has great potential as candidate drugs for lung cancer: A review.</a> Journal of Ethnopharmacology. 300, 115748 (2023).</li><li>Zhi, W., Liu, Y., Wang, X., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+of+traditional+Chinese+medicine+for+the+prevention+and+treatment+of+atherosclerosis.">Recent advances of traditional Chinese medicine for the prevention and treatment of atherosclerosis.</a> Journal of Ethnopharmacology. 301, 115749 (2023).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertensive+heart+disease+and+myocardial+fibrosis:+How+traditional+Chinese+medicine+can+help+addressing+unmet+therapeutical+needs.">Hypertensive heart disease and myocardial fibrosis: How traditional Chinese medicine can help addressing unmet therapeutical needs.</a> Pharmacological Research. 185, 106515 (2022).</li><li>Zhang, X. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+intervenes+ventricular+remodeling+following+acute+myocardial+infarction:+evidence+from+40+random+controlled+trials+with+3,659+subjects.">Traditional Chinese medicine intervenes ventricular remodeling following acute myocardial infarction: evidence from 40 random controlled trials with 3,659 subjects.</a> Frontiers in Pharmacology. 12, 707394 (2021).</li><li>Hao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+medicine+for+cardiovascular+disease:+evidence+and+potential+mechanisms.">Traditional Chinese medicine for cardiovascular disease: evidence and potential mechanisms.</a> Journal of the American College of Cardiology. 69 (24), 2952-2966 (2017).</li><li>Delgado-Montero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+stasis+imaging+predicts+cerebral+microembolism+during+acute+myocardial+infarction.">Blood stasis imaging predicts cerebral microembolism during acute myocardial infarction.</a> Journal of the American Society of Echocardiography. 33 (3), 389-398 (2020).</li><li>Lu, C. Y., Lu, P. C., Chen, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+trends+in+traditional+Chinese+medicine+for+acute+myocardial+infarction.">Utilization trends in traditional Chinese medicine for acute myocardial infarction.</a> Journal of Ethnopharmacology. 241, 112010 (2019).</li><li>Gao, Z. Y., Xu, H., Shi, D. Z., Wen, C., Liu, B. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+on+outcome+of+5284+patients+with+coronary+artery+disease:+the+role+of+integrative+medicine.">Analysis on outcome of 5284 patients with coronary artery disease: the role of integrative medicine.</a> Journal of Ethnopharmacology. 141 (2), 578-583 (2012).</li><li>Huang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crocetin+ester+improves+myocardial+ischemia+via+Rho/ROCK/NF-kappaB+pathway.">Crocetin ester improves myocardial ischemia via Rho/ROCK/NF-kappaB pathway.</a> International Immunopharmacology. 38, 186-193 (2016).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cell+number+by+hemocytometry+counting.">Estimation of cell number by hemocytometry counting.</a> Cold Spring Harbor Protocols. 2019 (11), (2019).</li><li>Zeng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+potential+value+and+mechanism+of+Kaji-Ichigoside+F1+on+arsenite-induced+skin+cell+senescence.">Assessing the potential value and mechanism of Kaji-Ichigoside F1 on arsenite-induced skin cell senescence.</a> Oxidative Medicine and Cellular Longevity. 2022, 9574473 (2022).</li><li>Chazotte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+mitochondria+with+JC-1.">Labeling mitochondria with JC-1.</a> Cold Spring Harbor Protocols. 2011 (9), (2011).</li><li>Kyrylkova, K., Kyryachenko, S., Leid, M., Kioussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+apoptosis+by+TUNEL+assay.">Detection of apoptosis by TUNEL assay.</a> Methods in Molecular Biology. 887, 41-47 (2012).</li><li>Yuan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+impairs+the+autophagic+flux+to+induce+p62-dependent+apoptosis+through+the+upregulation+of+CYLD+in+NRCMs.">Palmitate impairs the autophagic flux to induce p62-dependent apoptosis through the upregulation of CYLD in NRCMs.</a> Toxicology. 465, 153032 (2022).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting.">Western blotting.</a> Methods. 38 (4), 283-293 (2006).</li><li>Chen, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+flavonoids+of+rhizoma+drynariae+restore+the+MMP/TIMP+balance+in+models+of+osteoarthritis+by+inhibiting+the+activation+of+the+NF-&#954;B+and+PI3K/AKT+pathways.">Total flavonoids of rhizoma drynariae restore the MMP/TIMP balance in models of osteoarthritis by inhibiting the activation of the NF-&#954;B and PI3K/AKT pathways.</a> Evidence-Based Complementary and Alternative. 2021, 6634837 (2021).</li><li>Amin, A., Hamza, A. A., Bajbouj, K., Ashraf, S. S., Daoud, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron:+a+potential+candidate+for+a+novel+anticancer+drug+against+hepatocellular+carcinoma.">Saffron: a potential candidate for a novel anticancer drug against hepatocellular carcinoma.</a> Hepatology. 54 (3), 857-867 (2011).</li><li>Kamalipour, M., Akhondzadeh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+effects+of+saffron:+an+evidence-based+review.">Cardiovascular effects of saffron: an evidence-based review.</a> The Journal of Tehran Heart Center. 6 (2), 59-61 (2011).</li><li>Mani, V., Lee, S. K., Yeo, Y., Hahn, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+perspective+and+opportunities+in+pharmacologically+important+safflower.">A metabolic perspective and opportunities in pharmacologically important safflower.</a> Metabolites. 10 (6), 253 (2020).</li><li>Broadhead, G. K., Chang, A., Grigg, J., McCluskey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+saffron+supplementation:+current+clinical+findings.">Efficacy and safety of saffron supplementation: current clinical findings.</a> Critical Reviews in Food Science and Nutrition. 56 (16), 2767-2776 (2016).</li><li>Gao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+protective+effect+of+salidroside+against+H2O2-induced+injury+in+H9C2+cells.">Insight into the protective effect of salidroside against H2O2-induced injury in H9C2 cells.</a> Oxidative Medicine and Cellular Longevity. 2021, 1060271 (2021).</li><li>Chen, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+rhizoma+drynariae+targets+in+the+treatment+of+osteoarthritis+based+on+network+pharmacology+and+experimental+verification.">Prediction of rhizoma drynariae targets in the treatment of osteoarthritis based on network pharmacology and experimental verification.</a> Evidence-Based Complementary and Alternative. 2021, 5233462 (2021).</li><li>Reers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+monitored+by+JC-1+dye.">Mitochondrial membrane potential monitored by JC-1 dye.</a> Methods in Enzymology. 260, 406-417 (1995).</li><li>Radovits, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribose)+polymerase+inhibition+improves+endothelial+dysfunction+induced+by+reactive+oxidant+hydrogen+peroxide+in+vitro.">Poly(ADP-ribose) polymerase inhibition improves endothelial dysfunction induced by reactive oxidant hydrogen peroxide in vitro.</a> European Journal of Pharmacology. 564 (1-3), 158-166 (2007).</li><li>Song, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependence+of+parkin-mediated+mitophagy+and+mitochondrial+fission+in+adult+mouse+hearts.">Interdependence of parkin-mediated mitophagy and mitochondrial fission in adult mouse hearts.</a> Circulation Research. 117 (4), 346-351 (2015).</li><li>Gan, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+mechanism+of+PINK1.">Activation mechanism of PINK1.</a> Nature. 602 (7896), 328-335 (2022).</li><li>Nguyen, T. N., Padman, B. S., Lazarou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+molecular+signals+of+PINK1/Parkin+mitophagy.">Deciphering the molecular signals of PINK1/Parkin mitophagy.</a> Trends in Cell Biology. 26 (10), 733-744 (2016).</li><li>Yamada, T., Dawson, T. M., Yanagawa, T., Iijima, M., Sesaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SQSTM1/p62+promotes+mitochondrial+ubiquitination+independently+of+PINK1+and+PRKN/parkin+in+mitophagy.">SQSTM1/p62 promotes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy.</a> Autophagy. 15 (11), 2012-2018 (2019).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(4th+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).</a> Autophagy. 17 (1), 1 (2021).</li></ol>

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="...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin</td>
			<td>Gibco</td>
			<td>2323363</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>V900929</td>
			<td></td>
		</tr>
		<tr>
			<td>5x protein loading buffer</td>
			<td>Beijing Pulilai Gene Technology</td>
			<td>B1030-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad-mCherry GFP-LC3B adenovirus</td>
			<td>Beyotime</td>
			<td>C3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0514</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0513</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-free blocking solution</td>
			<td>CST</td>
			<td>15019s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Parkin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32282</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PINK1 antibody</td>
			<td>ABclonal</td>
			<td>A11435</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TOM20 antibody</td>
			<td>ABclonal</td>
			<td>A19403</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin&#160; antibody</td>
			<td>ABclonal</td>
			<td>AC026</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>KeyGEN Biotech</td>
			<td>KGP902</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood cell counting plate</td>
			<td>Servicebio</td>
			<td>WG607</td>
			<td></td>
		</tr>
		<tr>
			<td>CAT assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A007-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescence detection system</td>
			<td>Shanghai Qinxiang Scientific Instrument Factory</td>
			<td>ChemiScope 6100</td>
			<td></td>
		</tr>
		<tr>
			<td>CK assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A032-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme Q10 (CoQ 10)</td>
			<td>Macklin</td>
			<td>C6129</td>
			<td></td>
		</tr>
		<tr>
			<td>Crocetin</td>
			<td>Chengdu Ruifensi Biotechnology Co., Ltd.</td>
			<td>RFS-Z01802006012</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI-containing antifluorescence quenching tablets</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZLI-9557</td>
			<td></td>
		</tr>
		<tr>
			<td>DCFH-DA</td>
			<td>Beyotime</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Solarbio</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>8122091</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced Chemiluminescence (ECL) solution</td>
			<td>NCM Biotech</td>
			<td>P10100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Corning-Cellgro</td>
			<td>35-081-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7.0&#160;</td>
			<td>https://www.graphpad.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Px assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A005-1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 myocardial cells</td>
			<td>Beijing Dingguochangsheng Biotech Co., Ltd.</td>
			<td>CS0062</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-goat IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2305</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2301</td>
			<td></td>
		</tr>
		<tr>
			<td>JC-1 mitochondrial membrane potential assay kit</td>
			<td>LABLEAD</td>
			<td>J22202</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A020-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A003-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Aladdin</td>
			<td>A2114057</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS assay</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Perhydrol</td>
			<td>G-clone</td>
			<td>CS7730</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor</td>
			<td>CWBIO</td>
			<td>CW2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Beyotime</td>
			<td>C0351</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membranes</td>
			<td>Millipore</td>
			<td>ISEQ00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioimmunoprecipitation assay (RIPA) lysis buffer</td>
			<td>Solarbio</td>
			<td>R0010</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE gels</td>
			<td>Shanghai Epizyme Biomedical Technology</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer powder</td>
			<td>Servicebio</td>
			<td>G2018-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE transfer buffer powder</td>
			<td>Servicebio</td>
			<td>G2017-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>SOD assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A001-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline powder</td>
			<td>Servicebio</td>
			<td>G0001-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SLCC9172</td>
			<td></td>
		</tr>
		<tr>
			<td>TUNEL apoptosis assay kit</td>
			<td>Beyotime</td>
			<td>C1086</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Solarbio</td>
			<td>T8220</td>
			<td></td>
		</tr>
	</tbody>
</table>

protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy

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。

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?...

Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

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。

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

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

该协议为研究自噬细胞的技术方法提供了参考，并详细描述了整个实验程序。该技术的主要优点是它允许观察自噬通量变化的长度。在开始实验之前，确定有效的感染浓度并采取必要的安全措施。首先在室温下以179G离心中和的H9C2心肌细胞五分钟。弃去上清液，轻轻吹气将细胞混合在DMEM完全培养基中。将细胞分成九个相等的比例。将二甲基亚砜添加到对照组。然后，向其余组中添加不同浓度的藏红花素。在96孔板中每孔接种100微升细胞。孵育24小时后，弃去上清液。然后，用PBS洗涤细胞三次。向每个孔中加入100微升DMEM基础培养基，并在37摄氏度下孵育细胞4小时。现在，加入 20 微升 MTS 并按照所示再孵育两个小时。最后，测量490纳米处的吸光度并计算细胞活力。要测定 ROS，请用无血清培养基以 1：1000 的比例稀释 DCFH-DA。从先前接种有H9C2细胞的48孔板中丢弃细胞的上清液。然后，用无血清培养基洗涤细胞两次。向每个孔中加入150微升稀释的DCFH-DA，并在37摄氏度下孵育20分钟。接下来，用无血清培养基洗涤细胞三次。最后，使用荧光显微镜捕获图像。为了分析线粒体膜电位，将JC-1工作溶液添加到先前培养的细胞中并充分混合。将它们在 37 摄氏度下孵育 20 分钟。然后，用JC-1染色缓冲液洗涤细胞两次。向每个孔中加入完整的培养基，并使用荧光显微镜捕获照片。在室温下用细胞固定溶液固定细胞30分钟。然后，用PBS清洗一次。向每个孔中加入0.3%Triton-100，并在室温下孵育五分钟。两次PBS洗涤后，向每个孔中加入TUNEL检测工作溶液，并将细胞在37摄氏度的黑暗中孵育60分钟。添加含有抗荧光淬灭片的DAPI，并如图所示捕获图像。在48孔板的每个孔中用新鲜培养基替换先前培养的细胞中的一半培养基。添加mCherry GFP-LC3B腺病毒，然后加入聚溴乙烯以提高感染效率。每 24 小时更换一次培养基。同时使用共聚焦显微镜观察荧光蛋白表达以确认感染。感染后，按照手稿中的说明培养和捕获细胞。首先在室温下用4%多聚甲醛固定先前培养的细胞10分钟，然后加入0.1%Triton-100。接下来，用无动物阻断溶液封闭细胞一小时。然后，将它们与一抗在 4 摄氏度下孵育过夜。第二天，向细胞中加入荧光二抗，并在黑暗中孵育一小时。最后，在捕获图像之前添加含有抗荧光淬灭片的DAPI。在本研究中，藏红花素对细胞有显著的增殖作用。而浓度高于200微摩尔抑制H9C2细胞的增殖。过氧化氢处理后细胞活力大大降低。然而，藏红花素在一定程度上扭转了这种变化。过氧化氢处理后ROS水平的增强在一定程度上被藏红花素逆转。荧光结果也证实了藏红花素的ROS降低。此外，番红花素的添加恢复了由过氧化氢处理引起的线粒体膜电位塌陷。同样，藏红花素也逆转了细胞凋亡。此外，藏红花素预处理逆转了由于过氧化氢处理诱导的H9C2心肌细胞的自噬流。藏红花素预处理还减少了过氧化氢处理诱导的帕金线粒体易位。按照这一过程，电子显微镜也可用于直接观察微噬细胞，从而可以更直观地观察线粒体被自噬体吞噬。

Research

JoVE Journal

Medicine

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Acute Myocardial Infarction in Rats

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

多巴酚丁胺诱导的心脏压力测试和监控通过在小鼠心脏导管插入术

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Ultrasonic Assessment of Myocardial Microstructure

心肌微观结构超声波评估

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

人类心脏肌细胞模型中体温过低预科后心肌保护体外评估

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Utilizing Percutaneous Ventricular Assist Devices in Acute Myocardial Infarction Complicated by Cardiogenic Shock

经皮心室辅助装置治疗急性心肌梗死并发心源性休克

Cardiogenic shock is defined as persistent hypotension, accompanied by evidence of end organ hypo-perfusion. Percutaneous ventricular assist devices ...

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

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