NLRP3 inflammasome pathway

Accepted Manuscript Ghrelin protects the heart against ischemia/reperfusion injury via inhibition of TLR4/NLRP3 inflammasome pathway

Qin Wang, Ping Lin, Peng Li, Li Fen, Qian Ren, Xiaofeng Xie, Jing Xu PII: DOI: Reference:

S0024-3205(17)30378-8 doi: 10.1016/j.lfs.2017.08.004 LFS 15291

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

7 May 2017 3 August 2017 3 August 2017

Please cite this article as: Qin Wang, Ping Lin, Peng Li, Li Fen, Qian Ren, Xiaofeng Xie, Jing Xu , Ghrelin protects the heart against ischemia/reperfusion injury via inhibition of TLR4/NLRP3 inflammasome pathway, Life Sciences (2017), doi: 10.1016/ j.lfs.2017.08.004

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ACCEPTED MANUSCRIPT Ghrelin protects the heart against ischemia/reperfusion injury via inhibition of TLR4/NLRP3 inflammasome pathway Qin Wanga, Ping Lina*, Peng Lia, Li Fena, Qian Rena, Xiaofeng Xiea, Jing Xua Department of geriatrics, the 3rd Hospital of Hangzhou, Hangzhou,

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China

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* Corresponding author: Ping Lin

Address: No.38 Xihu Street, the 3rd Hospital of Hangzhou, Hangzhou,

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China, 310009

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E-mail address: [email protected]

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Tel: +86-571-87823126

ACCEPTED MANUSCRIPT Abstract Aims: The aim of this study was to investigate the cardioprotective effects of ghrelin against myocardial ischemia/reperfusion(I/R) injury and the underlying mechanism.

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Main methods: Sprague-Dawley rats were randomized into Sham,

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I/R and I/R + ghrelin groups. After 30 minutes ischemia, ghrelin (8

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nmol/kg) was injected intraperitoneally at the time of reperfusion in the I/R + ghrelin group. Then hemodynamic parameters were observed at 24

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h after reperfusion.

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Key findings: Ghrelin exhibited dramatic improvement in cardiac functions, as manifested by increased LVSP and ± dP/dtmax and decreased

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LVDP. At 24 h after reperfusion, ghrelin significantly attenuated the

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myocardial infarction area and apoptosis, accompanied with a decrease in the levels of the myocyte injury marker enzymes. Oxidative stress injury

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and inflammatory response were also relieved by ghrelin. Western blot

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showed that the expression of TLR4, NLRP3, and Caspase-1 were obviously increased in I/R group, while ghrelin significantly inhibited the I/R-induced TLR4, NLRP3, and Caspase-1 expression. Ghrelin could inhibit the increased protein levels of NLRP3, caspase-1, and IL-1β induced by lipopolysacharide in primary cultured cardiomyocytes of neonatal rats. Significance: Ghrelin protected the heart against I/R injury by

ACCEPTED MANUSCRIPT inhibiting oxidative stress and inflammation via TLR4/NLRP3 signaling pathway. Our results might provide new strategy and target for treatment of myocardial ischemia/reperfusion injury.

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Key Words: Ghrelin; Ischemia/Reperfusion Injury; Inflammation;

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NLRP3; Oxidative Stress

ACCEPTED MANUSCRIPT Introduction Acute myocardial infarction (AMI) is the major cause of death in the worldwide of modern society. Thrombolytic therapy or primary percutaneous coronary intervention (PCI) is currently the most effective

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strategy to improve the clinical outcome for the AMI patients. However,

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restoration of the coronary blood flow after a period of ischemia or lack

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of oxygen by these procedures may lead to ischemia/reperfusion (I/R) injury, resulting in additional damage to the myocardium.The

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pathogenesis of I/R injury involves the interplay of multiple mechanisms,

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including calcium overload, oxidative stress injury, cardiomyocyte autophagy and apoptosis, which are all contributing to the final damage

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inflicted on the heart[1-4].However, the mechanisms related to cardiac

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damage are not fully understood. It is known that the innate immune response to postischemic

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inflammation played a fundamental role in the pathophysiology of

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myocardial I/R injury[5]. Recently, a novel inflammasome signaling pathway which is responsible for initiating inflammation has been uncovered and the nod-like receptor protein 3(NLRP3) inflammasome may act as the key mediator in detecting cellular damage and mediating inflammatory responses after I/R injury[6]. After the activation of NLRP3 inflammasome, procaspase-1 clustering permits autocleavage and formation of the active caspase-1 which mediates the release of the

ACCEPTED MANUSCRIPT mature, biologically active cytokines such as interleukin-1β (IL-1β) and IL-18 to engage in immune defense[7]. More importantly, inhibiting NLRP3 could considerably prevent cardiomyocytes from cell death and attenuate I/R injury indifferent experiments[8-9].So it is urgent to develop

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more effective therapies to suppress NLRP3 inflammasome activation in

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myocardial I/R injury.

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Ghrelin, an octanoylated, 28-amino acid orexigenic peptide, is produced predominantly in the stomach, and also produced in small

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amounts throughout other parts of the body, including the heart, lungs,

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lymph nodes, muscle, and pancreas[10]. It is an endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a) which is widely

hormone-releasing

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growth

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distributed throughout the body. Ghrelin has been shown to possess properties

and

other

endocrine

and

non-endocrine activities, reflecting central and peripheral GHSR-1a

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distribution. In cardiovascular system, accumulating evidences showed

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that ghrelin could enhance endothelial and vascular function, prevent atherosclerosis, inhibit sympathetic drive, and decrease blood pressure due but not limited to regulating intracellular calcium concentration, inhibiting proapoptotic cascades, and protecting against oxidative damage[11-14]. In addition, recent studies found that ghrelin could preserve cardiac function, attenuate ventricular remodeling, and delay the progress of

heart

failure

after

myocardial

infarction[15].

Although

the

ACCEPTED MANUSCRIPT cardioprotective effects of ghrelin have been partially revealed by many studies, the action of ghrelin on myocardial I/R injury in vivo has not been fully developed, whereas the underlying mechanisms remain unknown.

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In the present study, we aimed to investigate the cardioprotective

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effects of ghrelin against myocardial I/R injury. Furthermore, we planned

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to explore whether ghrelin exertsthe anti-I/R injury effect via inhibition of

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the activation of the TLR4/ NLRP3 inflammasome pathway.

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Materials and Methods

Animals and experimental protocol

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Male Sprague-Dawley rats (250-280 g, 8 w) from Vital River

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Laboratories (Beijing, China) were housed under constant environmental conditions (12h light/dark cycle) in a temperature controlled (25 °C)

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facilityand unrestricted access to food and water. All animal experimental

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procedures were performed according to the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). After acclimatization for 1 week, rats were randomly divided into three different groups (n = 8, counting alive animals): Sham group; I/R group; I/R + ghrelin group. Ghrelin was administered intraperitoneally at a dose of 8 nmol/kg at the time of reperfusion. The sham group and the

ACCEPTED MANUSCRIPT I/R group were intraperitoneally administered the equivalent volume of saline at the same time points. Ischemia/reperfusion injury model The I/R model was induced by ligating the left anterior descending

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coronary artery (LAD) as previously described[16]. Briefly, the rats were

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anesthetized with an intraperitoneal injection of 10% chloral hydrate (300

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mg/kg). Ischemia was achieved by using a 6-0 silk suture around LAD that was tied into a slipknot for 30 min. Successful ligation of the LAD

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was confirmed by ST–segment shift on the ECG and the color change in

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the ischemic area (anterior ventricular wall and the apex) of the heart.The knot was then undone for 24 h, resulting in reperfusion. The ligature was

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placed under the LAD coronary artery without occlusion in the sham

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group.

Hemodynamic assessment

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At 24 h after reperfusion, the rats were re-anesthetized with 10%

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chloral hydrate (300 mg/kg, i.p.). A PE-50 catheter filled with heparin saline (500 U/ml) was carefully inserted into the left ventricle (LV) from the right carotid artery to monitor heart function, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), mean arterial pressure (MAP) maximum contraction velocity (+dp/dtmax), and maximum relaxation velocity (−dp/dtmax) in each group. And then the hearts were excisedand

ACCEPTED MANUSCRIPT stored for subsequent experimental analysis. The plasma was prepared by centrifuging the blood samples at 4000 rpm for 10 min and frozen at -80°C for subsequent experimental analysis. Determination of Infarct Size

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Evans’ Blue-2,3,5-triphenyltetrazolium chloride (TTC) double

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staining was introduced to measure the infarct size and the areas at risk as

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previously described[17]. Briefly, the LAD was re-occluded at 24 h after reperfusion. Two ml of 1% Evans’Blue dye (Sigma-Aldrich, St. Louis,

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USA) was injected intravenously to distinguish between the perfused and

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non-perfused zones (not colored zone). Then, the heart was rapidly excised and cross-sectioned into 1-2 mm thick slices, and incubated in a

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1% TTC solution (Sigma-Aldrich, St. Louis, USA) at 37 °C for 30 min to

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differentiate infarcted (pale) from viable (brick red) myocardial area. The slices were then photographed and the size of infarct (INF), size of area at

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risk (AAR), and size of left ventricular (LV) were quantified using Image

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J software by an observer blind to the study. Biochemical Analysis The plasma levels of lactate dehydrogenase (LDH),creatine kinase (CK), creatine kinase-MB (CK-MB), and aspartate transaminase (AST) were determined by automatic biochemical analyzer (Cobas 6000, Roche, Basel, Switzerland). TUNEL staining

ACCEPTED MANUSCRIPT At the end of reperfusion, myocardial apoptosis was analyzed by terminal deoxynu-cleotidyl transferase dUTP nick end labeling assay (TUNEL) using an in situ cell death detection kit (Roche Molecular Biochemicals) according the protocol of manufacturer. A double-staining

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technique was used, i.e., TUNEL staining for apoptotic cell nuclei and

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4′,6-diamino-2-phenylindole (DAPI) staining for all myocardial cell

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nuclei. Cardiomyocytes from at least four slides per block that were randomly selected were evaluated immunohistochemically to determine

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the number and percentage of cells exhibiting positive staining for

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apoptosis. For each slide, 10 fields were randomly chosen, and a total of 100 cells per field were counted by using a defined rectangular field area

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(20× objective). The index of apoptosis was determined [(number of

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apoptotic myocytes/total number of myocytes counted) × 100%] from a

manner.

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total of 40 fields per heart, and the assays were performed in a blinded

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Measurement of cytokines The plasma levels of the inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β were measured by using ELISA kits (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Measurement of myeloperoxidase (MPO) activity Ischemic heart tissues were assessed for the MPO activity as a

ACCEPTED MANUSCRIPT marker of neutrophil accumulation. Tissues were homogenized with 50 mmol/L potassium phosphate buffer. After centrifugation, the supernatant was used to measure the activities of MPO by using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China)

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according to the manufacturer’s instructions. The activity of MPO was

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standardized by protein content, determined by a bicinchoninic acid

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(BCA) protein assay kit (Beytime Institute of Biotechnology, Beijing, China).

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Measurement of reactive oxygen species (ROS) and anti-oxidant

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enzyme activities

After homogenized with 50 mmol/L potassium phosphate buffer,

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hydrogen peroxide (H2O2), malonydialdehyde (MDA) and glutathione

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(GSH) concentrations, as well as superoxide dismutase (SOD) and catalase (CAT) activities in ischemic heart tissues were measured with

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the corresponding assay kits (Jiancheng Bioengineering Institute, Nanjing,

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China) according to the manufacturer’s instructions. All the above indicators were standardized by protein content, determined using a BCA protein assay kit. Western blot analysis Protein extracted from left ventricle tissues were quantified using BCA reagent and protein samples (50 μg/lane) were subjected to 10% SDS-PAGE gels, transferred to polyvinylidene fluoride (PVDF)

ACCEPTED MANUSCRIPT membranes and blocked with 5% nonfat milk for 1 h. Then, the membranes were incubated with primary antibodies: TLR4, NLRP3, and Caspase-1 (1:1000, Abcam, Cambridge, United Kingdom) and GAPDH as internal control (1:2000, Cell Signaling Technology, Danvers, USA) at

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4°C overnight. After washing with TBST for three times, the membranes

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were then incubated with horseradish peroxidase-conjugated secondary

SuperSignal

West

Pico

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antibodies at room temperature for 1 h. Target bands were detected with Chemiluminescent

Substrate

(Thermo

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Scientific-Pierce, Waltham, USA). The band intensity was quantified by

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Image J software.

Primary cardiomyoctyes culture from neonatal rats

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Neonatal rat cardiac myocytes were isolated from 1- to 2-day-old SD

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rats. Briefly, the excised hearts were washed in Hanks balanced salt solution (HBSS; Ca2+-Mg2+ free) and the ventricular tissues were minced

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by use of fine scissors in HBSS containing trypsin (0.05%) and

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collagenase (0.055%), and digested at 37 ℃. Cells were isolated by multiple 10-min rounds of tissue digestion. After each incubation, the supernatant was added to an equal volume of DMEM containing 20% fetal bovine serum. The total cell suspensions were centrifuged at 1,000 rpm for 10 min. Supernatants were discarded and the cell pellets were resuspended in DMEM containing 10% fetal bovine serum. The cells were plated onto plastic culture dishes for 90 min so that most of the

ACCEPTED MANUSCRIPT non-myocytes attached to the dish, and the myocytes remained in the suspension. The myocytes were harvested and seeded onto 60-mm culture dishes at 105 cells per cm2. 5-Bromo-20-deoxyuridine (100 μmol/L) was added during the first 48 h to inhibit proliferation of nonmyocytes.

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Cardiomyocytes were challenged with lipopolysaccharide (LPS; 25

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ng/ml, for 4 h) and ATP (5 mM, for 30 min) to induce the NLRP3

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inflammasome formation. Ghrelin (10 μM) was treated 5 min before LPS challenge. The supernatants were collected to detect lactic dehydrogenase

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(LDH) activity by commercial kit (Jiancheng Bioengineering Institute,

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Nanjing, China), and the cardiomyocytes were collected to determine the protein levels of NLRP3, caspase-1 and IL-1β by Western blot.

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Statistical analyses

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Results were expressed as means±SEM. Statistical analysis was performed using an SPSS software package, version 13.0. The results for

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three or more groups were compared using one-way ANOVA followed

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by Student-Newman-Keuls test. Comparisons between two groups were made usingstudents’𝑡-test. 𝑃<0.05 was considered significant.

Results Ghrelin improved cardiac function after I/R injury Hemodynamic results demonstrated that there was a significantly decrease in LVSP, ±dp/dtmax and increase LVEDP after I/R injury as

ACCEPTED MANUSCRIPT compared with the Sham group. However, ghrelin treatments increased LVSP, ±dp/dtmax, but decreased LVEDP compared with I/R group (Fig. 1A-D). There were no significant differences in MAP and HR between

Ghrelin protected the heart from I/R injury

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the I/R and I/R + ghrelin group (Fig. 1E, F).

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Representative photographs for transverse sections of hearts after I/R

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were shown in Fig. 2A and differences of the ratios of INF versus AAR and AAR versus LV were shown in Fig. 2B-C. In I/R + ghrelin group, the

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white necrotic area (infarct size) was markedly reduced compared with

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that in I/R group. Between the two groups, however, percentages of AAR to LV were similar, indicating that ligature was reproducibly performed at

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the same level of the LAD. At 24 h after reperfusion, mortalities of rats

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during I/R were 33.3% (4 of 12) in I/Rgroup and 20.0% (2 of 10) in I/R + ghrelin group, respectively.

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The levels of LDH, CK, CK-MB and AST in plasma were measured

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at 24 h after reperfusion in the three groups (Fig. 2D-G). Compared to Sham group, the LDH, CK, CK-MB and AST concentrations in the plasma were much higher after I/R injury, while ghrelin treatments decreased their levels significantly. The above results clearly showed that ghrelin protected the heart from I/R injury. Detected by TUNEL staining, the apoptotic positive cardiomyocytes in I/R group (Fig. 3B) were significantly more than that in control group

ACCEPTED MANUSCRIPT (Fig. 3A). Ghrelin treatment intriguingly attenuated the increased apoptosis of cardiomyocytes induced by I/R injury (Fig. 3C). Ghrelin resisted oxidative stress after I/R injury Myocardial I/R injury induced a significant increase in oxidative

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stress reflected by the increased levels of H2O2 and MDA. A reduction in

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H2O2 and MDA levels were observed after ghrelin administration,

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indicating that ghrelin prevented oxidative stress injury induced by myocardial I/R injury (Fig. 4A-B).

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Concomitant to the increased H2O2 and MDA levels, a significant

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decrease in the activities of the antioxidant enzymes, SOD and CAT and concentrations of GSH, was observed in I/R group in comparison with the

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Sham group, demonstrating the damage to the endogenous antioxidant

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system occurring in myocardial I/R injury (Fig. 4C-E). Treatment with ghrelin significantly preserved the activities of SOD and CAT and

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concentrations of GSH.

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Ghrelin reduced myocardial inflammation after I/R injury MPO activity assay was performed to determine the neutrophil influx in the ischemic tissues. As shown in Fig. 5A, MPO activity was significantly higher at 24 h after reperfusion, while the increased MPO activity was reduced by treatment with ghrelin. To investigate the potential anti-inflammatory activity of ghrelin following myocardial I/R injury, the levels of cytokines associated with

ACCEPTED MANUSCRIPT inflammation, such as TNF-α, IL-6 and IL-1β were measured in the plasma. The levels of TNF-α, IL-6, and IL-1β increased in I/R group compared with those of Sham group, however, ghrelin could attenuate these changes significantly (Fig. 5B-D).

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Ghrelin inhibited NLRP3 inflammasome activation after I/R

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injury

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To further explore the potential molecular mechanisms behind the cardioprotective effects of ghrelin, the expression levels of TLR4, NLRP3,

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and caspase-1 in the ischemic myocardial tissues were measured by

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western blot analysis (Fig. 6A).Compared with the Sham group, the expression levels of TLR4, NLRP3, and Caspase-1 were obviously

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increased in I/R group. However, ghrelin significantly inhibited

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I/R-induced TLR4, NLRP3, and Caspase-1 expression (Fig. 6B-E) Ghrelin directly inhibited NLRP3 inflammasome induced by

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LPS in cardiomyocytes of neonatal rats

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Compared with control group, the protein levels of NLRP3 (Fig. 7A), caspase-1 (Fig. 7B) and IL-1β (Fig. 7C) in LPS-treated cardiomyocytes of neonatal rats were significantly increased. Interestingly, ghrelin treatment attenuated the increased protein levels of NLRP3, caspase-1 and IL-1β induced by LPS. Consistent with the alteration of protein levels, ghrelin also significantly reduced the increased LDH activity in cellular supernatant induced by LPS (Fig. 7D).

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Discussion In this study, we found that ghrelin significantly inhibited I/R-induced rat myocardial injury, as demonstrated by the decrease of

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infarct size, cardiomyocytic apoptosis and injury marker enzymes,

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companying with improvement of cardiac function. In addition, ghrelin

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administration relieved oxidative stress and inflammatory response after I/R challenge. Furthermore, we provided evidences showing that

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ghrelincould down-regulate the TLR4 expression, and then inhibit

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NLRP3 inflammasome activation after I/R injury, thus interferes in its downstream targets. These findings suggested that treatment with ghrelin

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might prevent I/R injury and improve the outcome.

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Early and successful reperfusion is most important to reduce infarct size and improve prognosis in patients with AMI. However, the

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restoration of blood flow causes further damage to the ischemic tissues,

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known as reperfusion injury, contributing to the overall infarct size. The mechanisms of myocardial I/R injury are multifaceted, including ATP depletion, Ca2+ overload, opening of themitochondrial permeability transition pore (mPTP) and increasing in ROS production which lead to apoptotic

and

necrotic

cardiomyocyte

death

[1-2,18-19]

.

Many

pharmacological agents have been shown to improve myocardial ischemia-reperfusion injury by affecting above mechanisms in preclinical

ACCEPTED MANUSCRIPT data[20-23], however, none of them has so far transferred to the clinics. Therefore, it is essential to further reveal the mechanisms of I/R injury and explore novel therapeutic strategies to attenuate its damage. In the present study, we investigated the cardioprotective effects of

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ghrelin against myocardial I/R injury. Ghrelin, a 28-amino acid growth

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hormone-releasing peptide was first identified in 1999 as an endogenous

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ligand for the GH secretagogue receptor (GHS-R) and is now implicated in a number of physiological and pathophysiological processes [24]. In

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cardiovascular system, ghrelin is shown to be a variety of benefit effects,

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including protection of endothelial cells, reduction of mean arterial blood pressure, increasing myocardial contractility, and improvement of cardiac and

exogenous

administration

of

ghrelin

suppresses

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function

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cardiomyocyte apoptosis, inhibits sympathetic nerve activity, reduces ventricular remodeling and protects from heart failure induced by infarction[14-15,

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myocardial

25-26]

.

In

clinical

studies,

exogenous

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administration of ghrelin has been substantiated to improve the cardiac function and prognosis in patients suffering from end-stage heart failure[27]. However, there is little research on the effect of ghrelinagainst myocardial I/R injury. In our study, we measured hemodynamic parameters, such as LVSP, ±dp/dtmax and LVEDP, to assess cardiac function in the rats after myocardial I/R injury. The results showed that there was a significantly decrease in LVSP, ±dp/dtmax and increase LVEDP,

ACCEPTED MANUSCRIPT indicating cardiac dysfunction after I/R injury. However, ghrelin presented a significant improvement of the above mentioned parameters. In addition, ghrelin could attenuate the levels of the myocyte injury marker enzymes, the infarct size and the mortalities of rats at 24 h after

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reperfusion.The above results clearly showed that ghrelin protected the

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heart from I/R injury.Previous in vitro experiment showed that ghrelin

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could produce a positive inotropic effect on ischemic cardiomyocytes and protect them from I/R by regulating of intracellular calcium to allow the

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maintenance or recovery of normal cardiac contractility[28].

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Within minutes of reperfusion, a surge of ROS and release of pro-apoptotic proteins from the matrix occur as a result of mitochondrial

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injury, then the activation of the NLRP3 inflammasome, leading to a

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secondary wave of inflammatory injury occurring minutes-to-hours following reperfusion.NLRP3 inflammasome is a multi-protein complex

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that is involved in the initiation and development of many diseases, such

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as atherosclerosis, Type 2 diabetes, metabolic syndrome, Alzheimer's disease, and AMI. Under the pathological conditions, inactive NLRP3 is generated in the cytoplasm by the recognition of Toll-like receptors (TLRs) in response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Liu et al[29] found that TLR4/NF-κB pathway was activated by high glucose stimulation in PC12 and significantly alleviated by the co-treatment of ghrelin. In our study,

ACCEPTED MANUSCRIPT we also found that the up-regulated TLR4 was significantly suppressed following the treatment with ghrelin after I/R, which was consistent with a recent study from Sun et al [30]. As TLRs is activated, there are three pathways involved in the

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process of NLRP3 inflammasome activation: potassium efflux triggered

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by ATP, the generation of ROS by the TLRs pathway, and cathepsins

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release after lysosomal destabilization. A large number of studies have demonstrated that an increased ROS is generated either during the

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ischemic phase or during the reperfusion period, whichplays an important

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role in the pathogenesis of myocardial I/R injury, including myocardial stunning, cardiomyocyte apoptosis, and reperfusion arrhythmias. Ghrelin

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or obestatin which were both encoded by the same gene and derived from

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the precursor protein proghrelin, was reported to improve I/R injury in rats via its antioxidant and anti-apoptotic effects in other tissues[31-32].

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Corresponding, our results showed that ghrelin significantly preserved

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concentrations of GSH and the activities of SOD and CAT and decreased levels of H2O2 and MDA. It indicated that ghrelin prevented oxidative stress injury induced by myocardial I/R injury. Excessive ROS contributes to the inflammasome assembly and activation of the NLRP3 inflammasome.In a recent work [33], combination of NADPH and NOX inhibitors to reduce the ROS generation could suppress the expression of inflammasome proteins including NLRP3 ASC,

ACCEPTED MANUSCRIPT caspase-1 in the ischemic stroke. TXNIP-mediated NLRP3 activation through oxidative stress was also found as a key signaling mechanism in the susceptibility to ischemic acute kidney injury in diabetic models [34]

.When activated, NLRP3 forms an inflammasome complex with the

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adaptor molecule ASC, thus controlling the activation of caspase-1, and

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subsequently promotes the secretion of pro-inflammatory cytokine such

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as IL-1β and IL-18 which eventually triggers cell damage and death. In view of the important role of NLRP3 in I/R injury, more and more studies

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are also focused on this field. Reperfusion therapy with recombinant

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human relaxin-2 (Serelaxin), a hormone that is produced during pregnancy and mediates the hemodynamic changes that occur during this

I/R

injury

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following

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time, attenuated myocardial infarct size and NLRP3 inflammasome via

eNOS-dependent

mechanism.[35].

Pharmacological inhibition of NLRP3 inflammasome also could attenuate

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myocardial I/R injury by activation of RISK pathway and improvement in

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mitochondrial function

[36]

. In our study, we found that the expression

levels of TLR4, NLRP3, and Caspase-1 were obviously increased in ischemic

myocardial

tissues

after

I/Rinjury.

However,

ghrelin

significantly inhibited I/R-induced TLR4, NLRP3, and Caspase-1 expression. Above results indicated that ghrelin could inhibit NLRP3 inflammasome formation via suppressing TLR4. Subsequently, the levels of cytokines associated with inflammation, such as TNF-α, IL-6 and

ACCEPTED MANUSCRIPT IL-1β were measured in the plasma to investigate the potential anti-inflammatory activity of ghrelin following myocardial I/R injury. Ghrelin could attenuate increased levels of TNF-α, IL-6, and IL-1β after I/Rinjury. Several lines of evidence have shown thatghrelin attenuates I/R

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injury in different types of organ damage, with a decrease in

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pro-inflammatory cytokine[37-39].

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To detect whether ghrelin could direct inhibit TLR4/NLRP3 inflammasome pathway, we used primary cultured cardiomyocytes from

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neonatal rats treated with LPS, generally considered as TLR4 agonist that

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could stimulate NLRP3 inflammasome [40-42]. Consistent with the published articles, our results showed that ghrelin could attenuate the

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LPS-induced inflammasome activation and cardiomyocytes injury. These

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results demonstrated the direct inhibited effect of ghrelin on TLR4/NLRP3 inflammasome pathway.

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One limitation of the study is that many of the P values are of

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borderline statistical significance. The borderline significant of P values might increase the risks of lack of reproducibility. Therefore, the cardioprotection of ghrelin and the underlying mechanisms should be further confirmed and reproduced in the future.

Conclusion In conclusion, our study suggested that ghrelin could protect the

ACCEPTED MANUSCRIPT heart against ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via TLR4/NLRP3 signaling pathway. Therefore, ghrelin might become a potential alternative treatment for patients

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presenting with I/R injury.

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Acknowledgments

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This study was supported by the science and technology bureau of

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Hangzhou (20140633B16).

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Conflicts of Interest

The authors declared that there is no conflict of interests regarding

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the publication of this paper.

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Figure legends Fig.1 Ghrelinimproved cardiac function after I/R injury: (A) The changes of maximum contraction velocity (+dp/dtmax). (B) The changes of

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maximum relaxation velocity (−dp/dtmax). (C) The changes of left

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ventricular systolic pressure (LVSP). (D) The changes of left ventricular

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end-diastolic pressure (LVEDP). (E) The changes of mean arterial pressure (MAP). (F) The changes of heart rate (HR). Results are means ±

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SEM. A 𝑃 of<0.05 was considered significant.

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Fig.2 Ghrelin protected the heart from I/R injury: (A) Representative cross sections of Evans blue and TTC-stained hearts at 24

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h after reperfusion.Blue area is nonischemic zone; red area is viable parts

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of the heart appear red; and white area is infracted tissue.Area at risk (AAR) includes the red and white parts. (B) Quantification of the infarct

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area (INF)/AAR. (C) Quantification of the AAR/ left ventricular size

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(LVS). (D) Lactate dehydrogenase (LDH) levels in the plasma. (E) Creatine kinase (CK) levels in the plasma. (F) Creatine kinase-MB (CK-MB) levels in the plasma. (G) Aspartate transaminase (AST) levels in the plasma. Results are means ± SEM. A 𝑃 of<0.05 was considered significant. Fig.3 Effect of ghrelin on cardiomyocytes apoptosis induced by I/R. A-C, TUNEL staining in control, I/R and ghrelin groups respectively.

ACCEPTED MANUSCRIPT D, quantitative analysis of percentage of cardiomyocyte apoptosis. Results are means ± SEM. A 𝑃 of<0.05 was considered significant. Fig.4 Ghrelin resisted oxidative stress after I/R injury: (A) Hydrogen peroxide (H2O2) levels in ischemic heart tissues. (B)

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Malonydialdehyde (MDA) levels in ischemic heart tissues. (C)

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Superoxide dismutase (SOD) activities in ischemic heart tissues. (D)

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Catalase (CAT) activities in ischemic heart tissues. (E) Glutathione (GSH) levels in ischemic heart tissues. Results are means ± SEM. A 𝑃 of<0.05

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was considered significant.

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Fig.5 Ghrelin reduced myocardial inflammation after I/R injury:(A) Myeloperoxidase (MPO) activities in ischemic heart tissues.

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(B) TNF-α levels in the plasma. (C) IL-6 levels in the plasma. (D) IL-1β

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levels in the plasma. Results are means ± SEM. A 𝑃 of<0.05 was considered significant.

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Fig.6 Ghrelin inhibited NLRP3 inflammasome activation after

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I/R injury: (A) Representative Western blots for TLR4, NLRP3, and caspase-1 expression in ischemic heart tissues. GAPDH was used as the internal control. (B-E) The quantitative analysis for TLR4, NLRP3, and caspase-1 protein expression in ischemic heart tissues. Fig.7 Ghrelin inhibited the increased protein levels of NLRP3 (A), caspase-1 (B), and IL-1β (C) and LDH activity (D).

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