This study was supported by the Beijing Natural Science Foundation (No. 7202119) and the National Natural Science Foundation of China (No. 82274380).

The experiments were performed in the Laboratory of Physiology at the Beijing University of Chinese Medicine, China. All study methods were performed in accordance with the relevant guidelines and regulations of Beijing University.
1. Cell culture
<ol>
	<li>Add 10% fetal bovine serum and 1% penicillin/streptomycin to Dulbecco&#39;s modified Eagle medium (DMEM) basic medium (with 4.5 g/L D-glucose, 4.g.g/L L-glutamine, and 110 mg/L sodium pyruvate; see Table of Materi...

<ol>
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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. 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The exploration of effective ingredients from complex compounds of natural drugs through advanced technology has been a hotspot of TCM research29, and can provide laboratory evidence for future drug development after verification. Safflower is a representative drug in the treatment of &#34;promoting blood circulation and minimizing blood stasis&#34; and is widely used in the treatment of myocardial infarction30,31. Saffron is believed to h...

Acute myocardial infarction (AMI) is a life-threatening myocardial necrosis caused by severe and persistent ischemia and hypoxia to coronary arteries1,2. Percutaneous coronary intervention (PCI) is one of the first-line therapeutic strategies for AMI, and usually protects cardiomyocytes from ischemic damage3,4. The distal myocardium will lack blood and oxygen supply if not promptly and effectively treated after AMI, which leads to ischemic necrosis and further cardiovascular complications5,6. Promoting cardiomyocyte recovery and minimizing irreversible myocardial damage after missing the PCI surgical opportunity has been a research hotspot. After AMI, cardiomyocytes are in a state of ischemia and hypoxia, resulting in the inhibition of mitochondrial oxidative phosphorylation, reduction of NAD+ to NADPH, and increased single electron reduction7. As a result, the incomplete reduction reaction of oxygen generates an excess of reactive oxygen species (ROS) and ultimately leads to oxidative stress damage to cardiomyocytes8. An excessive accumulation of ROS triggers lipid peroxidation, further disrupting the structure and function of mitochondrial membranes. The result is a continuous opening of mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential, inducing apoptosis and necrosis.
Angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), the inhibitors of &#946;-adrenoceptors, aldosterone antagonists, and other standard drugs in AMI can help enhance heart function after myocardial infarction and prevent the occurrence of malignant events, such as arrhythmias and left ventricular remodeling9. However, postinfarction survival and prognosis are greatly affected by infarct size, and satisfactory results have not been achieved for reducing cardiomyocyte apoptosis10,11. Thus, the development of drugs to promote cardiomyocyte recovery after myocardial infarction has become an urgent issue.
Traditional medicine has been a source of inspiration for modern pharmaceutical research for many years12,13,14,15. Traditional Chinese medicine (TCM) has a long history in the treatment of AMI, and a series of randomized control trials in recent years have confirmed that TCM can indeed improve the prognosis of patients16,17. According to TCM theory, AMI is caused by blood stasis18,19, so drugs for promoting blood circulation are usually used for the treatment of AMI in the acute phase20. Among them, saffron is believed to have a powerful effect on blood activation and stasis, and is often used in the acute treatment of AMI. Crocetin, a major component of saffron, may play a key role in protecting cardiomyocytes21.
In this study, H9c2 myocardial cells were induced by H2O2 to simulate myocardial ischemia/reperfusion, which causes a cardiomyocyte injury of AMI, and crocetin was used as an intervention to investigate its protective effect against oxidative stress-induced myocardial injury. The mechanism of crocetin protecting cardiomyocytes was further explored through mitophagy. More importantly, this article provides a reference for the technical approach to the study of mitophagy and describes the entire experimental procedure in detail.

<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

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Effects of crocetin on cell viability 
Crocetin at 0.1 &#181;M, 0.5 &#181;M, 1 &#181;M, 5 &#181;M, 10 &#181;M, 50 &#181;M, and 100 &#181;M had a significant proliferative effect on cells, while crocetin at concentrations above 200 &#181;M significantly inhibited the proliferation of H9c2 cells (Figure 1A). After 4 h of treatment with 400 &#181;M H2O2, the cell viability was reduced considerably, and crocetin could reverse this change to a certain e...

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

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

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

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

This protocol provides a reference for the technique approach to the study of autophage and it describes the entire experimental procedures in detail. The main advantage of this technique is that it allows the length observation of the change in autophagic flux. Before starting the experiment, determine the effective infection concentration and taking necessary safety measures.Begin by centrifuging neutralized H9C2 myocardial cells at 179G for five minutes at room temperature. Discard the supernatant and mix the cells in DMEM complete medium by gently blowing. Divide the cells into nine equal proportions.Add dimethyl sulfoxide to the control group. Then, add varying concentrations of crocetin to the remaining groups. Seed 100 microliters of cells per well in a 96-well plate.Once incubated for 24 hours, discard the supernatant. Then, wash the cells three times with PBS. Add 100 microliters of DMEM basal medium to each well and incubate the cells for four hours at 37 degrees Celsius.Now, add 20 microliters of MTS and incubate for another two hours as demonstrated. Finally, measure the absorbance at 490 nanometers and calculate the cell viability. To determine ROS, dilute DCFH-DA at a 1:1000 ratio with the serum-free medium.Discard the cell's supernatant from a 48-well plate, previously seeded with H9C2 cells. Then, wash the cells twice with a serum-free medium. Add 150 microliters of diluted DCFH-DA to each well and incubate it at 37 degrees Celsius for 20 minutes.Next, wash the cells three times with serum-free medium. Finally, capture images using a fluorescence microscope. To analyze mitochondrial membrane potential, add JC-1 working solution to the previously cultured cells and mix well.Incubate them at 37 degrees Celsius for 20 minutes. Then, wash the cells twice with JC-1 staining buffer. Add complete medium to each well and capture photographs using a fluorescence microscope.Fix the cells with cell fixation solution for 30 minutes at room temperature. Then, wash them with PBS once. Add 0.3%Triton-100 to each well and incubate at room temperature for five minutes.After two PBS washes, add TUNEL detection working solution to each well and incubate the cells at 37 degrees Celsius in the dark for 60 minutes. Add DAPI containing an anti-fluorescence quenching tablet and capture the images as demonstrated. Replace half of the medium from the previously cultured cells with fresh medium in each well of a 48-well plate.Add the mCherry GFP-LC3B adenovirus followed by polybrene to improve the infection efficiency. Replace the medium every 24 hours. Simultaneously using a confocal microscope, observe fluorescent protein expression to confirm infection.Once infected, culture and capture the cells as described in the manuscript. Begin by fixing previously cultured cells with 4%paraformaldehyde for 10 minutes at room temperature and add 0.1%Triton-100. Next, block the cells with an animal-free block solution for one hour.Then, incubate them with a primary antibody at four degrees Celsius overnight. The next day, add a fluorescent secondary antibody to the cells and incubate them in the dark for one hour. Finally, add the DAPI containing anti-fluorescence quenching tablets before capturing the images.In the present study, crocetin had a significant proliferative effect on cells. While a concentration above 200 micromolar inhibited the proliferation of H9C2 cells. The cell viability was reduced considerably after hydrogen peroxide treatment.However, crocetin reversed this change to a certain extent. The enhanced ROS level after hydrogen peroxide treatment was reversed by crocetin to some extent. The fluorescence results also confirmed reduced ROS with crocetin.Further, crocetin addition restored the mitochondrial membrane potential collapses caused by hydrogen peroxide treatment. Similarly, apoptosis was also reversed by crocetin. In addition, the crocetin pre-treatment reversed the autophagy flow of H9C2 cardiomyocytes, induced due to hydrogen peroxide treatment.Crocetin pre-treatment also reduced the translocation of Parkin mitochondria induced by hydrogen peroxide treatment. Following this procedure, electron microscopic can also be used to directly observe a microphage, allowing for a more intuitive view of mitochondria being engulfed by autophagosomes.

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Research

JoVE Journal

Medicine

897 조회수.  Beijing University of Chinese Medicine. 이 프로토콜은 자가파지 연구에 대한 기술 접근법에 대한 참조를 제공하고 전체 실험 절차를 자세히 설명합니다. 이 기술의 가장 큰 장점은 자가포식 플럭스의 변화를 길이 관찰할 수 있다는 것입니다. 실험을 시작하기 전에 효과적인 감염 농도를 결정하고 필요한 안전 조치를 취하십시오.중화된 H9C2 심근 세포를 실온에서 5분 동안 179G에서 원심분리하는 것으로 시작한?...