Protective Effect of Combined Metoprolol and Atractylenolide I in Rats with Acute Myocardial Infarction via Modulation of the SIRT3/β-CATENIN/PPAR-γ Signaling Pathway

ABSTRACT Herein, we examined the protective effect of metoprolol combined with atractylenolide I (Atr I) in acute myocardial infarction (AMI) by regulating the SIRT3 (silent information regulator 3)/β-catenin/peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling pathway. Briefly, 50 rats were randomly divided into the sham operation, model, metoprolol, Atr I, and combination metoprolol with Atr I groups (combined treatment group). The AMI model was established by ligating the left anterior descending coronary artery. After treatment, infarct size, histopathological changes, and cell apoptosis were examined using 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, and the TUNEL assay. The left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), and left ventricular mass index (LVMI) were detected by echocardiography. Endothelin-1 (ET-1), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) levels were detected using enzyme-linked immunosorbent assays. Furthermore, we measured lactate dehydrogenase (LDH), creatine kinase (CK) isoenzyme (CK-MB), and CK levels. Western blotting was performed to determine the expression of SIRT3, β-catenin, and PPAR-γ. Herein, the combined treatment group exhibited increased levels of LVEF, LVFS, and NO, whereas LVMI, ET-1, TNF-α, IL-6, LDH, CK-MB, and CK levels were decreased. Importantly, the underlying mechanism may afford protection against AMI by increasing the expression levels of SIRT3, β-catenin, and PPAR-γ.


INTRODUCTION
Acute myocardial infarction (AMI), a type of myocardial necrosis, is caused by acute and persistent ischemia and hypoxia of the coronary arteries (Chen et al., 2016;Sheu et al., 2019).Most clinical manifestations are severe, presenting as persistent retrosternal pain accompanied by elevated serum myocardial enzyme activity and potentially complicated by arrhythmia, shock, or heart failure (Posma et al., 2019).AMI is a widespread disease that impairs the safety of human life (Zhao et al., 2020).Importantly, the incidence of AMI in China has shown a notable upward trend.

Protective Effect of Combined Metoprolol and Atractylenolide I in Rats with Acute Myocardial Infarction via Modulation of the SIRT3 /β-CATENIN/PPAR-γ Signaling Pathway
Page 2/13 Braz.J. Pharm.Sci.2023;59: e21639 Weijian Zhou, Jing Liu, Zhongli Sun, Yongpeng Dong, Meiming Zhu, Li Li Currently, it is estimated that approximately 2 million individuals suffer from AMI in China, with at least half a million new patients diagnosed annually (Wu et al., 2021).AMI is a pathophysiological process involving myocardial tissue structure, ventricular size, morphology, and functional changes (Yang et al., 2021).It is the main pathophysiological mechanism underlying progressive changes in cardiac function post-myocardial infarction and is mainly related to the overexpression of inflammatory cytokines, neuroendocrine regulation, and excessive activation of oxidative stress (Meng et al., 2021).Currently, the principal drugs employed to treat AMI include rosuvastatin, enteric-coated aspirin tablets, and dopamine (Fei et al., 2016;Sun et al., 2020;Zhou et al., 2021).However, these therapeutic agents are known to be associated with serious side effects, thereby reducing their clinical efficacy.
Considering the changes observed in the SIRT3/ β-catenin/PPAR-γ signaling pathway during the development of AMI, we examined the role and mechanism of metoprolol and Atr I in myocardial infarction in the present study using AMI model rats to establish an experimental basis for rational and efficient clinical drug utilization.
Male Sprague Dawley rats (6-7 week-old, 200 ± 20 g) were supplied by the Laboratory Animal Center.The rats were reared adaptively for one week prior to experimentation, with free access to food and water during the experiment.All applicable international, national, and institutional guidelines for the care and use of animals were followed.This study was approved by the Ethics Committee of the Zibo Central Hospital.

Modeling and administration methods
The AMI model was established by ligating the left anterior descending coronary artery.Rats were intraperitoneally administered anesthesia using 3 mL/ kg chloral hydrate.Disinfection was performed using iodophor.The skin of the neck was incised, and the subcutaneous tissue and muscles were separated.The fourth rib was cut, the chest was opened, the pericardium was cut, and the heart was exposed.The anterior descending coronary artery was ligated at approximately 2 mm from the lower edge of the left atrial appendage.After 20 min, the left ventricular anterior wall was pale, and contractility was weakened.Considering the sham operation group, the thread was placed without ligation.Post-surgery, penicillin (40,000 U) was administered intramuscularly for three days to prevent wound infection.
Fifty rats were randomly separated into five groups: the sham operation group, AMI group, metoprolol group, Atr I group, and combined treatment group.Metoprolol, Atr I, and combined treatment group rats were orally administered metoprolol (8 mg/kg), Atr I (4 mg/kg), and metoprolol (8 mg/kg) + Atr I (4 mg/kg).The sham and AMI rats were administered physiological saline continuously for 28 days.

2,3,5-Triphenyltetrazolium chloride (TTC) staining determined myocardial infarct size
After euthanizing the rats, the heart was harvested quickly, and the aorta was intubated.The heart was then flushed with phosphate-buffered saline (PBS), and TTC was injected into the aorta, followed by incubation at 37°C for 30 min.The necrotic myocardium was white, and the non-necrotic myocardium was red.Prepared specimens were stored at -80℃ for 10 min, sliced (1.0 mm thick), and fixed overnight with 10% formaldehyde solution.ImageJ software (National Institutes of Health, Bethesda, MD) was used to calculate the left ventricular myocardial necrosis area and total left ventricular area.Percentage of myocardial infarction area = left ventricular myocardial necrosis area/total left ventricular area × 100%.

Echocardiographic examination
Af ter t reat ment, rats were anesthetized intraperitoneally with 1% pentobarbital sodium (40 mg/ kg) and fixed on their backs.High-resolution ultrasound (Shanghai Yuyan Scientific Instrument Co., Ltd) was used to detect left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) to further evaluate cardiac function.Next, the right ventricular wall and atrium were excised, and the left ventricle and ventricular septum were preserved.The left ventricular mass index (LVMI) was calculated by precise weighing with a balance.

Hematoxylin and eosin (HE) staining
Following ultrasound examination, the rats were euthanized.The heart was harvested by thoracotomy.The left heart tissue was cut into 5-mm thin sections and fixed with paraformaldehyde for 10 min.Slices were decolorized with ethanol, dehydrated with xylene, and sealed with neutral glue.Histopathological changes were observed under a microscope.

TUNEL assay
After the final administration, the rats were sacrificed, and their hearts were dissected to separate the myocardial tissue at the infarct site.The left heart tissue was fixed with 4% paraformaldehyde and then embedded in paraffin.The specimens were dehydrated and embedded in 30% sucrose.After TUNEL staining, fluorescence microscopy was used to observe and obtain images.

Determination of myocardial enzyme content
After the final treatment, heart tissue was harvested and ground in PBS.The supernatant was centrifuged at 12000 rpm for 10 min and stored at -80°C.The respective kit was used to detect LDH, CK, and CK-MB levels.

Enzyme-linked immunosorbent assay (ELISA) detection
Twenty-four hours after the last administration, blood was collected from the retroorbital venous plexus of rats.After standing for 30 min, the whole blood was centrifuged at 12000 rpm for 10 min.After absorbing the supernatant, the levels of ET-1, NO, TNF-α, and IL-6 were measured using ELISA kits.

Western blot detection
Briefly, myocardial tissue was harvested from the infarct site.Non-myocardial tissues, including blood vessels and fat, were removed to isolate the left ventricle.Total protein was extracted using RIPA lysate and quantified.The total protein (50 μg) from each protein sample was used Weijian Zhou, Jing Liu, Zhongli Sun, Yongpeng Dong, Meiming Zhu, Li Li for western blot analysis.Protein samples were added to a polyacrylamide gel for electrophoresis and film transfer.Subsequently, the nitrocellulose membrane was placed in 5% skim milk, sealed at room temperature for 2 h, and thrice washed with TBST (Tris-buffered saline, 0.1% Tween 20).SIRT3 (1:100), anti-β-catenin (1:200), and PPAR γ (1:1000) antibodies were incubated overnight at 4°C.Next, the corresponding horseradish peroxidase-labeled secondary antibody (1:1000) was diluted and incubated at room temperature for 1 h.The specimens were developed in a darkroom using ECL reagent, and the image was scanned using a Bio-Rad gel electrophoresis image analysis system.The relative intensities of protein bands were analyzed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
Data were statistically analyzed using SPSS software (version 20.0;IBM Corp., Armonk, NY, USA).Data are presented as x ± s. One-way analysis of variance (ANOVA) was used for multi-group comparisons.A p-value of <0.05 was deemed a significant difference.

Assessment of cardiac function
Herein, cardiac function was examined using echocardiography.The model group exhibited markedly reduced LVEF and LVFS, whereas LVMI was substantially increased, thereby indicating the occurrence of left ventricular remodeling to a certain extent after AMI (Figure 1, p <0.01).LVEF and LVFS were dramatically enhanced, and LVMI was notably reduced in the metoprolol, Atr I, and combined treatment groups (p <0.05, p <0.01).Moreover, the impact of combined treatment was superior to that of metoprolol or Atr I administration (p <0.05).The effect of combined treatment was slightly better than that of Atr I. Considering LVEF and LVFS levels, there was no significant difference between the combined treatment and Atr I groups (Figure 1A-1 B).

Detection of areas of myocardial infarction
The sham-operated group displayed no myocardial infarction.The infarct size was substantially reduced in the Atr I and combined treatment groups (Table I, p <0.05 or p <0.01).No significant difference was observed between the metoprolol and AMI groups.The therapeutic effect of combined treatment was similar to that of Atr I.In the TTC experiment, the non-infarcted areas of the myocardium were notably brick-red, whereas infarcted areas appeared predominantly yellow-white.The combined treatment group exhibited better alleviation of myocardial infarction than the metoprolol or Atr I groups (Figure 2).The effects of Atr I were superior to those of metoprolol.Most importantly, the combined treatment group exhibited the smallest infarct area, which was restored to the level of the sham-operated group.

Detection of cell apoptosis using TUNEL
Next, we examined cardiomyocyte apoptosis using TUNEL staining.Black arrows indicate apoptotic cells, and yellow arrows indicate apoptotic cells.Based on the TUNEL analysis, the apoptosis rate of cells surrounding the myocardial infarct was elevated in the model group after AMI.Interestingly, metoprolol, Atr I, and combined treatment could reduce the apoptosis rate of cells surrounding the myocardial infarct (Figure 4).Moreover, the effect of the combined treatment was superior to that of metoprolol and Atr I alone.In addition, there were no significant differences in cardiomyocyte apoptosis between the metoprolol and AMI groups.

Histopathological changes in myocardial tissues
In the HE staining experiment, black arrows represent cardiomyocytes, and red arrows indicate vacuoles.In the sham-operated group, myocardial cells were closely arranged without notable inflammatory cell infiltration.In the model group, the arrangement of myocardial cells was disordered, accompanied by inflammatory cell infiltration.The metoprolol, Atr I, and combined treatment could relieve the degree of myocardial necrosis (Figure 3).The combined treatment exhibited the strongest effect against cardiomyocyte injury.The effect of Atr I was slightly better than that of metoprolol.No prominent differences were detected between the metoprolol and AMI groups.

Serum levels of inflammatory cytokines
The model group presented substantially elevated serum levels of ET-1, TNF-α, and IL-6, along with a marked reduction in NO levels (Figure 5, p <0.01).Herein, metoprolol, Atr I, and combined treatment could suppress ET-1, TNF-α and IL-6 levels and enhance NO levels (Figure 5, p <0.01).There was no significant difference between the combined treatment group and Atr I groups considering ET-1 levels (Figure 5A).Notably, the combined treatment afforded the strongest effect.

Western blotting analysis
We performed western blotting to measure expression levels of SIRT3, β-catenin, and PPAR-γ in rats.As shown in Figure 7A and 7B, expression levels of SIRT3, β-catenin, and PPAR-γ were decreased in the model group (p < 0.01).Compared with the model group, the combined treatment group exhibited markedly increased expression levels of SIRT3, β-catenin, and PPAR γ, exceeding those of metoprolol or Atr I groups (p < 0.01 or p < 0.05).The effect of Atr I was better than that of metoprolol considering SIRT3 expression, while metoprolol afforded a better effect than Atr I considering β-catenin expression.

Effect of myocardial tissue on myocardial enzymes
As shown in Figure 6, levels of LDH, CK, and CK-MB were significantly elevated in the AMI group (p <0.01).Metoprolol, Atr I, and combined treatment substantially ameliorated LDH, CK, and CK-MB levels (p <0.05, p <0.01).We detected almost no difference in the CK content between the combined treatment and Atr I groups (Figure 6B).The CK-MB content exhibited an identical trend (Figure 6C).Following combined treatment, the levels of these factors were similar to those in the sham-operated group.

DISCUSSION
AMI is a common cardiovascular emergency (Sun et al., 2019;Sun et al., 2019;Zhao et al., 2019) characterized by myocardial necrosis, attributed to acute and continuous ischemia and hypoxia of the coronary arteries (Abel, Clark, 2021;Ishikura et al., 2021).Typically, this clinical syndrome occurs secondary to thrombosis due to unstable plaque rupture, erosion, and endothelial injury of the coronary artery, resulting in myocardial ischemia, injury, and necrosis (Bosco et al., 2021;Gao et al., 2021;Lin, Zheng, Jiang, 2021;Metwalli et al., 2021).Although AMI is most common in Western countries, its incidence in China is increasing annually (Tai et al., 2018;Lin, Feng, Xu, 2019).The World Health Organization predicted that AMI would be the world's largest cause of death by 2021 (Gheini, Pooria, Pourya, 2020).β-blockers are commonly used to treat AMI (Liu et al., 2019;Chen et al., 2020) and are known to antagonize the competitive binding of catecholamines and beta-adrenoceptors, thereby blocking excitatory synaptic transmission (Pesaro et al., 2010;DiNicolantonio et al., 2015).In the present study, we revealed that combined treatment with metoprolol and Atr I could afford cardioprotection in rats by activating the SIRT3/β-catenin/PPAR-γ signaling pathway, exhibiting superior effects to metoprolol and Atr I monotherapy.
Clinically, ultrasonic electrocardiography is used to examine the degree of myocardial infarction (Wang et Weijian Zhou, Jing Liu, Zhongli Sun, Yongpeng Dong, Meiming Zhu, Li Li al., 2020).LVEF, LVFS, and LVMI can reflect cardiac function and adjudge abnormal changes in cardiac systolic function (Ma et al., 2017;Rong et al., 2018;Hou et al., 2020).Herein, we observed that combined treatment could ameliorate cardiac function by enhancing LVEF and LVFS and reducing LVMI.During myocardial infarction, coronary blood flow is disrupted, accompanied by reduced myocardial contractility (Veltman et al., 2021).It has been reported that myocardial contractility is closely related to the size of myocardial infarction (Zhao et al., 2019).In the present study, the combined treatment showed better abatement of myocardial infarction than treatment with metoprolol or Atr I alone.Importantly, the infarct area in the combined treatment group was restored to normal.Furthermore, our results revealed that the combined treatment could reduce cardiomyocyte apoptosis.
Vascular endothelial dysfunction affects fibroblast proliferation and promotes the development of myocardial infarction by secreting vasoactive substances, such as ET-1, TNF-α, IL-6, and NO (Wang et al., 2017;Li, Yan, 2018); these factors are deemed crucial myocardial inflammatory markers (Somasuntharam et al., 2016).Following myocardial infarction, these inflammatory factors indirectly reflect the inflammatory status of myocardial cells.Herein, combined treatment with metoprolol and Atr I could dramatically reduce the release of ET-1, TNF-α, and IL-6, while enhancing NO levels, thereby alleviating the AMI-induced inflammatory reaction.
Cellular active enzymes, such as LDH, CK, and CK-MB, have been employed as indicators to determine the degree of cardiomyocyte injury (Qi et al., 2020).An increase in these factors is known to be directly proportional to cardiomyocyte injury (Liu et al., 2020).Combined treatment with metoprolol and Atr I markedly reduced LDH, CK, and CK-MB levels.These results suggest that combined treatment could alleviate the degree of myocardial injury in AMI rats by suppressing the activity of myocardial enzymes.
It is well-established that myocardial tissue consists of parenchymal and stromal cells, and stromal cells are primarily cardiac fibroblasts (Zhou et al., 2017).SIRT3, a deacetylase, is known to regulate energy metabolism, biosynthesis, and apoptosis (Paget et al., 2011).In addition, SIRT3 has been closely associated with cardiovascular diseases such as myocardial hypertrophy and myocardial infarction (Wei et al., 2019;Maghbooli et al., 2020).Reportedly, PPAR-γ can ameliorate heart failure and participates in ventricular remodeling (Xu et al., 2017).As a key factor in the Wnt signaling pathway, β-catenin promotes the transformation of cardiac fibroblasts and leads to myocardial fibrosis (Albrecht-Schgoer et al., 2012).Furthermore, both β-catenin and PPAR-γ can regulate each other and can be directly related to PPAR-γ (Yin et al., 2011).In the present study, combined treatment with metoprolol and Atr I markedly enhanced the expression of SIRT3, and SIRT3 could activate PPAR-γ, thus protecting cardiomyocytes.This was mainly reflected in two aspects: combined treatment not only alleviated myocardial histopathological changes, but also suppressed cardiomyocyte apoptosis.
In summary, combined treatment with metoprolol and Atr I could inhibit the inflammatory response in rats with AMI and suppress cardiomyocyte apoptosis, thereby exerting a cardioprotective role in AMI.The underlying mechanism may be related to the activated SIRT3/β-catenin/PPAR-γ signaling pathway in AMI, which will afford a new research direction for preventing and treating myocardial infarction.

FIGURE 2 -
FIGURE 2 -TTC staining of myocardial tissue.(A) Sham-operated group, (B) AMI group, (C) metoprolol group, (D) Atr I group, and (E) Combined treatment group.Black arrows indicate the infarct area, and red arrows indicate the non-infarction area.AMI, acute myocardial infarction; Atr I, atractylenolide I.
Protective Effect of Combined Metoprolol and Atractylenolide I in Rats with Acute Myocardial Infarction via Modulation of the SIRT3 /β-CATENIN/ PPAR-γ Signaling Pathway

TABLE I -
Percentage of myocardial infarction ** p < 0.01 or # p < 0.05, ## p < 0.01 contrast to Sham operation group or AMI group, a p < 0.05 contrast to metoprolol.