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

Summary

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.

Abstract

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 µM) 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.

Introduction

Acute myocardial infarction (AMI) is a life-threatening myocardial necrosis caused by severe and persistent ischemia and hypoxia to coronary arteries^1,2. Percutaneous coronary intervention (PCI) is one of the first-line therapeutic strategies for AMI, and usually protects cardiomyocytes from ischemic damage^3,4. 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 complications^5,6. 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 β-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 apoptosis^10,11. 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 years^12,13,14,15. 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 patients^16,17. According to TCM theory, AMI is caused by blood stasis^18,19, 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.

Protocol

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

1. Add 10% fetal bovine serum and 1% penicillin/streptomycin to Dulbecco'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 Materials) to prepare DMEM complete medium.
2. Thaw the liquid nitrogen-frozen H9c2 myocardial cells (see Table of Materials) in warm water at 37 °C with a quick and uniform stir until the ice melts.
3. Transfer the cells to a centrifuge tube and add four times the volume of the DMEM complete medium. Centrifuge at 358 x g for 5 min at room temperature and discard the supernatant using a pipette.
4. Dilute the obtained cell suspension with culture medium, blow gently, and inoculate the cells in a culture flask. Culture in an incubator at 37 °C with 5% CO₂.

2. Determination of cell viability

1. Dissolve crocetin (see Table of Materials) in dimethyl sulfoxide (DMSO) to concentrations of 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, and 200 mM.
2. Dissociate the H9c2 myocardial cells by trypsin, then neutralize the mixture with DMEM complete medium.
3. Transfer the cells to a centrifuge tube and centrifuge at 179 x g for 5 min at room temperature. Discard the supernatant and mix the cells in DMEM complete medium by gently blowing.
4. Count the cells using a blood cell counting plate²² (see Table of Materials) and dilute them to 5 × 10⁴ cells/mL with DMEM complete medium.
5. Divide the cells into nine equal portions. Add DMSO (diluted in a 1:1,000 ratio with DMEM complete medium) to the control group and add different concentrations of crocetin to the remaining groups at a ratio of 1:1,000.
6. Seed the cells in 96-well plates at 100 µL per well. Discard the supernatant after incubation for 24 h and wash the cells three times with PBS.
7. After adding 100 µL of DMEM basal medium, incubate the cells for 4 h and add 20 µL of MTS (see Table of Materials).
8. Incubate the cells for another 2 h, measure the absorbance at a wavelength of 490 nm, and calculate the cell viability.
   ​NOTE: Cell viability = (OD treated - OD blank) / (OD control - OD blank) × 100.

3. Determination of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH Px), and catalase (CAT)

1. Seed the H9c2 cells in a 6-well plate at a density of 1 × 10⁵ cells. Collect the supernatant after the intervention and detect LDH and SOD levels, according to the manufacturer's instructions (see Table of Materials).
2. Collect the supernatant and wash it with phosphate-buffered saline (PBS) once. Add the cell lysate to the culture dish and allow it to sit on ice for 20 min. Collect the liquid from the culture dish into a centrifuge tube.
3. Detect CK, MDA, GSH-Px, and CAT levels according to the manufacturer's instructions²³ (see Table of Materials and Supplementary File 1).
4. Detect the total protein concentration of the lysis by the bicinchoninic acid (BCA) method to correct the concentration of CK, MDA, GSH-Px, and CAT²⁴(see Table of Materials).

4. Determination of ROS

1. Seed the H9c2 cells in a 48-well plate at a density of 5 × 10³ cells. Dilute DCFH-DA (see Table of Materials) at a 1:1,000 ratio with the serum-free medium. Discard the cell supernatant and wash the cell twice with a serum-free medium.
2. Add 150 µL of diluted DCFH-DA into each well and incubate at 37 °C for 20 min. Wash the cells three times with a serum-free medium and capture images under a fluorescence microscope (see Table of Materials).

5. Detection of mitochondrial membrane potential

1. Detect the mitochondrial membrane potential using a JC-1 mitochondrial membrane potential assay kit²⁵ (see Table of Materials). Discard the culture medium and wash them once with serum-free medium.
2. Add JC-1 working solution to each tube and mix them well. Incubate at 37 °C for 20 min and wash the cells twice with JC-1 staining buffer. Add complete medium to each well and capture photographs under a fluorescence microscope.

6. TUNEL staining assay

1. Use a TUNEL apoptosis assay kit (see Table of Materials) to determine apoptosis rates²⁶. Discard the supernatant and wash the pellet once with serum-free medium.
2. Add cell fixation solution and wash them with PBS once after incubating at room temperature for 30 min.
3. Add 0.3% triton-100 to each well and incubate at room temperature for 5 min. Wash the cells with PBS twice. Add TUNEL detection working solution and incubate at 37 °C in the dark for 60 min.
4. Add an anti-fluorescence quenching sealing tablet containing 4′,6-diamidino-2-phenylindole (DAPI; see Table of Materials) and capture photographs under a fluorescence microscope.

7. Monitoring autophagic flow by transfection of mCherry GFP-LC3B adenovirus

1. Replace half of the medium in each well with fresh medium. Add Ad-mCherry GFP-LC3B adenovirus (multiplicity of infection [MOI] of 2) to the culture medium and add 5 µg/mL polybrene (see Table of Materials) to improve the infection efficiency.
2. Replace the fresh medium after 24 h and observe the expression of the fluorescent protein under a confocal microscope.
3. Culture the cells with the same conditions as section 1 after confirming successful virus infection and capture images under the confocal microscope²⁷.
   ​NOTE: More than 20% of cells showed green fluorescent protein (GFP) fluorescence, indicating a successful infection.

8. Western blot analysis

1. Collect cells for whole protein extraction and lyse them (RIPA, protease inhibitor, and phosphatase inhibitor = 100:1:1; see Table of Materials) on ice for 30 min.
2. Add 5x protein loading buffer to the protein sample after protein quantification and boil the samples for 10 min.
3. Electrophorese the samples that contain equal amounts of protein with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels²⁸. Then, transfer the samples onto polyvinylidene difluoride (PVDF) membranes.
4. Block the PVDF membranes for 1 h with 5% skim milk powder and incubate with the primary antibody (PINK1, Parkin; see Table of Materials) at 4 °C overnight and the secondary antibody at room temperature for 1 h.
5. Detect the target bands using enhanced chemiluminescence (ECL) solution and the chemiluminescence detection system. Use Image J software to quantify the bands via densitometry²⁸.

9. Detection of Parkin's mitochondrial translocation by immunofluorescence

1. Observe the colocalization of Parkin with mitochondria by double immunofluorescence staining. Discard the cell supernatant from each group and wash them three times with PBS.
2. Fix the cells for 10 min at room temperature with 4% paraformaldehyde and add 0.1% triton-100 in PBS.
3. Block with animal-free block solution (see Table of Materials) for 1 h to prevent non-specific binding between proteins and antibodies and reduce the fluorescence background.
4. Incubate with a primary antibody (Parkin and Tom20; see Table of Materials) at 4 °C overnight.
5. Add a fluorescent secondary antibody (see Table of Materials) and incubate in the dark for 1 h.
6. Add the DAPI-containing anti-fluorescence quenching tablets and capture images under the confocal microscope.

10. Statistical analysis

1. Perform statistical analysis using graphing and analysis software (see Table of Materials).
2. Compare continuous variables between groups using one-way ANOVA. p < 0.05 was considered statistically significant.

Results

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

Discussion

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 "promoting blood circulation and minimizing blood stasis" and is widely used in the treatment of myocardial infarction^30,31. Saffron is believed to h...

Materials

Name	Company	Catalog Number	Comments
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