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staining results revealed that morphine overt-ly reversed decreased expressions of TLR4 and p65 induced by I/R in rats (p<0.05). Furthermore, Western blotting found that morphine signifi-cantly inhibited the protein expressions of TLR4 and phosphorylated p65.

CONCLUSIONS: Morphine clearly alleviates I/R-induced myocardial injury in rats. The possi-ble mechanism may be associated with the inhi-bition on TLR4/NF-κB signaling pathway.

Key Words:Morphine, Myocardial ischemia/reperfusion, TLR4.

Introduction

Myocardial infarction (MI) is a leading cause of death worldwide, which is also a major pub-lic health problem1. Currently, the most effective treatment for MI is early percutaneous coronary intervention (PCI)2,3. However, continuous reper-fusion after ischemia usually leads to secondary damage to the myocardium, namely myocardial ischemia/reperfusion (I/R) injury4. I/R injury is known as an inevitable pathophysiological phe-nomenon in patients with ischemic heart disease and undergoing cardiac thoracic surgery. Mean-while, it is capable of leading to reperfusion ar-rhythmia, transient mechanical dysfunction, myo-cardial stunning and other pathological changes5. Therefore, suppressing myocardial I/R injury is of great significance for the prevention and treat-ment of ischemic cardiomyopathy. Myocardial I/R injury is closely correlated with various fac-tors, including massive formation of oxidative free radicals, changes in cardiac hemodynamics, overload of calcium in myocardial cells, inflam-mation, necrosis and apoptosis of myocardial cells6, 7. In myocardial I/R injury, a large number of pro-inflammatory factors such as interleukin-6

Abstract. – OBJECTIVE: The aim of this study was to investigate the effect of morphine on myocardial ischemia/reperfusion (I/R) injury in rats and its underlying mechanism, thereby pro-viding a reference for the prevention and treat-ment of myocardial I/R injury in clinical practice.

MATERIALS AND METHODS: A total of 60 male Sprague-Dawley (SD) rats were random-ly divided into 3 groups, including: Sham group (n=20), I/R group (n=20) and I/R + morphine group (n=20) using a random number table. The left an-terior descending coronary artery (LAD) of rat was ligated and re-canalized, and the I/R model was established in rats. Rats in I/R + sevoflurane (SEV) group were pretreated with 2.5% SEV. In-farction area of heart in each group was detect-ed using triphenyltetrazolium chloride (TTC) test. Ejection fraction % (EF%) and fraction shorten-ing % (FS%) were determined by echocardiogra-phy. Hematoxylin-eosin (HE) staining assay was performed to detect the morphological changes of cardiac myocardial cells in each group. Mean-while, terminal deoxynucleotidyl transferase-me-diated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was adopted to detect the apoptosis of myocardial cells and fibroblasts. In addition, the expression levels of toll-like re-ceptor 4 (TLR4) and p65 in heart samples of rats in each group were measured via immuno-histo-chemical staining. Finally, the influence of mor-phine on TLR4/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway was detected using Western blotting.

RESULTS: Morphine significantly alleviat-ed I/R-induced cardiac dysfunction in rats, whereas significantly increased EF% and FS% (p<0.05). In addition, morphine evidently inhib-ited myocardial infarction caused by I/R injury. Meanwhile, it reduced the infarction area from [(59.61±3.41) %] to [(26.67±3.62) %] (p<0.05). The results of HE staining showed that compared with I/R group, I/R + morphine group exhibited remarkably tidier cardiac myofilament, less deg-radation and necrosis, as well as significantly relieved cellular edema. Immuno-histochemical

European Review for Medical and Pharmacological Sciences 2019; 23: 8616-8624

Y. WANG1, L. WANG2, J.-H. LI3, H.-W. ZHAO1, F.-Z. ZHANG1

1Department of Anesthesioiogy, People’s Hospital of Rizhao, Rizhao, China2Department of Anesthesioiogy, Rizhao Central Hospital, Rizhao, China3No. 1 Department of Operation Room, People’s Hospital of Rizhao, Rizhao, China

Corresponding Author: Fazhan Zhang, MM; e-mail: zfll78@163.com

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(IL-6), IL-10, tumor necrosis factor-α (TNF-α) and IL-8 are released, directly damaging the heart8. Meanwhile, these inflammatory factors further recruit the infiltration of other inflamma-tory cells in the myocardium. This can indirectly damage myocardial cells and vascular endothelial cells9. Han et al10 have revealed that during MI, Toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated. Eventually, this transcribes and activates multifarious pro-inflam-matory factors, causing damage to the heart.

Morphine is a typical analgesic. In recent years, its pharmacological effects on cardiovascular system, tumor and digestive tract have been re-vealed11. For instance, in nasopharyngeal carcino-ma, low-dose of morphine can induce resistance of tumor cells to cisplatin-based chemotherapy drugs12. In addition, morphine represses cell cycle and induces the apoptosis of breast cancer MCF-7 cells, ultimately inhibiting breast cancer progres-sion13. However, the role of morphine in cardiac I/R injury has not been elucidated. In this study, the cardiac I/R rat model was established, and a certain dose of morphine was used for pretreat-ment. Moreover, its effect on cardiac I/R injury was detected, so as to provide a reference for the treatment of MI in clinical practice in the future.

Materials and Methods

Grouping and Treatment of Experimental Animals

A total of 60 male Sprague-Dawley (SD) rats weighing (285.61±10.66) g and aged 12-14 weeks old were randomly divided into 3 groups [includ-ing: sham operation group (Sham group, n=20), I/R group (n=20) and I/R + morphine group (n=20)]. There were no statistically significant differences in basic data, such as age and body weight, among rats of the three groups. Rats in I/R + morphine group were intraperitoneally in-jected with a certain dose of morphine (20 mg/kg/d). This study was approved by the Animal Ethics Committee of People’s Hospital of Rizhao Animal Center.

Specific operation procedures were as follows: rats in each group were anesthetized with 50 mg/kg pentobarbital intraperitoneally. A cannu-la was then inserted into the left carotid artery, and blood pressure of rats was measured. Limb leads II of Electrocardiograph (ECG) was used to detect heart rate. The thorax was opened from

the fourth intercostal space, and the pericardium was removed to expose the heart. The left anterior descending coronary artery (LAD) was ligated 2 mm above the left auricle using a 6-0 silk suture to induce local MI. After 30 min of ischemia, the silk suture was loosened for 2 h of reperfusion. Rats in Sham group underwent the same surgery without silk suture ligation. After reperfusion, rats were sacrificed. Left ventricular anterior wall myocardial tissues were removed and placed in a refrigerator at -80°C. After blood was washed off with normal saline, the tissues could be used (Figure 1).

Echocardiography To examine the heart function of rats in each

group, Mylab 30CV ultrasound system (Esaote S.P.A, Genoa, Italy) and 10-MHz linear ultra-sound transducer were used in this study. Subse-quently, echocardiogram of rats in each group was detected. After shaving off the hair on the chest, rats were anesthetized and placed on a hot plate at 37°C with the left side facing up. Next, param-eters including ejection fraction (EF%), fraction shortening (FS%) and heart rate were measured.

Triphenyltetrazolium Chloride (TTC) Staining

(1) Fresh myocardial tissues were placed into a rat heart section mould and then frozen in a re-frigerator at -20°C for 30 min for sectioning. (2) Myocardial tissues were cut into about 2 mm thick sections, and the number of sections per tissue did not exceed 6. (3) Sections cut were placed in fresh TTC solution (2%) (Solarbio, Beijing, China) for full contact and at least 0.5 h of incubation. (4) Sections were taken out at 0.5 h, fixed with 4% paraformaldehyde and photographed.

Immunohistochemical StainingMyocardial tissue sections were baked in an

oven at 60°C for 30 min. Then the sections were dewaxed with xylene (5 min × 3 times) and dehy-drated with 100% ethanol, 95% ethanol and 70% ethanol separately. 3% methanol hydrogen perox-ide was added to each section to inhibit the ac-tivity of endogenous peroxidases. After blocking with goat serum for 1 h, the sections were incu-bated with anti-TLR4 and p65 antibodies [diluted with phosphate-buffered saline (PBS) at a ratio of 1:200] at 4°C overnight. The sections were then washed with PBS on a shaker for 4 times. Subsequently, secondary antibodies were added, followed by color development with diaminoben-

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zidine. 6 samples were randomly selected from each group, and 5 fields were randomly selected for each sample. Photography was finally per-formed using 200× and 400× light microscopes.

Hematoxylin-Eosin (HE) StainingHearts obtained from each group were placed

in 10% formalin overnight. On the next day, the tissues were dehydrated and embedded with par-affin blocks. All myocardial tissues were then cut into thin sections with thickness of 5 μm, fixed on a glass slide, dried and stained. According to the instructions, the sections were immersed in xylene, ethanol with gradient concentrations and hematoxylin, respectively. Subsequently, the sections were mounted with resin. After drying naturally, the sections were observed and photo-graphed with a light microscope. Finally, the mor-phologies of myocardial cells, cardiac interstitial tissues and myofilaments were observed.

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate-Biotin Nick end Labeling (TUNEL) Staining

Cut myocardial tissue sections were first baked in an oven at 60°C for 30 min. Next, they were dewaxed with xylene (5 min × 3 times), and dehy-drated with 100% ethanol, 95% ethanol, and 70% ethanol, respectively. Subsequently, the sections were incubated with protein kinase K for 0.5 h and rinsing with PBS. Next, terminal deoxyribonu-cleotidyl transferase and luciferase-labeled dUTP were added for reaction at 37°C for 1 h. Horserad-ish peroxidase (HRP) labeled specific antibodies were added for incubation in a 37°C incubator for

another 1 h. Thereafter, diaminobenzidine (DAB) (Solarbio, Beijing, China) was used as a substrate for reaction at room temperature for 10 min. Af-ter cell nuclei were stained with hematoxylin, the cells were photographed and counted under a flu-orescence microscope.

Western Blotting AssayHeart tissues of rats in each group were fully

ground in lysis buffer and subjected to ultrason-ic lysis. Then the lysis solution was centrifuged, and the supernatant was aspirated and separately sub-packaged into EP tubes. Next, the concentra-tion of extracted protein was measured via bicin-choninic acid (BCA) method (Pierce, Rockford, IL, USA) and ultraviolet spectrometry. After the concentration of all protein samples was the same, the proteins were sub-packaged and stored in a refrigerator at -80°C. Subsequently, total protein samples were separated by sodium dodecyl sul-phate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene di-fluoride membranes (Roche, Basel, Switzerland). Then the membranes were incubated with prima-ry antibody at 4°C overnight. On the next day, the membranes were incubated with goat anti-rabbit secondary antibody for 1 h in dark. Finally, Od-yssey membrane scanner was used to scan and quantify protein bands. Glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) was utilized to correct the level of target protein.

Statistical AnalysisStatistical Product and Service Solutions

(SPSS) 22.0 software (IBM, Armonk, NY, USA) was used for all statistical analysis. Measurement

Figure 1. Flow chart of treatment in rats of each group. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group.

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data were expressed as mean ± standard devia-tion. t-test was employed to compare the differ-ence between two groups. p<0.05 was considered statistically significant.

Results

Effects of Morphine on Cardiac Function in Rats With Cardiac I/R

Echocardiography results showed that there was no statistically significant difference in heart rate among the three groups. Therefore, the differ-ences in EF% and FS% among each group were not caused by heart rate. Compared with con-trol group, I/R group exhibited enlarged ventri-cles and thinned heart wall. However, morphine (20 mg/kg) pretreatment significantly improved I/R-induced abnormal changes in ventricular and wall structures in rat heart. Furthermore, the lev-els of FS% and EF% in rats of each group were ex-amined in this study. It was found that morphine pretreatment remarkably reversed the decrease of

FS% and EF% in I/R group (p<0.05). These re-sults indicated that morphine pretreatment could improve cardiac function induced by reperfusion in rats (Figure 2).

Morphine Pretreatment Reduced Infarction Area In Rat Heart

TTC staining was used in this study to assess the infarction area in rat heart of each group. As shown in Figure 3, the results indicated that statistically significant differences were found in the infarction area of rats in the three groups [(1.23±0.52) vs. (58.93±1.45) vs. (22.19±1.47)] (p<0.05). This suggested that morphine (20 mg/kg) effectively reduced the infarction area in myocardium in rats with myocardial I/R.

Influence of Morphine Pretreatment on Cardiac I/R-Induced Pathological Injury

To evaluate the changes of microstructure of myocardial cells in the cross sections of rat heart, HE staining was performed. The results showed that I/R group showed evident edema,

Figure 2. Effects of morphine pretreatment on cardiac function in rats. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group. *Compared with Sham group. #Compared with I/R group, p<0.05.

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disordered myofilament, degradation and vary-ing degrees of necrosis in myocardial cells. Af-ter morphine pretreatment, myocardial tissue edema and myofilament abnormality in rats were significantly alleviated. These findings implied that morphine alleviated myocardial damage caused by I/R (Figure 4).

Role of Morphine Pretreatment in Myocardial Apoptosis in Rats

The level of myocardial apoptosis in rats of the three groups was examined via TUNEL staining in this study. The results (Figure 5) revealed that the apoptosis of myocardial cells in rats after I/R injury was significantly [about (4.02±1.78) times] higher than that of control group (p<0.05). How-ever, the number of apoptotic myocardial cells af-

ter morphine pretreatment was (2.42±0.83) times lower than that of control group (p<0.05). This indicated that morphine pretreatment could evi-dently inhibit myocardial apoptosis in rats.

Immunohistochemical Staining of TLR4 and p65 in Myocardial Tissues of Rats in Each Group

Furthermore, immunohistochemistry was used in this study to determine the expressions of TLR4 and p65 in heart of each group. The results demonstrated that compared with Sham group, the expressions of TLR4 and p65 in I/R group were significantly elevated (p<0.05). However, after treatment with morphine, the expressions of TLR4 and p65 in rat heart tissues were clearly suppressed (p<0.05). These results suggested that

Figure 3. Effects of morphine pretreatment on infarction area in rats of each group. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group. *Compared with Sham group. #Compared with I/R group, p<0.05.

Figure 4. Effect of morphine pretreatment on rat heart structure (Magnification x 40) in each group. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group.

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morphine pretreatment could remarkably reduce the expressions of TLR4 and p65 in rat myocardi-al tissues (Figure 6).

Expression of TLR4/NF-κB signaling Pathway in Myocardial Tissues of Rats in Each Group

To further quantify the protein changes in TLR4/NF-κB signaling pathway, Western blot-ting was utilized in this study to detect the protein

expressions of TLR4 and NF-κB in rat myocardi-al tissues. The results (Figure 7) revealed that the expression levels of TLR4 and phosphorylated p65 in rat myocardial tissues were significantly up-reg-ulated in I/R group (p<0.05). However, the protein expressions of the two molecules were significant-ly inhibited after morphine pretreatment (p<0.05). Therefore, we hypothesized that morphine played an important role in protecting the heart by inhibit-ing TLR4/NF-κB signaling pathway.

Figure 5. Role of morphine pretreatment in myocardial apoptosis in rat heart tissues (Magnification x 40) in each group. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group. *Compared with Sham group. #Compared with I/R group, p<0.05.

Figure 6. Impacts of morphine pretreatment on TLR4 and p65 in rat heart tissues (Magnification x 40) in each group. Sham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group. *Compared with Sham group. #Compared with I/R group, p<0.05.

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Discussion

Mechanical or drug intervention is the most ef-fective strategy to rapidly recover the blood flow in occlusive coronary artery, limit the infarction area of myocardium and improve the clinical out-come after acute MI14. However, reperfusion it-self can lead to additional cardiac cell death and increase infarction area in the heart. The main factors leading to reperfusion injury include oxi-dative stress, inflammation and apoptosis. During myocardial ischemia, ATP consumption in myo-cardial cells reduces the ability of sarcoplasmic reticulum to uptake Ca2+. This can cause massive accumulation15,16 of mitochondrial Ca2+. During reperfusion, oxygen re-enters into myocardial cells, resulting in impaired mitochondrial elec-tron transport chains and increased production of reactive oxygen species (ROS)17. Furthermore, increased mitochondrial Ca2+ overload and ROS production promote the opening of mitochondri-al membrane permeability transition pores. This can eventually lead to cellular energy disturbance and even irreversible necrosis and apoptosis of cells18,19. Therefore, inhibition of myocardi-al apoptosis, inflammation and oxidative stress during reperfusion is capable of effectively alle-viating cardiac dysfunction caused by I/R injury and reducing infarction area.

TLR family is a class of important molecules in innate immunity. The activation of corre-

sponding TLRs can induce a series of inflam-matory responses, alerting the host to microbial invasion and initiating immune responses20. Sev-eral members of the TLR family have been found to induce endogenous inflammatory factors re-leased by damaged tissues. This suggests that tissue damage can be detected in the host. Hep-aran sulfate, hyaluronic acid and other endoge-nous molecules induce inflammatory pathways by activating TLR421. Researches have revealed that in the case of heart failure, the expression level of TLR4 in myocardial cells is significant-ly reduced. At the same time, natural immunity of the body is activated22. Meanwhile, compared with wild-type mice, TLR4 knockout mice ex-hibit obviously relieved cardiac dysfunction and myocardial damage caused by ischemia. The possible underlying mechanism may be related to the regulatory effects of TLR4 on AMPK and ERK signaling pathways23. In addition, TLR4 knockout can alleviate cardiac dysfunction due to high-fat diet by inhibiting NF-κB and JNK-de-pendent autophagy24. Inhibition of TLR4 expres-sion cannot change blood pressure in rats, while it can inhibit myocardial remodeling induced by spontaneous hypertension25. These findings suggest that TLR4 as a target and endogenous knockout of TLR4 or exogenous inhibition of its expression play important roles in the develop-ment and progression of cardiovascular diseases.

Figure 7. Effect of morphine pretreatment on TLR4/NF-κB signaling pathway in rat heart tissues in each group.mSham: sham operation group, I/R: I/R group, and I/R + morphine: I/R + morphine pretreatment group. *Compared with Sham group. #Compared with I/R group, p<0.05.

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In this study, rats with I/R were constructed and intraperitoneally injected with a certain amount of morphine for successive 7 d. On the 7th day, cardiac function of rats in each group was eval-uated via two-dimensional ultrasound. It was found that morphine treatment significantly im-proved the cardiac function in I/R rats, which was reflected in the improvement of EF and FS. In addition, HE staining was employed to ob-serve the microstructure of myocardial tissues in rats of each group. The results showed that morphine could inhibit I/R-induced pathologi-cal changes in the myocardium. Moreover, the number of apoptotic cells in myocardial tissues of each group was quantified through TUNEL staining. Results demonstrated that morphine overtly reduced the number of apoptotic myocar-dial cells induced by I/R. Further, it was found that morphine repressed the expressions of TLR4 and NF-κB in myocardial tissues. Nonetheless, this study still had some limitations: (1) No cel-lular experiments were designed for verification, (2) TLR4 knockout mice were selected to vali-date whether the protective effect of morphine on myocardium was dependent on TLR4.

Conclusions

We showed that morphine can relieve myocar-dial I/R injury in rats by suppressing TLR4 sig-nal, so morphine may become a new drug for the treatment of MI.

Conflicts of interestThe authors declare no conflicts of interest.

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