Parkinson’s disease protein 7 protected against oxidative stress of myocardial infarction direct through p47phox and nicotinamide adenine dinucleotide phosphate oxidase 4

Abstract

In the present study, we aimed to investigate the role and mechanism of Parkinson’s disease protein 7 (Park7) in myocardial infarction (MI). The Park7 expression in the serum and tissues was down-regulated in mice with MI. Recombinant Park7 protein protected against MI-induced injury and reduced oxidative stress in mice model. Conversely, knockout Park7 increased injury of MI and promoted oxidative stress in MI mice model. In embryonic rat cardiac myoblasts H9c2 cells, over-expression of Park7 reduced reactive oxygen species (ROS)-induced oxidative stress, while down-regulation of Park7 increased ROS-induced oxidative stress. Park7 combined nicotinamide adenine dinucleotide phosphate (NADPH) oxidase cytoplasmic subunit p47phox protein had direct effect on inducing NADPH activator. The inhibition of p47phox reduced the effects of Park7 in ROS production of H₂O₂-treated H9c2 cells. The regulation of NADPH participated in the effects of Park7 on ROS production of in both MI mice model and H₂O₂-treated H9c2 cells. Our data demonstrated that Park7 protects against oxidative stress in MI model direct through p47phox and NADPH oxidase 4.

Introduction

At present, cardiovascular disease is the main cause of death of urban residents all over the world. ¹ Among cardiovascular diseases, myocardial infarction has a very high disability rate and mortality rate. ² In the early stage of myocardial ischemia (MI), myocardial infarction due to hypoxia and ischemia myocardial tissue in oxidation and antioxidant imbalance leading to myocardial apoptosis and oxidative stress. ³ Myocardial apoptosis and oxidative stress can accelerate ventricular remodeling and increase the risk of death in chronic myocardial infarction. ⁴ Hypoxia is a pseudo-stimulus to myocardial tissue, and the imbalance between reactive oxygen species (ROS) production and antioxidant defense further leads to oxidative stress.^5,6

There are several sources of ROS in heart and blood vessel tissues, including mitochondrial electron transport chain, xanthine oxidase, uncoupled endothelial nitric oxide synthase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ⁷ NOX is a type of protein, whose main function is to generate ROS. ⁸ It is of great importance to clarify the expression and role of NOX in common cardiovascular diseases for the implementation of clinical antioxidant therapy. ⁹ Among them, NOX is an important source of active oxygen in the body. And p47phox plays a crucial role in activating NOX. ¹⁰

Parkinson disease 7 (Park7), a marker of nerve damage, is involved in the regulatory processes of cell transformation, antioxidant response, molecular chaperones and apoptotic inhibition, which is massively expressed in astrocytes in the infarct area. ¹¹ Clinical studies have shown that Park7 is specifically expressed in patients with acute cerebral infarction and can be used as an early diagnostic indicator for patients with ischemic stroke.^13–15 However, the mechanism of Park seven in myocardial infarction is unclear. Therefore, in this study, we aimed to investigate the role and mechanism of Park7 in the oxidative stress of myocardial infarction.

Materials and methods

Animals and treatment

The animal experiments were carried out in strict accordance with the recommendations for Care and Use of Laboratory Animals of Dongzhimen Hospital Affiliated with Beijing University of Traditional Chinese Medicine (No. KIJP-L0035032). Male C57/BL mice, including mice treated with recombinant Park7 protein and Park7-knockout mice, were obtained from the Laboratory Animal Center of Dongzhimen Hospital Affiliated with Beijing University of Traditional Chinese Medicine. They were housed in standard metabolic cages and maintained under standard condition at room temperature of 23–25°C and humidity of 55–60% with a 12:12 h light and dark cycle. The protocol was approved by the Committee on the Ethics of Laboratory Animals of Dongzhimen Hospital affiliated with Beijing University of Chinese Medicine. All mice were randomly divided into sham and MI model groups.

As described in the previous study, the MI model was established. ¹² A total of 40 mice (20 recombinant Park7 protein-treated mice and 20 Park7-knockout mice) were intraperitoneally injected with 35 mg/kg pentobarbital sodium (Sigma Chemical Co., St. Louis, MO, USA), the rodent ventilator was switched on, and the left chest hair were deprived and subjected to routine disinfection. Thoracic cavity was entered via the intercostal space with the strongest cardiac impulse. Pericardium was removed and the left anterior descending coronary artery (LAD) was located. LAD was then ligated using myocardium sing the 8/0 atraumatic suture.

Echocardiography and myocardial histological examination

At the fifth week after the operation, 10 mice were randomly selected from each group. They were anesthetized with 35 mg/kg pentobarbital sodium (Sigma Chemical Co., St. Louis, MO, USA) and fixed on an ultrasound test table. Mice were deprived with left chest hair, and smeared using ultrasound coupler (Shenzhen Hongxin optoelectronics Co., Ltd, Shenzhen, China). A Canada VEVO 2100 High-frequency Animal Ultrasound System (Toronto, Canada) was used to perform echocardiography examination.

Heart tissues were cut into 4-μm sections and stained with hematoxylin and eosin (H&E, Sigma Chemical Co., St. Louis, MO, USA). Tissue sample were observed under an Olympus BX43 F fluorescence microscope (Olympus, Tokyo, Japan).

Reverse transcription-polymerase chain reaction (RT-PCR)

mRNA expression of Park7 was detected by RT-PCR method. Total RNA was extracted from mouse myocardial tissues by using TRIzol reagent (Tiangen, Beijing, China) and converted into cDNA using the TIANScript RT kit (Tiangen, Beijing, China). PCR conditions were set as follows: 30 s at 95°C for denaturation, 40 s at 56°C for annealing, and 20 s at 72°C for extension for 40 cycles. The fold change of the relative mRNA expression for each sample was calculated through relative quantification (2^-∆∆Ct). mRNA expressions were subjected to the internal gene β-actin.

Enzyme-linked immunosorbent assay (ELISA)

The levels of ROS, malondialdehyde (MDA) and superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-PX) were determined by ELISA kits (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., Nanjing, China) according to the manufacturer’s instruction. The colorimetric reaction was measured at 450 nm by microplate reader (ThermoFisher, MA, USA).

Western blotting

Heart tissues were homogenized in lysis buffer containing protease inhibitor cocktail (MedChem Express, Princeton, NJ, USA) and protein were collected. Total proteins in cells were extracted using lysis buffer containing protease inhibitor cocktail (MedChem Express, Princeton, NJ, USA). The protein concentration was determined by the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Total protein samples (50 μg) were loaded separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore, MO, USA). The transferred membranes were blocked with 5% bovine serum albumin (BSA) for 60 min and incubated with anti-Park7, anti-p47phox, anti-NOX4 and anti-β-actin (Abcam, San Jose, CA, USA). After washing in Tris-buffered saline Tween 20 (TBST) for 15 min, membranes were incubated with goat anti-rabbit or goat anti-mouse secondary antibodies for 1 h at 37°C. After further three washes with TBST, the bands were added to Infrared Imaging System (Odyssey Sa; Li-COR, Lincoln, NE, USA) with ECL western blotting substract reagent (Tsea biotech Co., Shanghai, China) for detection.

Cell experiments

The embryonic rat cardiac myoblast H9c2 cell (China Center for Type Culture Collection of Wuhan University, Wuhan, china) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher, MA, USA) containing 10% (vol/vol) fetal bovine serum (FBS; Gibco, Australia) at 37°C in a humidified atmosphere of 5% CO₂. H9c2 cells treated with H₂O₂ (1000 μM) were transfected with Park7, si-p47phox, si-Park7, p47phox and negative mimics using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h of transfection, cells were cultured with DMEM containing 10% FBS at oxygen training for 2 h.

Immunofluorescence and Proximity Ligation Assay (PLA)

After experiment, cell was seeded into 12 well-plates with glass bottom inserts, and were washed with phosphate-buffered saline (PBS), and fixed with cold polysorbate for 15 min at room temperature. Cell was added with 0.1% Triton X-100 (ThermoFisher, MA, USA) in PBS for 15 min, and then blocked with 5% bovine serum albumin (BSA, ThermoFisher, MA, USA) in PBS buffer for 1 h at room temperature. Then, cell was incubated with anti-Park7 and anti-p47phox antibodies at 4°C overnight. The cells were then incubated with DyLight 595 AffniPure Donkey anti-rabbit IgG (H + L) or 488 anti-mouse IgG (H + L) (Dako, Carpinteria, CA, USA) at room temperature for 2 h. The nuclei were counter-stained by 4,6-diamidino-2 phenylindole (DAPI) for 5 min after washing with PBS. Images were captured by an Olympus BX43 F fluorescence microscope (Olympus, Tokyo, Japan).

Proximity ligation assay experiment was done using PLA (DUO92007, Sigma-Aldrich, Saint Louis, MO, USA). Cells were incubated with the appropriate probes (DUO92002 and DUO92004, Sigma-Aldrich, Saint Louis, MO, USA) for 1 h at 37°C after incubating with primary antibodies. Probes were then ligated for 1 h at room temperature and stained with DAPI for 10 min.

Statistical analysis

All experiments were repeated three times with the same sample. Statistical analysis was made by Statistical Product and Service Solutions (SPSS) software version 24.0 (International Business Machines, corp., Armonk, NY, USA). Data were expressed as means ± standard deviation (SD). p value was calculated by one-way ANOVA followed by Bonferroni correction test afterwards. In addition, the differences between the two groups were compared by student t test. GraphPad Prism 5.0 software (La Jolla, California, USA) was carried out to draw figures. The criterion for statistical differences was p < .05 for all comparisons.

Results

The Park7 expression in MI mice model

To confirm the importance of Park7 in MI, we analyzed the gene expressions in mice with MI and looked for special gene. Heat map showed that Park7 gene expression were down-regulated in MI mice model (Figure 1(a), (b)). Compared to the control, the mRNA (Figure 1(c)) and protein (Figure 1(d), (e)) expressions of Park7 were also reduced in mice with MI (p < .05).

Figure 1.

The expression of Park7 in myocardial infarction of mice model. Heat map (a), statistical results (b), Park7 mRNA and protein expression in mice model of myocardial infarction (c, d and e). Sham: sham control group; MI: Kawasaki Disease-Related Vasculitis Model mice group. ##p < .01 compared with sham control group.

Park7 protected against injury in MI mice model

Next, we confirmed the function of Park7 in MI mice injected with recombinant Park7 or Park7^−/− MI mice. It was shown that Park7 protein over-expression reduced left ventricular internal diameter in systole and diastole (Figure 2(a)), increased left ventricular ejection fraction (Figure 2(b)), left ventricular fractional shortening (Figure 2(c)), and left ventricular stroke volume (Figure 2(d)), prevented myocardial fibrosis (Figure 2(e)) and inhibited left levels of hydroxyproline (Figure 2(f)) in mice model of MI (all p < .05). Conversely, knockdown of Park7 increased left ventricular internal diameter in systole and diastole (Figure 2(g)), the inhibition of left ventricular ejection fraction (Figure 2(h)), left ventricular fractional shortening (Figure 2(i)), and left ventricular stroke volume (Figure 2(j)), the aggravation of myocardial fibrosis (Figure 2(k)) and the increased left levels of hydroxyproline (Figure 2(k)) in MI mice (all p < .05).

Figure 2.

Park7 protect against ROS production of myocardial infarction in mice model. Left ventricular internal diameter in systole and diastole (a), left ventricular ejection fraction (b), left ventricular fractional shortening (c), myocardial fibrosis (d), left ventricular stroke volume (e), and left levels of hydroxyproline (f) in mice model by Park7 protein; Left ventricular internal diameter in systole and diastole (g), left ventricular ejection fraction (h), left ventricular fractional shortening (i), myocardial fibrosis (j), left ventricular stroke volume (k), and left levels of hydroxyproline (l) in Park7^−/− mice model. MI model: Kawasaki Disease-Related Vasculitis Model mice group; MI model+Park7: Kawasaki Disease-Related Vasculitis Model mice treated with Park7 protein group; WT+MI model: Kawasaki Disease-Related Vasculitis Model WT mice group; Park7^−/−+MI model: Kawasaki Disease-Related Vasculitis Model Park7^−/− mice group. ##p < .01 compared with Kawasaki Disease-Related Vasculitis Model mice group or Kawasaki Disease-Related Vasculitis Model WT mice group.

Park7 decreased oxidative stress in H9c2 cells

To evaluate effects of Park7 on oxidative stress, H9c2 cells were treated with Park7 mimics (Figure 3(b)) and siRNA (Figure 3(h)). It was found that over-expression of Park7 reduced ROS production (Figure 3(a)) and MDA levels (Figure 3(c)), and increased the levels of SOD (Figure 3(d)), GSH (Figure 3(e)) and GSH-PX (Figure 3(f)) in H9c2 cells (all p < .05). Next, down-regulation of Park7 increased ROS production (Figure 3(g)) and MDA levels (Figure 3(i)), and promoted the levels of SOD (Figure 3(j)), GSH (Figure 3(k)) and GSH-PX (Figure 3L) in H₂O₂-treated H9c2 cells (all p < .05).

Figure 3.

Park7 increased ROS production in vitro model of myocardial infarction. ROS production (a and b), MDA, SOD, GSH and GSH-PX levels (c, d, e and f) in vitro model by over-expression of Park7; ROS production (g and h), MDA, SOD, GSH and GSH-PX levels (i, j, k and l) in vitro model by down-regulation of Park7. Negative: negative mimic group; Park7: over-expression of Park7 group; si-Park7: down-regulation of Park7 group. ##p < .01 compared with negative mimic group.

Park7 regulated p47phox expression direct through NADPH activation in H₂O₂-treated H9c2 cells

To explain the mechanism of Park7 in MI, the Park7 expression in H₂O₂-treated H9c2 cells was measured. Heat map (Figure 4(a)), analysis diagram (Figure 4(b)) and volcanic map (Figure 4(c)) showed that Park7 expression was down-regulated, and NOX4 and p47phox expression was down-regulated in H₂O₂-treated induced by over-expression of Park7. Immunofluorescence analysis showed that over-expression of Park7 induced p47phox expression in H₂O₂-treated H9c2 cells (Figure 4(d)). Western blotting analysis showed that over-expression of Park7 increased Park7 and p47phox protein expression (Figure 4(e), (g)), and suppressed NOX4 protein expression (Figure 4(h)) in H₂O₂-treated H9c2 cells (all p < .05). Down-regulation of Park7 suppressed the protein expression levels of Park7 (Figure 4(i)) and p47phox (Figure 4(j)), and induced NOX4 protein expression (Figure 4(k)) in H₂O₂-treated H9c2 cells (p < .05).

Figure 4.

Park7 regulated p47phox expression to direct NADPH activation was important signal in the function of Park7 in myocardial infarction in vitro model. Heat map and microarray data (a), Volcanic map (b), result analysis (c), Park7 and p47phox expression (Immunofluorescence, d), Park7, p47phox, NOX4 protein expression in vitro model by over-expression of Park7 (e, f, g and h); Park7, p47phox, NOX4 protein expression in vitro model by down-regulation j and k). Negative: negative mimic group; Park7: over-expression of Park7 group; si-Park7: down-regulation of Park7 group. ##p < .01 compared with negative mimic group.

Next, Park7 protein hyperlinked with p47phox protein (Figure 5(a)), and p47phox protein hyperlinked with Park7 protein (Figure 5(b)). Using PLA experiments, this study showed a proximity of Park7 protein with p47phox protein in MI mice model (Figure 5(c), (d)). Immunoprecipitations results confirmed the interaction between Park7 protein and p47phox protein (Figure 5(e), (f)).

Figure 5.

Park7 regulated p47phox expression was important signal in the function of Park7 in myocardial infarction in vitro model. Park7 protein hyperlinked with p47phox protein (a), p47phox protein hyperlinked with Park7 protein (b), this study showed that a proximity of Park7 protein with p47phox protein in murine using PLA experiments (c and d), Park7-p47phox protein using PLA experiments (e), the interaction of Cathepsin B only occur with NLRP3 (f). ##p < .01 compared with control group.

Furthermore, in H9c2 cells, Park7 over-expression induced p47phox protein expression and suppressed NOX4 protein expression (Figure 6(a)-(c)). Knockout Park7 suppressed p47phox protein expression and induced NOX4 protein expression (Figure 6(c)-(e), all p < .05). Park7 over-expression decreased MDA levels (Figure 6(f)), and increased the levels of SOD (Figure 6(g)), GSH (Figure 6(h)) and GSH-PX (Figure 6(i)) in H₂O₂-treated H9c2 cells (all p < .05). Knockout of Park7 increased MDA levels, and reduced the levels of SOD, GSH and GSH-PX in H₂O₂-treated H9c2 cells (Figure 6(j)-(m), all p < .05).

Figure 6.

Park7 regulated p47phox expression to direct NADPH activation was important signal in the function of Park7 in myocardial infarction in vivo model. p47phox and NOX4 protein expression (a, b and c), MDA, SOD, GSH and GSH-PX levels (d, e, f and g) in mice model by Park7 protein; p47phox and NOX4 protein expression (h, i and j), MDA, SOD, GSH and GSH-PX levels (k, l, m and n) in Park7^−/− mice model. MI model: Kawasaki Disease-Related Vasculitis Model mice group; MI model+Park7: Kawasaki Disease-Related Vasculitis Model mice treated with Park7 protein group; WT+MI model: Kawasaki Disease-Related Vasculitis Model WT mice group; Park7^−/−+MI model, Kawasaki Disease-Related Vasculitis Model Park7^−/− mice group. ##p < .01 compared with Kawasaki Disease-Related Vasculitis Model mice group or Kawasaki Disease-Related Vasculitis Model WT mice group.

p47phox participated in the function of Park7 on oxidative stress in H₂O₂-treated H9c2 cells

We further explore the role of p47phox in the function of Park7 in H₂O₂-treated H9c2 cells. It was found that transfection of p47phox plasmid obviously increased p47phox protein expression (Figure 7(a), (c); p < .05), suppressed NOX4 protein expression (Figure 7(b), (c); p < 0.05), reduced MDA levels (Figure 7(d)) and ROS production (Figure 7(e), (f)), and increased the levels of SOD (Figure 7(g)), GSH (Figure 7(h)) and GSH-PX (Figure 7(i)) in H₂O₂- treated H9c2 cells following down-regulation of Park7 expression (all p < .05). Down-regulation of p47phox suppressed p47phox protein expression (Figure 8(a), (c); p < .05), induced NOX4 protein expression (Figure 8(b), (c); p < .05), increased MDA levels (Figure 8(d)) and ROS production (Figure 8(e), (f)), and inhibited the levels of SOD (Figure 8(g)), GSH (Figure 8(h)) and GSH-PX (Figure 8(i)) in H₂O₂-treated H9c2 cells following up-regulation of Park7 expression (Figure 8; all p < .05).

Figure 7.

p47phox participated in the function of Park7 on ROS production in myocardial infarction. p47phox and NOX4 protein expression (a, b and c), MDA (d), ROS production (e and f), SOD, GSH and GSH-PX levels (g, h and i). Negative: negative mimic group; si-Park7: down-regulation of Park7 group; si-Park7+ p47phox: down-regulation of Park7 and over-expression of p47phox group. ##p < .01 compared with negative mimic group; ###p < .01 compared with down-regulation of Park7 group.

Figure 8.

The inhibition of p47phox participated in the function of Park7 on ROS production in myocardial infarction. p47phox and NOX4 protein expression (a, b and c), MDA (d), ROS production (e and f), SOD, GSH and GSH-PX levels (g, h and i). Negative: negative mimic group; Park7: over-expression of Park7 group; Park7+ si-p47phox: over-expression of Park7 and down-regulation of p47phox group. ##p < .01 compared with negative mimic group; ###p < .01 compared with over-expression of Park7 group.

NADPH activation was an important target for the function of Park7 in H₂O₂-treated H9c2 cells

Lastly, NOX4 plasmid was transfected in H₂O₂-treated H9c2 cells. It was suggested that NOX4 over-expression induced NOX4 protein expression (Figure 9(a), (b)), increased MDA levels (Figure 9(c)), and ROS production (Figure 9(g), (h)), and reduced the levels of GSH-PX (Figure 9(d)), GSH (Figure 9(e)) and SOD (Figure 9(f)) in H₂O₂-treated H9c2 cells following up-regulation of Park7 (all p < .05). Si-NOX4 plasmid suppressed NOX4 protein expression (Figure 9(i), (j)), inhibited MDA levels (Figure 9(k)) and ROS production (Figure 9(m), (l)), and increased the levels of GSH-PX (Figure 9(n)), GSH (Figure 9(o)), SOD (Figure 9(p)) in H₂O₂-treated H9c2 cells following down-regulation of Park7 expression (all p < .05).

Figure 9.

NADPH activation was important targets for the function of Park7 in myocardial infarction. NOX4 protein expression (a and b), MDA, SOD, GSH and GSH-PX levels (c, d, e and f), ROS production (g and h) in vitro model of myocardial infarction following up-regulation of Park7 expression; NOX4 protein expression (i and j), MDA (k), ROS production (l and m), SOD, GSH and GSH-PX levels (n, o and p), in vitro model of myocardial infarction following down-regulation of Park7 expression. Negative: negative mimic group; si-Park7: down-regulation of Park7 group; si-Park7+ NOX4: down-regulation of Park7 and over-expression of NOX4 group; Park7: over-expression of Park7 group; Park7+ NOX4: over-expression of Park7 and down-regulation of NOX4 group. ##p < .01 compared with negative mimic group; ###p < .01 compared with over-expression of Park7 group or down-regulation of Park7 group.

Discussion

AMI is a common and severe type of coronary heart disease and is mainly caused by thrombotic obstruction due to the rupture of coronary atherosclerotic plaque. ¹⁶ The main clinical manifestations include persistent severe pain behind the sternum, acute circulatory dysfunction, arrhythmia, abnormal myocardial enzyme spectrum and corresponding changes in electrocardiogram.^16,17

Ischemia can lead to tissue damage and even cell death, and reperfusion after ischemia can cause new damage, namely the so-called reperfusion injury. Myocardial ischemia-reperfusion injury experiences two unique and related pathological processes: endothelial triggering and neutrophil amplification. ¹⁸ The main pathophysiological change in the early stage of reperfusion is the dysfunction of endothelial cells, which is characterized by a significant decrease in endothelial nitric oxide release. This change of endothelial function can lead to a series of pathophysiological changes and start the pathological process of reperfusion injury, which is called “endothelial trigger mechanism”. This is followed by the second major pathophysiological change during reperfusion, namely “neutrophil amplification”. This effect is the product of leukocyte endothelial interaction. It is characterized by neutrophils (PMNs) adhering to endothelial cells through cell adhesion molecules and migrating across the endothelium. In this process, PMNs release mediators, which diffuse to myocardial tissue through endothelial cells, causing myocardial cell damage.

In this study, we demonstrate that the mRNA and protein expressions of Park7 were also reduced in myocardial infarction of mice model. Park7 protect against ROS production of myocardial infarction in mice model. Dongworth et al. proposed that DJ-1 protects against cell apoptosis and represent a novel therapeutic target for cardio protection, ¹⁹ which in turn result showed that Park7 protect myocardial infarction by the inhibition of ROS-induced oxidative stress.

Numerous studies have shown that AMI can cause necrosis and dissolution in a large number of cardiomyocytes as well as infiltration of inflammatory cells, leading to declined heart function. ²⁰ AMI leads to abnormal cellular internal environment caused by insufficient energy supply, and the compensatory increase in cardiac contraction can lead to increased level of ROS caused by NADPH in the cell membrane. ²¹ More seriously, the increases levels of ROS trigger mitochondria to produce a large amount of ROS. Oxidative stress can not only attack cell membrane and organelles, but also lead to the occurrence of inflammatory response through the mutual enhancement of inflammatory factors, further aggravating myocardial damage caused by myocardial infarction. ²² Therefore, increasing the level of antioxidant enzymes and decreasing the content of ROS are generally considered as one of the important therapeutic ideas for AMI.^23,24 In fact, we demonstrated that NADPH activation was important targets for the function of Park7 in myocardial infarction. Liu et al. shows that Park7 has a protective role against sepsis by NADPH oxidase activation. ²⁵ Therefore, it is believed that Park7 could be essential regulators of NADPH oxidase activation in myocardial infarction.

p47phox is a subunit of NADPH. After external stimuli, the cytoplasmic subunit p47phox is phosphorylated and activated to catalyze the production of ROS and to promote the occurrence of oxidative stress.^26,27 Therefore, NOX is an important biosynthetic enzyme that promotes the synthesis of ROS, and p47phox subunit can regulate its activity.^28–30 Therefore, these results of this study indicate that Park7 regulated p47phox expression to direct NADPH activation was important signal in the function of Park7 in myocardial infarction in vivo or vitro model. Liu et al. have found that Park7 has a protective role against sepsis by controlling macrophage activation, NADPH oxidase activation and inflammation responses. ³¹ Their results are similar to ours in the role of Park7 combined with p47phox in ROS production and inflammatory response. However, the differences are that the model is different and the methods are different. Cheng et al. also have reported that Park seven is a novel therapeutic target in sepsis-induced immunosuppression by 47phox, ³² suggesting that the enhanced interaction between Park7 and p47phox may be due to suppress NADPH activation in myocardial infarction.

However, there are also some limitations in this study. For example, we have not proved whether other inflammatory markers or other pathways related to park seven are involved in the formation of MI. In the future study, we will further assess the mechanism of MI.

Conclusion

In conclusion, our results showed that Park7 has a protective effect on oxidative stress in myocardial infarction. We demonstrated that Park7 regulated p47phox expression directly through NADPH activation, which decreased ROS-induced oxidative stress injury in myocardial infarction. These promising results suggest that Park7 may be an emerging potential target in the treatment of myocardial infarction.