The protective effect of Ghrelin peptide on doxorubicin hydrochloride induced heart failure in rats

Abstract

Background

To investigate the protective effect and mechanism of Ghrelin on Doxorubicin (Dox) hydrochloride induced heart failure (HF) and myocardial injury in rats.

Methods

45 rats were randomly divided into control group, HF group and Ghrelin group. Dox hydrochloride was injected intraperitoneally to establish the model of HF in rats of HF group and Ghrelin group. Rats in the Ghrelin group were given intraperitoneal injection of Ghrelin twice a day, and rats in the HF group and control group were given equal volume of normal saline for a total of 6 weeks. The changes of echocardiography, cardiac hemodynamics, myocardial histology and plasma inflammatory factors were observed.

Results

After the Ghrelin intervention, compared with the HF group, the left ventricular end-diastolic diameter (LVDD) and left ventricular end-systolic diameter (LVSD) in the Ghrelin group was markedly reduced (P < 0.05), and left ventricular ejection fraction (LVEF) was significantly increased (P < 0.05). Compared with HF group, the left ventricular systolic pressure (LVSP), maximum rate of increase in left ventricular pressure (+ dP/dtmax) and maximum rate of decrease in left ventricular pressure (− dP/dtmax) of Ghrelin group was remarkedly increased (P < 0.05), left ventricular diastolic pressure (LVDP) decreased (P < 0.05). In the Ghrelin group, the degree and extent of cardiomyocyte degeneration and necrosis were remarkedly reduced compared with the HF group. The levels of TNF-α and iNOS in Ghrelin group were notably lower than those in HF group (P < 0.05), the IL-10 level increased markedly (P < 0.05).

Conclusion

Ghrelin may reduce Dox-induced myocardial injury and improve cardiac function in rats by regulating inflammation and oxidative stress.

Introduction

Heart Failure (HF) is widespread in clinical practice, and the incidence is increasing, the annual treatment cost due to HF is also increasing. HF is an end-stage cardiac disease caused by the chronic progression of coronary heart disease, hypertension, heart valve diseases, myocardial infarction and other diseases [1]. According to worldwide statistics, nearly 1–2 out of every 100 people suffer from HF. With the progress of aging population, HF is increasing worldwide [2]. Chronic Heart Failure (CHF) has a higher incidence in clinical practice, and the life quality of patients is severely reduced due to complications such as reduced activity tolerance, lower limb oedema, and gastrointestinal and digestive stasis and oedema [3]. Among the causes of CHF, myocardial infarction is one of the more frequent ones [4]. The venous stasis will lead to oedema of many organs, resulting in eventual organ hypoplasia and serious impairment of normal physiological functions [5, 6]. Therefore, the search for factors or drugs that can reduce myocardial inflammation and improve cardiac function to counteract the cardiotoxicity induced by antineoplastic drug therapy is of great clinical significance to improve the therapeutic effect of oncology patients [7].

Doxorubicin (Dox) is a common broad-spectrum anti-tumor drug. During the synthesis of RNA and DNA, Dox is inserted into the DNA chain to destroy the synthesis process, thus playing a therapeutic role in destroying tumor cells and inhibiting the spread of tumor cells [8]. Although the drug has a wide range of clinical applications, it can be used in combination with other chemotherapy drugs to treat various tumor diseases. However, most studies have found that Dox has side effects such as myelosuppression, cardiotoxicity, and digestive reactions, with cardiotoxicity being one of the most serious side effects of anthracyclines [9]. The short-term use of the drug in large quantities can cause acute cardiac injury, mainly inflammation. The long-term application can also lead to irreversible chronic cardiac injury, and the most common ventricular remodeling is cardiomyocyte apoptosis, myocardial fibrosis and cardiac atrophy [10, 11]. Ghrelin was first discovered in 1999 by Japanese scientists Kojima et al. in the stomach cells of animal bodies, and its research has been increasing since then [12]. At first, it was only found that Ghrelin belongs to a kind of growth hormone secretagogues, which regulates energy metabolism of the organism [13]. In recent years, more researchers have found that Ghrelin’s role in the body goes beyond this, and that its role in cardiovascular and cerebrovascular diseases is also more extensive, and its cardiovascular protective effects have led to more possibilities for clinical application in many chronic and intractable diseases, such as coronary heart disease and HF [14]. It has been found that Growth Hormone Secretagogue Receptor (GHSR) has a wide distribution in living organisms, including cardiac cells, blood vessel wall cells, and the organs of Ghrelin secretion are not only the digestive organs such as the stomach, small intestine, colon, pancreas [15]. Ghrelin is also secreted in the hypothalamus, pituitary gland, thyroid gland and adrenal gland, suggesting that Ghrelin has a certain amount of secretion in the body and has a wide range of ligands. It has been found that Ghrelin binds to specific sites in cardiomyocytes, inhibits apoptosis and myocardial remodeling. It has also been observed that Ghrelin protects cardiomyocytes against acute infarction myocardial tissue remodeling by inhibiting the synthesis of collagenous substances and inhibiting the production of inflammatory substances and the activation of inflammatory mediators, such as TNF-α in the tissue [16]. Studies have shown that Ghrelin can increase myocardial contractility, dilate blood vessels and protect the formation of heart failure after myocardial infarction [14]. Further studies have shown that Ghrelin and its receptor GHSR-la can be expressed in the heart and have a high affinity with the binding site on the heart [17, 18]. This suggested that the cardiovascular protective effect of Ghrelin exists independently of growth hormone, but its specific cellular and molecular mechanisms have not been fully clarified. In this study, the protective effect of Ghrelin on Dox induced myocardial injury and HF in rats was investigated to explore the protective effect and possible mechanism of Ghrelin in drug-induced cardiomyopathy, filling the gap of the effect of Ghrelin on heart failure.

Materials and methods

Reagents and equipment

The ultrasound machine and ultrasound probe for cardiac examination of animals were Visual Sonics, Canada, Dox hydrochloride was purchased from Pfizer Co. CM1900 frozen microtome was Leica, Germany. Enzyme-labeled apparatus (Multiskan SkyHigh) was Thermo, Finland. The TNF-α, IL-10 and iNOS ELISA kits purchased from Sigma Corporation, USA. The antibody was purchased from Abcam in the UK (TNF-α: ab6671; IL-10: ab34843; iNOS: ab15323;Actin: ab197345).

Animal grouping and model preparation

45 healthy adult 10-week-old male Sprague-Dawley rats, body weight 250–300 g. The rats were fed in separate cages, with 5 rats per cage. The rats were fed with ordinary feed formula, the room temperature of the animal house was controlled at about 24℃, and the rats were fed under light-dark cycle for 12 h.

45 rats were randomly divided into three groups. Rats in the HF group (n = 15) and Ghrelin group (n = 15) were intraperitoneally injected with Dox solution 2 mg/kg to establish an animal model of HF [19]. Control group (n = 10) rats were injected with an equal volume of 9% sodium chloride injection by the same method. The mental state, activity level, body mass and other general conditions of the rats were observed regularly during the experiment for 8 weeks. The rats were killed by decapitated method. Myocardial tissue was removed, partially treated with liquid nitrogen, and frozen at -80℃. The other part was incubated in paraformaldehyde.

Animal model evaluation

The rats were anesthetised by intraperitoneal injection of ketamine hydrochloride. The dose of rats in the control group was 100 mg/kg, and the dose of rats in the HF and Ghrelin groups was 70 mg/kg. After anesthesia, the hair was removed with 8% Na₂S, and the fixed animals were examined by ascending echocardiography. All experimental data are completed by the same researcher to ensure the uniformity of research results. The study was approved by the Jiangxi Provincial People’s Hospital, the First Affiliated Hospital of Nanchang Medical College.

Echocardiographic examination

After modeling, the Ghrelin group was given 50 nmol/kg Ghrelin, the HF group and the control group were given equal volume normal saline, respectively, for a total of 6 weeks. They were given intraperitoneal injection at 8:00 am and 4:30 PM, respectively. After dry preconditioning, echocardiography was performed again in Ghrelin group, HF group and control group. Left ventricular end-diastolic diameter (LVDD), left ventricular end-systolic diameter (LVSD), left ventricular ejection fraction (LVEF), and left ventricular short axis shortening rate (LVFS) were measured and stored within 3 cardiac cycles.

Hemodynamic determination

The MPA-V multi-channel biological analysis system was used for detection. The rats were anaesthetized by intraperitoneal injection of ketamine hydrochloride and fixed in supine position. The rats’ neck fur was removed with 8% Na₂S and disinfected, and the two common carotid arteries were exposed by making an incision in the middle of the neck. The right common carotid artery was isolated, the distal end was ligated and the proximal end was temporarily clamped off, then the polystyrene catheter filled with heparin saline was inserted retrogradely into the left ventricle. The pressure-volume curves were observed and the following hemodynamic parameters are recorded, including: left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), maximum rate of increase in left ventricular pressure (+ dP/dtmax) and maximum rate of decrease in left ventricular pressure (− dP/dtmax).

Myocardial HE staining

After the myocardial tissue was fixed with 4% paraformaldehyde for 72 h, conventional paraffin embedding, Sect. (5 μm thick), xylene dewaxing, and gradient alcohol hydration were performed. HE staining was performed according to kit instructions, and the staining degree was observed under microscope. After dyeing, gradient alcohol was dehydrated, xylene transparent, neutral gum was sealed, observed and photographed under an optical microscope.

Serum levels of TNF-α, IL-10 and iNOS

4 mL of blood was taken from rats at the time of execution, centrifuged at 1500 r/min for 10 min at 4℃, and the supernatant was extracted and frozen at -80℃ in a freezer. The serum TNF-α, IL-10 and iNOS levels were measured by ELISA.

Detection of protein expression levels

The total protein was extracted from the myocardial tissue. Protein concentration was measured and quantified by BCA method to prepare protein samples. The protein was separated by 10% SDS-polyacrylamide gel electrophoresis. The protein was transferred to PVDF membrane by semi-dry method, and the protein was placed in 5% skim milk powder and sealed at room temperature for 2 h, and the primary and secondary antibodies of the egg white were added to incubate for 2 h. Actin was used as the internal reference protein, and the relative expression of each protein was calculated by absorbance analysis after color development with the chromogenic solution.

Immunohistochemical staining

The paraffin sections of rat heart tissue were prepared, soaked in xylene for 10 min twice. The sections were deparaffinised in 100%, 95%, 85% and 75% ethanol to water, and the antigen was thermally repaired. 3% hydrogen peroxide was used for incubation for 10 min, and the primary antibodies of TNF-a, 1 L-10 and iNOS (1:200) were added dropwise at 4℃ overnight, and the HRP-labelled IgG (1:200) was added dropwise at 4℃. The sections were incubated at 37℃ for 30 min, and then the DAB work-up was added dropwise to develop the colors, and the sections were stained with hematoxylin, then dehydrated with ethanol (60-100%) and blocked. After staining with hematoxylin, gradient dehydration with ethanol (60-100%) was performed, and the slices were sealed. The samples were observed and photographed under a microscope. The samples were analyzed by Image Pro Plus 6.0 image analysis software to determine their mean optical density (MOD).

Statistical methods

SPSS 27.0 analysis software was applied. The measurement data conform to normal distribution and are expressed as ($\:\stackrel{-}{x}$± SD). T test was used to compare the mean of two samples, and one-way analysis of variance was used to compare the mean of multiple groups. LSD post hoc test was performed on the data. P < 0.05 indicating that the difference was statistically significant.

Results

Changes of echocardiography in rats after Ghrelin intervention

Figures 1 and 2 showed that all rats survived the modelling period. After 8 weeks of Dox modelling, the echocardiograms were performed before Ghrelin intervention. Compared with the control group, the left ventricle of rats in the HF group was apparently enlarged, ventricular wall activity was obviously weakened, LVDD and LVSD were increased (P < 0.05), and LVEF and LVFS were decreased (P < 0.05), indicating that HF was established. After the Ghrelin intervention, compared with the HF group, the LVDD and LVSD in the Ghrelin group was markedly reduced (P < 0.05), and LVEF was significantly increased (P < 0.05), indicating that Ghrelin significantly improved cardiac function in HF rats.

Fig. 1

The echocardiogram in different groups

Fig. 2

Comparison of echocardiographic parameters in different groups. Note: LVDD, Left ventricular end-diastolic diameter; LVSD, Left ventricular end-systolic diameter; LVEF, Left ventricular ejection fraction; LVFS, Left ventricular short axis shortening rate. Compared with HF group, * P < 0.05; Compared with control group, ^#P < 0.05

Results of hemodynamic detection

Figure 3 demonstrated that compared with control group, LVSP, +dP/dtmax, − dP/dtmax decreased obviously in HF group (P < 0.05), LVDP increased (P < 0.05). Compared with HF group, the LVSP, +dP/dtmax and − dP/dtmax of Ghrelin group was remarkedly increased (P < 0.05), LVDP decreased (P < 0.05), indicating that Ghrelin significantly improved the hemodynamic status of rats with HF.

Fig. 3

Comparison of cardiac hemodynamics in different groups. Note: LVSP, Left ventricular systolic pressure; LVDP, Left ventricular diastolic pressure; +dP/dtmax, maximum rate of increase in left ventricular pressure; − dP/dtmax, maximum rate of decrease in left ventricular pressure. Compared with HF group, * P < 0.05; Compared with control group, ^#P < 0.05

Myocardial morphological and histological changes

Figure 4 presented that under the light microscope, in the control group, the cardiomyocytes were arranged neatly, the nuclei were clear, the transverse lines were clear, the cell space was normal, there was no cell degeneration and necrosis, and there was no inflammatory exudation in the interstitial tissue. In the HF group, the cardiomyocytes lost normal morphological structure, the cardiomyocytes were arranged irregularly, and some of them showed degeneration, dissolution and necrosis, the gap between cardiomyocytes widened, and inflammatory cells infiltrated in the interstitium. The myocardial tissue injury in Ghrelin group was significantly less than that in HF group.

Fig. 4

The HE staining image of myocardium (400 ×). Note: The black indicates infiltration of inflammatory cells, the green indicates uneven alignment of cardiomyocytes and widening of gaps

Comparison of serum levels of TNF-α, IL-10 and iNOS

Figures 5, 6 and 7 depicted compared with control group, the serum TNF-α and iNOS levels of HF group rats were prominently increased, IL-10 decreased (P < 0.05). The levels of TNF-α and iNOS in Ghrelin group were notably lower than those in HF group (P < 0.05), the IL-10 level increased markedly (P < 0.05), indicating that Ghrelin significantly improved the inflammatory response in rats with HF.

Fig. 5

Comparison of serum TNF-α, IL-10 and iNOS level in different groups. Note: Compared with HF group, * P < 0.05; Compared with control group, ^#P < 0.05

Fig. 6

The protein expression of TNF-α, IL-10 and iNOS.. Note: Compared with HF group, * P < 0.05; Compared with control group, ^#P < 0.05

Fig. 7

The immunohistochemical results of TNF-α, IL-10 and iNOS (200 ×). Note: Compared with HF group, * P < 0.05; Compared with control group, ^#P < 0.05

Discussion

Dox is an anthraquinone-based antitumor chemotherapeutic agent, which is clinically used in the treatment of various solid tumors and hematological tumors. However, its clinical application is limited by its severe cardiotoxic effects [20]. The pathological features of Dox-induced cardiomyopathy are similar to those of dilated cardiomyopathy, including apoptosis, mitochondrial swelling, disturbances in energy metabolism in cardiomyocytes and accumulation of oxygen free radicals (ROS). However, it was found that the inhibition of ROS aggregation did not ameliorate the toxic effects of Dox on the heart, so it was hypothesized that multiple factors and pathways would be required to achieve a primary effect [21, 22]. Our study indicated that Ghrelin had a certain protective effect on Dox-induced myocardial injury in rats, which could improve the contractile function of the heart, inhibit the necrosis of cardiomyocytes and regulate the expression of circulating inflammatory factors to a certain extent.

Ghrelin is synthesized and secreted mainly from the digestive tract, but its receptors are expressed in various tissues and organs of the body. Ghrelin plays a key role in the regulation of food intake and energy balance [23]. In addition, Ghrelin can act on myocardial tissue in both healthy and cardiac individuals. It has been shown that plasma Ghrelin levels are obviously higher in patients with CHF than in healthy individuals, and the Ghrelin levels decrease after heart transplantation. Meanwhile, Ghrelin may improve cardiac function and cardiac output in CHF patients by promoting the release of growth hormone from the pituitary gland and directly stimulating the GHS-R [24, 25]. In a study of healthy adults, it was confirmed that the administration of double human physiological dose of Ghrelin can apparently improve cardiac index (CI) and cardiac stroke volume index (SVI), and reduce peripheral vascular resistance. The previous studies in animal models also found that the administration of exogenous Ghrelin in rats with dilated cardiomyopathy can reduce left ventricular end-diastolic volume and increase LVEF, and improve the 30-day survival rate of rats [26]. Our study also found that Ghrelin partially improved cardiac hemodynamic parameters in rats with HF, which was consistent with the results of Çetin’s study [27]. On the other hand, Ghrelin inhibited sympathetic nerve activity and activated parasympathetic nerve activity in healthy subjects, which may partly explain why Ghrelin improved survival in rats with dilated cardiomyopathy [28]. Our study also demonstrated that exogenous Ghrelin administration in rats with drug-toxic cardiomyopathy histologically ameliorated cardiomyocyte injury, reduced LVDD and LVSD, and increased LVEF.

CHF is a chronic inflammatory state manifested by elevated plasma levels of the inflammatory factor TNF-α and activation of the renin-angiotensin system [29]. Ghrelin inhibits the excessive production of oxygenation stress products by vascular endothelial cells and activation of the enzyme NADPH oxidase, as well as the production of pro-inflammatory cytokines by inhibiting the activation of NF - κB [30]. In a study of heart-lung transplant recipients, Ghrelin was found to be cardioprotective by decreasing circulating levels of TNF-α, IL-6 and myeloperoxidase activation. These inflammatory cytokines of TNF-α and IL-6 can activate the expression of iNOS in myocardial tissue [31, 32]. As an anti-inflammatory cytokine, IL-10 can inhibit the inflammatory cytokines produced in the process of congestive HF, which can directly combat the damage of myocardium mediated by inflammatory cytokines and has a protective effect on the heart [33]. In our study, we found that Ghrelin increased the expression of IL-10, decreased the expression of TNF-α and iNOS, suggesting that Ghrelin may play a role in improving cardiac function in rats with HF, which was consistent with the results of Erhardsson’s study [25]. Recently, Ghrelin was found to inhibit the development of ischemia-reperfusion-induced hepatic fibrosis by regulating the matrix metalloproteinase tissue inhibitor pathway and inducing the degradation of hepatic extracellular matrix (ECM), and it is hypothesized that Ghrelin may have a similar beneficial effect on the heart, which has yet to be confirmed in further studies. Previous studies have suggested that Ghrelin attenuated Dox-induced cardiotoxicity in mice by affecting the AMPK and p38-MAPK signaling pathways [34, 35]. Therefore, our study suggested that Ghrelin played a protective role in Dox induced myocardial injury and HF through various mechanisms. Our study has some limitations. For example, the molecular mechanism is not explored deeply enough. In future studies, single-cell sequencing of Ghrelin and HF groups could be performed to better understand how different heart cells respond to Ghrelin treatment.

Conclusion

Ghrelin protects Dox from cardiac muscle damage and improves cardiac function in rats. This provides a new way to study the beneficial effects of Ghrelin on the prevention and treatment of cardiomyopathy caused by drug toxicity.