Short-Term Hesperidin Pretreatment Attenuates Rat Myocardial Ischemia/Reperfusion Injury by Inhibiting High Mobility Group Box 1 Protein Expression via the PI3K/Akt Pathway

Acute myocardial infarction (AMI) is a global health problem. In China, the number of patients with MI was 2.5 million in 2012, and this number is expected to increase over the next ten years [1]. For patients with AMI, early and successful myocardial reperfusion, such as thrombolytic therapy or primary percutaneous coronary intervention, is the most effective strategy for reducing infarct size and improving clinical outcome. However, the process of restoring blood flow can induce local myocardial apoptosis, inflammation and oxidative stress, which paradoxically reduce the beneficial effects of myocardial reperfusion, resulting in irreversible myocardial cell damage and increased infarct size. This phenomenon is termed myocardial ischemia/reperfusion (I/R) injury [2]. Although patients with AMI may receive optimal reperfusion therapy, the mortality is still 10% because of myocardial I/R injury [3]. Therefore, the prevention and treatment of myocardial I/R injury has become one of the most important issues in AMI research.

Hesperidin, a flavanone glycoside abundant in citrus fruits, has been reported to have multiple bioactivities such as anti-inflammatory [4], anti-oxidative [5], radioprotective [6] and anticancer effects [7]. Recently, Gandhi et al. [8] showed that a continuous 15-day hesperidin pretreatment could protect against myocardial I/R injury in rats by suppressing inflammation and oxidative stress. Meanwhile, Rong et al. [9] demonstrated that a 3-day hesperidin pretreatment attenuated hypoxic-ischemic brain injury in neonatal rats. Hence, we speculated that a 3-day hesperidin pretreatment may also protect against myocardial I/R injury. However, the mechanism underlying hesperidin protection against I/R injury remains elusive.

High mobility group box 1 protein (HMGB1) is a ubiquitous, non-chromosomal nuclear protein that has been identified as a novel pro-inflammatory mediator in several cardiovascular diseases, including myocardial I/R injury [10,11,12,13]. Previous studies have demonstrated that HMGB1 may contribute to the pathophysiological process of myocardial I/R injury [12,14]. Furthermore, our prior studies confirmed that the downregulation of HMGB1 by several drugs, such as minocycline and ethyl pyruvate, can reduce myocardial I/R injury in rats [15,16]. In addition, Kawaguchi et al. [17] demonstrated that hesperidin suppressed HMGB1 expression in an endotoxin shock model. Thus, we speculate that the cardioprotective effects of hesperidin may be related to its inhibitory effects on HMGB1 expression.

Phosphoinositide 3-kinase (PI3K) plays an important role in the control of cell growth, proliferation, survival and migration [18]. Activation of Akt, which is downstream of PI3K, may ameliorate I/R injury [19]. The PI3K/Akt pathway has been identified as a key component of the protective mechanism of ischemic preconditioning that inhibits myocardial I/R injury [20,21]. Furthermore, Wolfrum et al. [22] showed that LY294002, a PI3K inhibitor, could abolish the cardioprotective effects of simvastatin on I/R injury. Wang et al. [23] reported that the PI3K pathway may be associated with the inhibition of HMGB1 expression in a rat model of myocardial I/R injury. In addition, Nones et al. [24] demonstrated that the PI3K pathway may mediate the protective effects of hesperidin in neural crest cell survival. Hence, we hypothesize that hesperidin-induced HMGB1 expression inhibition depends on the PI3K/Akt pathway.

In this study, we investigated whether a short-term hesperidin pretreatment could alleviate myocardial I/R injury and whether the cardioprotective effects of hesperidin involve HMGB1 inhibition and the PI3K/Akt pathway.

Eighty male Sprague-Dawley rats (200-250g) were obtained from the animal experiment center of Wuhan University, China. All experimental protocols conformed to the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Wuhan University (Approval Number: 2015-0536).

After being anesthetized with an intraperitioneal (i.p.) injection of sodium pentobarbital (45 mg/kg, Sigma, St. Louis, USA), the rats were ventilated artificially with a volume-controlled rodent respirator and monitored with an electrocardiogram using a computer-based EP system (LEAD2000B, Jinjiang Ltd., Chengdu, China). Then, the myocardial I/R rat model was established with a 30-min left anterior descending coronary artery (LAD) occlusion and a 4-h reperfusion, as previously described [23]. At the end of the reperfusion period, the animals were euthanized, and their blood and hearts were collected for various biochemical analyses.

Hesperidin (HPLC>98%, Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) was dissolved in 0.5% sodium carboxymethyl-cellulose (CMC-Na) and administered orally to rats by gavage for 3 days [9]. To determinate the optimal dose for the 3-day hesperidin administration, we designed a fixed-dose study in which thirty rats were randomly assigned to one of the following five groups (n=6 per group): (i) Sham-operated (SO): 0.5% CMC-Na; (ii) I/R: 0.5% CMC-Na; (iii) Hesperidin (100 mg/kg) + I/R (Hesp100-I/R); (iv) Hesperidin (200 mg/kg) + I/R (Hesp200-I/R); or (v) Hesperidin (400 mg/kg) + I/R (Hesp400-I/R): After 3 days, rats in the SO group were subjected to surgical manipulation without ligature of the LAD, but rats in the other four groups were subjected to LAD occlusion for 30 min followed by reperfusion for 4 h.

To further clarify the protective effects of hesperidin on myocardial I/R injury, we conducted another experiment in which 48 rats were randomly assigned to one of the four following groups (n=12/group): (i) SO; (ii) I/R; (iii) Hesp200-I/R; or (iv) LY294002 + Hesperidin (200 mg/kg) + I/R (LY-Hesp200-I/R): After anesthesia, rats in LY-Hesp200-I/R group were treated with LY294002 (a specific PI3K inhibitor, 0.3 mg/kg, Sigma-Aldrich, St. Louis, USA) [23] via the caudal vein 30 min before LAD occlusion, and other three groups were subjected to 30 min occlusion and 4 h reperfusion except for the SO group.

Myocardial tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) to observe myocardial damage. To quantify the histological cardiac damage, 6 fields at ×200 magnification were randomly selected from two sections in each group and scored in a blinded manner by two individuals, as previously described [25]. The scores were as follows: 0, no damage; 1 (mild), interstitial edema and focal necrosis; 2 (moderate), diffuse myocardial cell swelling and focal necrosis; 3 (severe), necrosis with the presence of contraction bands and inflammatory cell infiltrate; and 4 (highly severe), widespread necrosis with the presence of contraction bands, inflammatory cell infiltrate, and hemorrhage.

To assess creatine kinase (CK) and lactate dehydrogenase (LDH) levels, blood samples were collected and centrifuged at 3000 rpm for 15 min. Supernatants were stored at -20°C for later analyses. Standard techniques were performed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. LDH and CK were used as indices of myocardial injury.

Infarct size was determined using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, USA) staining as previously described [8,26]. In brief, after reperfusion, the LAD was occluded again and 2 ml of 1% Evans blue dye (Sigma-Aldrich, St. Louis, USA) was injected via the femoral vein. Each frozen heart (-80°C, 15 min) was then cut (approximately 2 mm) from the apex to the base. The slices were incubated in 1% TTC for 20 min at 37°C and then fixed in 4% paraformaldehyde. The infarct area (white) and the risk area (red) from each section were measured using image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Silver Spring, USA); six rats in each group were used for myocardial infarct size measurement. Infarct size was expressed as the following percentage: infarct area / (risk + infarct) area.

Pulverized, frozen ischemic areas of the left ventricle were washed in ice-cold 0.9% NaCl (w/v), blotted on absorbent paper, and weighed. Each sample was then minced in ice-cold 0.9% NaCl and homogenized at a ratio of 1:10 (w/v). After the cardiac muscle samples were centrifuged at 5000g for 10 min at 4°C, enzyme-linked immunosorbent assays (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used according to the manufacturer's instructions to measure the levels of TNF-α and IL-6 in the supernatant. The TNF-α and IL-6 levels were used as indices of the inflammatory response.

Myocardial SOD activity and MDA content were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. These markers were used as indices of oxygen free radical and lipid superoxide levels in the myocardium, respectively.

Pulverized, frozen ischemic areas of the left ventricle samples were analyzed by western blot analysis, as previously described [27]. In brief, equal amounts of protein were electrophoresed in a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% non-fat dry milk for 2 h, the membrane was subsequently incubated with primary antibodies at 4°C overnight. Then, the membrane was washed and incubated with secondary antibodies (Boster, Wuhan, China) at 37°C for 2 h. The protein bands were visualized using an enhanced chemiluminescence system, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Boster, Wuhan, China) was used as an internal control. The following primary antibodies were used for western blot analysis: anti-Bax (1:1000), anti-Bcl-2 (1:1000), anti-t-Akt (1:1000), anti-p-Akt (Ser473, 1:2000) (Cell Signaling Technology, Danvers, USA), and anti-HMGB1 (1:200, Boster, Wuhan, China). Five hearts in each group were used for the western blot analysis.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to determine the extent of apoptosis in the myocardium using a commercial kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer's instructions. Only the nuclei of the apoptotic cells were stained brown. Normal nuclei were stained blue. To determine the extent of myocardial apoptosis, 6 fields at ×200 magnification were randomly selected from two sections in each group, and the AI was calculated using Image-Pro Plus 6.0. Apoptosis index (AI) was defined as the following percentage: number of apoptotic cells / total number of cells counted.

Quantum dot-tagging fluorescence methods were used to observe HMGB1 expression in the myocardial tissue. Paraffin sections were incubated with the anti-HMGBl antibody (1:200) at 37°C for 2 h and washed three times with TBS-T. Then, the sections were incubated with a biotin-labeled secondary antibody (1:400, Wuhan Jiayuan Quantum Dots Co. Ltd., Wuhan, China) at 37°C for 30 min. After being washed three times in TBS-T and incubated with quantum dot-conjugated streptavidin (1:150, Wuhan Jiayuan Quantum Dots Co. Ltd., Wuhan, China) at 37°C for 30 min, the sections were visualized using an Olympus BX51 fluorescence microscope (excitation 488 nM, emission 605 nM). To quantify HMGB1 fluorescence density, 6 fields at ×200 and ×400 magnifications were randomly selected from two sections in each group and the mean fluorescence density of HMGB1 in each field was analyzed using Image-Pro Plus 6.0.

SPSS 17.0 was used for data analysis. All data are expressed as the mean ± SD. A one-way analysis of variance or a Welch test was used for comparisons among groups and the Student-Newman-Keuls or Dunnett' s T3 test was used for post hoc multiple comparisons. P values < 0.05 were considered statistically significant.

To determine the optimal dose of the 3-day hesperidin pretreatment, we evaluated histopathological changes using H&E staining and detected biomarkers of myocardial damage (CK and LDH) in serum following treatment with different doses of hesperidin. As shown in Fig. 1A, the myocardial structure in the SO group exhibited a regular arrangement, normal cardiac muscle fibers and no necrosis. Compared with the SO group, the I/R group demonstrated ruptured cardiac muscle fibers, necrosis with inflammatory cell infiltration, edema and hemorrhage. The myocardial damage in the hesperidin groups was less than that in the I/R group. In the hesperidin groups, we observed less myocardial fiber disruption, necrosis and inflammatory cell infiltration. We also examined the damage score and serum CK and LDH levels to assess myocardial injury (Fig. 1B, 1C and 1D). Compared with the SO group, the I/R group exhibited a markedly increased damage score as well as serum CK and LDH levels (p < 0.05). All groups pretreated with hesperidin (100, 200 or 400 mg/kg) showed lower damage scores as well as decreased serum CK and LDH levels, suggesting that a short-term hesperidin pretreatment could attenuate myocardial I/R injury. However, only groups pretreated with 200 or 400 mg/kg of hesperidin showed significant differences compared with the I/R group. As no significant differences were observed between the groups receiving 200 and 400 mg/kg of hesperidin, the 200 mg dose was used for subsequent experiments.

To further confirm the protective effects of hesperidin against myocardial I/R injury, the myocardial infarction size and the serum levels of CK and LDH were measured using TTC staining and an automatic biochemistry analyzer. Compared with the I/R group, the hesperidin group exhibited a significant decrease in infarct size; however, the addition of LY294002 abolished the hesperidin-mediated decrease in infarct size (p < 0.05, Fig. 2A and 2B). Additionally, serum levels of CK and LDH were significantly higher in the I/R group than in the SO group (p < 0.05, Fig. 2C and 2D). Oral administration of hesperidin significantly lowered the serum levels of CK and LDH, which were increased in the I/R group. These results suggested that hesperidin could attenuate myocardial I/R injury. However, administration of LY294002 partially reversed the cardioprotective effects of hesperidin (p < 0.05).

To investigate the effects of hesperidin on myocardial apoptosis in I/R injury, the AI and the expression of apoptosis-related proteins (the Bcl-2/Bax ratio, Bcl-2 and Bax) in the myocardium were measured using TUNEL assays and western blot analysis, respectively. As shown in Fig. 3, compared with the SO group, the AI and the level of Bax expression were markedly increased in the I/R group; in addition, both Bcl-2 expression and the Bcl-2/Bax ratio were significantly lower in the I/R group than in the SO group (p < 0.05). The hesperidin pretreatment group exhibited striking decreases in the AI and Bax expression and increases in the Bcl-2/Bax ratio and Bcl-2 expression compared with those of the I/R group (p < 0.05), indicating that hesperidin could reduce myocardial apoptosis. Conversely, administration of LY294002 partially reversed the anti-apoptotic effects of hesperidin (p < 0.05).

To evaluate the effect of hesperidin on the inflammatory response in myocardial I/R injury, the levels of TNF-α and IL-6 in the myocardium were measured. Compared with the SO group, the cardiac levels of TNF-α and IL-6 were substantially increased in the I/R group. The levels of TNF-α and IL-6 were significantly lower in the hesperidin group than in the I/R group (p < 0.05), demonstrating the anti-inflammatory effects of hesperidin. However, LY294002 completely abolished these effects (p < 0.05, Fig. 4).

To assess the effects of hesperidin on oxidative stress in myocardial I/R injury, myocardial SOD activity and MDA content were measured. Compared with the SO group, SOD activity was significantly decreased and MDA content was significantly increased in the I/R group (p < 0.05). Hesperidin treatment markedly inhibited the decrease in SOD activity and the increase in MDA content (p < 0.05), illustrating the anti-oxidative effects of hesperidin; however, LY294002 reversed these effects (p < 0.05, Fig. 5).

To investigate the possible mechanism underlying the effects of hesperidin during myocardial I/R injury, the levels of HMGB1, p-Akt and t-Akt expression in the myocardium were measured using quantum dot-tagging fluorescence technology and western blot analysis. As shown in Fig. 6A, the HMGB1 (red) signals detected in the I/R group were much higher than those in the SO group. Hesperidin substantially reduced the I/R-induced HMGB1 signal accumulation, but its effect was abrogated by LY294002. Meanwhile, compared with the SO group, the mean densitometry values and HMGB1 expression were significantly increased and p-Akt expression was markedly decreased in the I/R group (p < 0.05). Hesperidin markedly suppressed the increased mean densitometry values and HMGB1 expression as well as the decreased p-Akt expression induced by I/R (p < 0.05, Fig. 6B, 6C and 6D), indicating that hesperidin could activate the PI3K/Akt pathway and inhibit HMGB1 expression. However, administration of LY294002 dramatically reversed these effects (p < 0.05). In addition, no significant difference in t-Akt expression was observed among the four groups (p > 0.05, Fig. 6E).

A growing number of epidemiological studies have consistently shown that diets rich in herbs, fruits and spices can reduce the risk of cardiovascular diseases [28,29]. Hesperidin is a major flavonoid found in citrus fruit, and it is highly abundant in orange juice. Morand et al. [30] reported that hesperidin (294 mg in 500 ml of orange juice) contributed to the cardiovascular protective effects of orange juice in healthy, middle-aged, moderately overweight men by decreasing diastolic blood pressure and increasing endothelium-dependent microvascular reactivity. Using the dose conversion between humans and rats (human:rat, 1:6.17, by body surface area comparisons) [31], hesperidin dose of 200 mg/kg/day in rats is equal to 1944 mg/day for a 60-kg man, which is almost 8-fold greater than 294 mg/day. Therefore, the dose of hesperidin used in the present study is far greater than a nutritional dose.

Gandhi et al. [8] reported that a 15-day hesperidin pretreatment at 100 mg/kg/day could protect against myocardial I/R injury and limit infarct size. Agrawal et al. [32] demonstrated that a 2-week hesperidin pretreatment at 100 mg/kg/day attenuated myocardial I/R injury and reduced infarct size in diabetic rats. These studies suggested that relatively long-term hesperidin administrations could attenuate myocardial I/R injury. Rong et al. [9] reported that a 3-day hesperidin pretreatment alleviated hypoxic-ischemic brain injury in neonatal rats. Selvaraj et al. [5] showed that a 7-day hesperidin pretreatment could inhibit lipid peroxidation and increase antioxidant status at 200 and 400 mg/kg/day in an isoproterenol-induced model of myocardial infarction. Consistent with previous studies, we found that a 3-day hesperidin pretreatment at 200 or 400 mg/kg/day significantly promoted histopathological changes and decreased both myocardial damage as well as infarct size caused by I/R injury. These results suggest that short-term hesperidin conditioning could protect against myocardial I/R injury.

Myocardial I/R injury is a complex pathophysiological process in which myocardial apoptosis, the inflammatory responses and oxidative stress all play important roles [2,33,34]. Apoptosis, a form of programmed cell death, is the early and predominant form of cell death in myocardial I/R injury, which leads to the loss of myocardium and functional abnormalities [34]. Apoptosis is regulated by various signals, including those of the anti-and pro-apoptosis proteins of the Bcl-2 family. Bcl-2 (an anti-apoptosis protein) and Bax (a pro-apoptosis protein) both belong to the Bcl-2 family. The ratio of Bcl-2/Bax plays crucial roles in determining the extent of apoptosis in I/R heart [35]. Agrawal et al. [36] reported that hesperidin inhibited myocardial apoptosis by upregulating the ratio of Bcl-2/Bax in an isoproterenol-induced model of myocardial infarction in diabetic rats. Consistent with these findings, we found that hesperidin inhibited myocardial apoptosis and reduced myocardial damage during myocardial I/R injury, suggesting that hesperidin could inhibit myocardial apoptosis and further attenuate myocardial I/R injury.

Meanwhile, the inflammatory response and oxidative stress have also been identified as key processes involved in myocardial I/R injury [2]. The inflammatory response, including inflammatory cell infiltration and pro-inflammatory cytokine production, contributes to myocardial damage after I/R, whereas anti-inflammatory treatments reduce myocardial I/R injury [37]. Free radicals, the main products of oxidative stress, are actively involved in myocardial I/R injury and can be assessed by MDA, a marker of oxidative stress, and SOD, an antioxidant enzyme [38]. The anti-inflammatory effects of hesperidin in a mouse model of allergic asthma have been well documented [4]. Sevajar et al. [5] reported the powerful antioxidant and anti-lipid peroxidative properties of hesperidin on isoproterenol-induced oxidative stress in myocardial infarction. Similarly, our data showed that hesperidin inhibited the infiltration of inflammatory cells and decreased the levels of pro-inflammatory markers, such as TNF-α and IL-6. In addition, hesperidin suppressed oxidative stress and deceased myocardial infarct size. These results suggest that the cardioprotective effects of hesperidin are partly due to its anti-inflammatory and anti-oxidative effects.

HMGB1, a novel pro-inflammatory cytokine, plays an important role in myocardial I/R injury [14]. A series of studies have demonstrated that HMGB1 can upregulate the expression of TNF-α and IL-6, while the same cytokines promote HMGB1 released in a positive-feedback manner, exacerbating the inflammatory response [11,39,40,41,42]. Hu et al. [15] reported that HMGB1 can promote the apoptosis of myocytes in a dose-dependent manner. Additionally, Zhang et al. [43] reported that high HMGB1 expression level is associated with oxidative damage in severe acute pancreatitis. In the present study, increased HMGB1 expression was detected using both quantum dots-tagging fluorescence methods and western blot analysis in the I/R group, thus suggesting that high levels of HMGB1 may contribute to cardiomyocyte apoptosis, the inflammatory response and oxidative stress that occur during myocardial I/R injury. We also found that hesperidin significantly decreased HMGB1 expression and alleviated I/R injury. Kawaguchi et al. [17] reported that hesperidin can suppress the expression of HMGB1 in an endotoxin shock model. Our findings suggest that hesperidin may attenuate I/R injury by inhibiting HMGB1 expression.

The PI3K/Akt pathway, a major regulator of cell growth and survival, is activated in response to various physiological and stress stimuli such as growth factors [44], hypoxia [45] and oxidative stress [46]. It has been reported that the PI3K/Akt pathway was activated in the first minutes of reperfusion following the index ischemia [47] and was involved in the cardioprotection of simvastatin pretreatment [22] and postconditioning [48] against myocardial I/R injury. Zhao also et al. [49] documented that SO₂ preconditioning protected against myocardial I/R injury through PI3K/Akt pathway activation. These studies demonstrated that PI3K/Akt pathway activation could ameliorate I/R injury in the heart. Notably, Rong et al. [9] demonstrated that hesperidin can activate the PI3K/Akt pathway to protect against neonatal hypoxic-ischemic brain injury. Furthermore, Wang et al. [23] reported that PI3K pathway activation inhibited HMGB1 expression and attenuated myocardial I/R injury. In the present study, we found that hesperidin activated the PI3K/Akt pathway and decreased HMGB1 expression, while the LY294002 treatment reversed these effects of hesperidin. Furthermore, LY294002 also partially abrogated the cardioprotective, anti-inflammatory anti-apoptotic and anti-oxidative effects of hesperidin during I/R. These results indicate that hesperidin can protect against myocardial I/R injury by inhibiting HMGB1 via PI3K/Akt pathway activation.

In the present study, we demonstrated that a short-term pretreatment with hesperidin attenuated myocardial I/R injury by reducing myocardial apoptosis, the inflammatory response and oxidative stress. We also showed that these protective effects of hesperidin were predominantly due to HMGB1 expression inhibition via PI3K/Akt pathway activation.

The main alterations in the PI3K/Akt pathway [47] and in myocardial oxidative stress [50] have been reported to occur during the first few minutes of reperfusion following ischemia. In the present study, we only detected the levels of p-Akt, SOD and MDA in the myocardium at a single time (after 4 h of reperfusion); therefore, we cannot comment on the kinetics of Akt phosphorylation or oxidative stress during reperfusion.

None.

X. Li and X. Hu contributed equally to this work.