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

Hindawi BioMed Research International Volume 2021, Article ID 9931885, 7 pages https://doi.org/10.1155/2021/9931885

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

Zhenjun Wu ,1 Xuewen Li,2 Xiuyue Li,3 and Lihua Yu4

1Cardiology Department, Tianjin Huaxing Hospital, Tianjin 300270, China 2Cadre’s Ward, Characteristic Medical Center of Chinese People’s Armed Police Force 300, Tianjin 300171, China 3Cardiology Department, Xianshuigu Hospital of Jinnan District, Tianjin 300350, China 4Beijing Chaoyang Integrative Medicine Emergency Medical Center, Beijing 100022, China

Correspondence should be addressed to Zhenjun Wu; [email protected]

Received 26 March 2021; Accepted 29 June 2021; Published 9 July 2021

Academic Editor: Junyan Liu

Copyright © 2021 Zhenjun Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

1. Introduction effects. [3] This condition is known as myocardial ischemia-reperfusion (MI/R) injury that refers to the deteri- Ischemic heart disease (IHD), the most common cardiovas- oration of tissue damage after reperfusion of the ischemic cular disease in human, is one of the leading causes of death heart and is a major event hindering the treatment of cardio- in the world [1].) The most effective method to reduce the vascular diseases at present [4]. infarct size and maintain cardiac function after acute myo- The Janus kinase/signal transducer and activator of tran- cardial infarction is myocardial reperfusion [2] that requires scription (JAK/STAT) signal transduction pathway is an blood reperfusion of ischemic myocardium to restore the important cellular signal transduction pathway transducing oxygen supply to the myocardium and improve the biological signals from the plasma membrane to nuclear cells and play- function of ischemic myocardium. However, additional cell ing a key role in regulating cardiac protection against I/R death and severer damage to the myocardium may be caused injury [5, 6]. Besides, the JAK2/STAT3 signaling pathway by myocardial reperfusion, thus weakening its beneficial has been proven to be able to prevent I/R injury [7]. In 2 BioMed Research International addition, the JAK/STAT signaling pathway participates in Table 1: Survival rate of rats at 7 d after MI. the growth, development, proliferation, apoptosis, and n necrosis of cells [8]. The JAK/STAT signal transduction Group Survival rate pathway activates many cytokines under the control of the Sham group 10 100% following drugs after MI/R injury. In particular, Model group 10 70%# ∗ phosphorylation-activated JAK2 and STAT3 mediate the Low-dose group 10 90% apoptosis of myocardial cells, which is regarded as an impor- ∗ 90% tant pathological mechanism of apoptosis after MI/R injury High-dose group 10 ∗ [9, 10]. Note: p <0:05 vs. the model group, #p <0:05 vs. the sham group. Aspirin, an indispensable drug in the secondary prevention of cardiovascular disease and also the most popular drug hours of reperfusion, including ejection fraction (EF%), frac- in modern medicine, induces platelet dysfunction by irre- tional shortening (FS%), left ventricular end-systolic pressure versibly repressing the cyclooxygenase-1 enzyme in platelets (LVESP%), and left ventricular end-diastolic pressure [11], which has been successfully applied to prevent cardio- (LVEDP%). Finally, the rats were euthanized, and then, vascular complications in high-risk patients. In addition, blood samples were taken for biochemical examination, aspirin can also be used to treat patients undergoing coro- myocardial tissues were collected, and the left ventricle was nary artery bypass surgery (CABG). During the perioperative used for subsequent experiments. period, the administration of aspirin intravenously decreases graft failure [12, 13]. Besides, it is confirmed that aspirin 2.2.3. Echocardiography. After MI/R, the rats were anesthe- fl reduces perioperative cardiovascular complications to reduce tized with iso urane and then subjected to M-mode echocar- morbidity and mortality rates [14]. diography in an echocardiography imaging system equipped In this study, rat models of MI/R were established, before with a 15 MHz linear transducer, with cardiac function which aspirin was given to the rats, to evaluate the effect and parameters measured digitally on M-mode traces. Left ven- underlying molecular mechanism of aspirin on MI/R injury. tricular end-diastolic dimension and left ventricular end- The understanding of such a mechanism provided important systolic dimension were recorded to calculate EF and FS. information for the application of aspirin in the treatment of 2.2.4. Tetrazolium Chloride (TTC) Staining. The heart was MI/R injury. cut into five pieces on average and placed in a culture dish. ° The sections were incubated in an incubator at 37 C for 10- 2. Materials and Methods 15 min and stained with TTC. Then, Image-Pro Plus 6.0 soft- 2.1. Materials. A total of 60 clean-grade 1-month-old rats ware was employed to assess infarct size. were selected, fed in an air-conditioned room with a constant 25 ± 1° 2.2.5. TUNEL Staining. After the rats were euthanized by temperature of C and a 12 : 12 h light/dark with water intraperitoneal injection of sodium pentobarbital, their chest and a free standard diet for one week, randomly grouped, and cavity was opened, and 10 mL of phosphate-buffered saline weighed. (PBS) was retrogradely injected via the aorta, followed by 2.2. Methods reperfusion with 4% paraformaldehyde. Then, their heart was taken out, fixed in 4% paraformaldehyde for 24 h, con- 2.2.1. Major Reagents and Instruments. Primary anti-B-cell ventionally dehydrated, embedded in paraffin, cut into 10 ° lymphoma 2 (Bcl-2) antibodies, Bcl-2-associated X protein consecutive sections (3 μm in thickness), and baked at 50 C (Bax) antibodies, anti-phosphorylated JAK2 (p-JAK2) anti- for 1 h. Thereafter, the sections were added with immuno- bodies, and anti-p-STAT3 antibody (Abcam), an immuno- staining permeabilization buffer (0.1% Triton X-100 in histochemistry kit, a terminal deoxynucleotidyl transferase- 0.1% sodium citrate solution), subjected to ice bath for 2 (TdT-) mediated dUTP nick end labeling (TUNEL) apopto- min of incubation, washed with PBS, and incubated with ° sis kit, an AceQ quantitative polymerase chain reaction 50 μL of TUNEL solution in a wet box at 37 C for 60 min (qPCR) SYBR Green Master Mix kit, a light microscope, and then 50 μLof4′,6-dimidyl-2-phenylindole (DAPI) stain- ° and a fluorescenceqPCR instrument were used. ing reagent in the dark at 37 C for 5 min. After that, a fluorescence microscope was used to photograph and count 2.2.2. Modeling. The animals were randomly divided into apoptotic cells. three groups, including the (1) sham, (2) MI/R group, and (3) MI/R+aspirin group (aspirin group). We ensure that 2.2.6. Real-Time (RT) qPCR. The messenger ribonucleic acid there are 6 valid experimental data for each group. We estab- (mRNA) expression levels were measured via RT-qPCR lished a rat model of I/R injury by ligating for 45 min and using the 2× Power Taq PCR Master Mix (BioTeke, Beijing, subsequently releasing the mouse left anterior descending China) and SYBR Green (BioTeke, Beijing, China) and an (LAD) coronary artery. Among them, the LAD coronary ExicyclerTM 96 fluorescence quantitative analyzer. Total arteries were not ligated in sham-operated rats and ligated RNAs were extracted from myocardial tissues using TRIzol in aspirin-treated rats, resulting in 30 min ischemia followed (Invitrogen) and reversely transcribed and synthesized into by release of the silk suture and tail vein injection of 60 mg/kg complementary deoxyribonucleic acids (cDNAs). Then, the aspirin 10 min before reperfusion. The ultrasound system concentration and purity of RNAs were detected using was used to collect hemodynamic parameters after three Thermo NanoDrop 2000. The synthesis of cDNAs was BioMed Research International 3

100 60 0 ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ 80 40 ## 60 ##

LVEF % LVEF 40 % LVEF 20 20 0 0 Sham Model Low-dose High-dose Sham Model Low-dose High-dose group group group group group group group group (a) (b) 10 10 ⁎ ## ⁎ 8 8 ⁎⁎ ⁎⁎ 6 6 ## 4 4 LVIDs (mm) LVIDs (mm) LVIDs 2 2

0 0 Sham Model Low-dose High-dose Sham Model Low-dose High-dose group group group group group group group group (c) (d)

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.

50 Sham ## group 40

30 ⁎⁎ ⁎⁎ Model 20 group

Infarct volume (%) volume Infarct 10

0 Low-dose group Sham group Model group Model High-dose Low-dose group Low-dose group group High-dose

(a) (b)

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. conducted using 1 μg of total RNAs, reverse transcriptase PCR instrument. The recommended reaction conditions ° (Fermentas), and Oligo-dT primers. In PCR amplification, are as follows: denaturation at 94 C for 20 s, annealing at ° ° a50μL of PCR system was utilized, which contained a 55 C for 30 s, extension at 72 C for 30 s, 30 cycles, and ° reaction buffer, Taq DNA polymerase, dNTPs, 1 μL of for- extension at 72 C for 30 s. PCR was performed using an ward primer, 1 μL of reverse primer, and 3 μL of template 7900 QPCR system (Applied Biosystems), with the follow- ° ° cDNAs (resulting products of reverse transcription reac- ing cycle parameters: 95 C for 5 min, 1 cycle, and 95 C for ° tion). The samples were loaded, shaken for blending, and 10 s and 60 C for 20 s, 40 cycles. The sequences are shown transiently centrifuged, followed by amplification using a in Table 1. 4 BioMed Research International

Sham group Model group Low-dose group High-dose group 300 ## )

2 200 ⁎⁎ ⁎⁎

(/mm 100

50μm cell TUNEL-positive 0 Sham group Model group Model Low-dose group Low-dose High-dose group High-dose (a) (b)

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.

Sham group Model group Low-dose group High-dose group 100 ## 80 60 ⁎⁎ ⁎⁎ 40 20 0 Intracellular ROS (%) ROS Intracellular 50μm Sham group Model group Model Low-dose group Low-dose High-dose group High-dose (a) (b)

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.

2.2.7. Western Blotting. The myocardial tissues were crushed, ences among groups; p <0:05 suggested that the difference lysed in RIPA lysis buffer containing 1 mM phenylmethane- was significant. sulfonyl fluoride on ice, ground in a tissue homogenizer at ° 4 C, and placed on ice for 30 min. Next, the lysis solution 3. Results was centrifuged at 12,000 rcf for 10 min to obtain proteins. Then, the proteins were quantified using the BCA protein 3.1. Results of Echocardiography. Compared with those in the assay kit based on standard protein concentration curves, pre- MI/R group, the EF and FS were overtly increased, while the pared and separated by sodium lauryl sulfate-polyacrylamide left ventricular internal diameter (LVID)was evidently gel electrophoresis. Thereafter, the separated bands were decreased in the MI/R+aspirin group (Figure 1), suggesting transferred to a polyvinylidene fluoride membrane (Millipore, that aspirin can significantly improve the EF, FS, and LVID Billerica, MA, USA) and incubated with p-JAK2 (1 : 1000), p- at end-systole (LVIDs) of the MI/R heart. STAT3 (1 : 1000), and GAPDH (1 : 25,000). After that, the membrane was washed with Tris-buffered saline Tween (10 3.2. Myocardial Infarction Size. The myocardial infarction μ μ size was greater in the MI/R group and MI/R+aspirin group mM Tris-base, 100 mM sodium chloride, and 0.1% fi Tween-20, pH = 7:50) and incubated with an appropriate sec- than that in the sham operation group, con rmed by TTC ° staining results (p <0:01), and it was significantly reduced ondary antibody at room temperature (20-25 C) for 30 min. p <0:01 Lastly, Image Lab software system (Bio-Rad, Hercules, CA, after treatment with aspirin ( ) (Figure 2). USA) was utilized to detect protein bands. 3.3. Results of Immunohistochemical Staining. Based on Figure 3, the apoptosis rate was higher in the MI/R group 2.3. Statistical Analysis. All data were expressed as mean ± than that in the sham operation group, while it was remark- standard deviation (χ ± s) and analyzed by SPSS 11.0 soft- ably lower in the MI/R+aspirin group than that in the MI/R ware. One-way ANOVA was applied to compare the differ- group (p <0:05). BioMed Research International 5

Sham Model Low-dose High-dose group group group group

Caspase3

Bax

Bcl-2

GAPDH

(a) 2.5 1.5 ## 2.0 ⁎⁎ 1.0 1.5 ⁎⁎ ⁎⁎ ## ⁎⁎ 1.0 0.5 0.5

Protein ratio of Bcl-2/Bax of ratio Protein 0.0 0.0 Sham Model Low-doseHigh-dose Protein ratio of caspase/GAPDH of ratio Protein Sham Model Low-doseHigh-dose group group group group group group group group (b) (c)

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.

3.4. PCR and Western Blot Test Results. Compared with those 4. Discussion in the sham operation group, the mRNA expression of Bax was clearly raised, while that of Bcl-2 was distinctly lowered IHD has a high incidence rate and becomes the leading cause in the MI/R group (p <0:05). Compared with the MI/R of high mortality rate in developed countries, and its inci- group, the MI/R+aspirin group exhibited an overtly dence rate is high and shows an increasing trend in develop- decreased mRNA expression of Bax and a significantly ing countries [15]. Timely intervention can restore blood increased mRNA expression of Bcl-2, and the differences flow of coronary arteries, but reperfusion will induce myo- were statistically significant (p <0:05) (Figure 4, Bax and cardial injury [16], known as MI/R injury that is the main Bcl-2 expressions in myocardial tissue). Figure 4(a) shows cause of heart remodeling and subsequent heart failure the Bax mRNA expression in each group. Figure 4(b) shows (HF). MI/R-related deaths are on the rise worldwide [17]. the Bcl-2 mRNA expression in each group compared with Therefore, the effective prevention and treatment strategies the MI/R group; the mRNA expression of Bax in the aspirin alleviating or reversing MI/R-induced remodeling are of group was significantly decreased, whereas Bcl-2 was signifi- important clinical value for IHD patients. cantly increased, and the difference was statistically signifi- The JAK/STAT pathway is a stress response mechanism, cant, ∗p <0:05 compared with the sham operation group which regulates gene expressions by transmitting signals and #p <0:05 compared with the MI/R group. Figure 4(c) from the cell surface to the nucleus [18]. Recent studies have shows the protein expression of Bax and Bcl-2 in myocardial found that the JAK/STAT pathway can be rapidly activated tissue. through MI/R [19]. Considering the crucial regulatory role of the JAK/STAT signaling pathway in the apoptosis after 3.5. Results of Western Blotting. The expression levels of p- MI/R injury, in this study, the role of aspirin in mediating STAT3 and p-JAK2 were distinctly raised in the MI/R+as- MI/R in rats through the JAK/STAT signaling pathway was pirin group compared with those in the MI/R group investigated. (p <0:01) (Figure 5), implying that the JAK2/STAT3 sig- Aspirin is a typical nonsteroidal anti-inflammatory drug naling pathway is activated by pretreatment with aspirin (NSATD), which, in comparison with other NSATDs, has after MI/R. certain advantages like reducing the risk of occlusive 6 BioMed Research International cardiovascular events [20]. In the traditional concept, Ethical Approval NSATDs have analgesic, antipyretic, and anti-inflammatory effects, as well as typical side effects including gastric intoler- The study was approved by the ethics committee of Xian- ance and inhibition of blood clotting by repressing platelet shuigu Hospital of Jinnan District. activation [21, 22]. Moreover, aspirin has been verified to be very effective in the secondary prevention of cardiovascu- Consent lar diseases. However, its mechanism in MI/R injury is unclear. Consent is not necessary. In the present study, we first explored the protective effects of aspirin in a heart model of MI/R and found that Conflicts of Interest ff aspirin e ectively protected the heart against MI/R by acti- The authors declare that they have no competing interests. vating the JAK2/STAT3 signaling pathway. Aspirin pretreatment confers cardioprotection during reperfusion in the rat Authors’ Contributions heart. These cardioprotective effects of aspirin are mediated, at least in part, through activation of the JAK2/STAT3 signal- ZW wrote the manuscript. ZW and XWL helped with animal ing pathway. In this study, it was observed that the expres- modeling. XYL and LY were responsible for TTC staining sion of proapoptotic gene Bax was obviously upregulated, and TUNEL staining. All authors read and approved the final while antiapoptotic gene Bcl-2 was obviously decreased after manuscript. myocardial ischemia/reperfusion injury. This may eventually trigger apoptosis in myocardial tissue and increase the apo- References ptotic rate of cardiomyocytes. However, after aspirin intervention, the expression level of proapoptotic gene Bax was [1] A. S. Go, D. Mozaffarian, V. L. Roger et al., “Executive sum- obviously downregulated, while antiapoptotic gene Bcl-2 mary: heart disease and stroke statistics–2013 update: a report was obviously upregulated. 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