Cardioprotective effect of isorhamnetin against myocardial ischemia reperfusion (I/R) injury in isolated rat heart through attenuation of apoptosis

Abstract In this study, we investigated the effects of isorhamnetin on myocardial ischaemia reperfusion (I/R) injury in Langendorff‐perfused rat hearts. Isorhamnetin treatment (5, 10 and 20 μg/mL) significantly alleviated cardiac morphological injury, reduced myocardial infarct size, decreased the levels of marker enzymes (LDH and CK) and improved the haemodynamic parameters, reflected by the elevated levels of the left ventricular developed pressure (LVDP), coronary flow (CF) and the maximum up/down velocity of left ventricular pressure (+dp/dtmax). Moreover, isorhamnetin reperfusion inhibited apoptosis of cardiomyocytes in the rats subjected to cardiac I/R in a dose‐dependent manner concomitant with decreased protein expression of Bax and cleaved‐caspase‐3, as well as increased protein expression of Bcl‐2. In addition, I/R‐induced oxidative stress was manifestly mitigated by isorhamnetin treatment, as showed by the decreased malondialdehyde (MDA) level and increased antioxidant enzymes activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH‐Px). These results indicated that isorhamnetin exerts a protective effect against I/R‐induced myocardial injury through the attenuation of apoptosis and oxidative stress.

injury treatment will definitely improve therapy outcome for patient with AMI.
Although the phenomenon of myocardial I/R injury existed for many decades, scientists never stop exploring the pathophysiology mechanisms behind myocardial I/R injury and developing novel drugs to treat or alleviate it. 5 Myocardial I/R injury is an intricate pathophysiological process that involves multifactorial mechanisms, such as increased inflammatory response, intracellular calcium overload, oxidative stress, myocardial necrosis and apoptosis. [6][7][8] Among these contributors, a substantial body of evidence has suggested that apoptosis plays a critical role in myocardial I/R injury, which impairs cardiac function. 9 Apoptosis is a continually occurring programmed cell death that maintains tissue homoeostasis, and the importance of apoptosis contributed to myocardial I/R injury has been well documented by many research groups. 10,11 Long periods of myocardial ischaemia result in an increase of necrosis, but paradoxically, reperfusion leads to an enhancement in apoptosis. 12 Myocardial apoptosis accelerates the development of necrosis which might determine the degree of myocardial injury. More importantly, oxidative stress during I/R-induced myocardial damage is closely associated with cell apoptosis 13 and usually occurs when the generation of ROS prevails over antioxidant system function in the organism. 14,15 Excessive ROS can cause damage to the structure of myocardial mitochondria and then trigger mitochondrial-mediated cell apoptosis. 16,17 It is evident that suppressing myocardial oxidative stress and cell apoptosis may attenuate I/R-induced cardiac injury 18 and can be considered as a pivotal target for therapeutic intervention for this diseases. Therefore, seeking antioxidant and anti-apoptotic agent from plants is necessary for the treatment of myocardial I/R injury.
Natural flavonoids, which are natural polyphenols universally found in herbal drugs and foods, have attracted considerable attention for their biological and physiological importance. Interestingly, increasing epidemiological studies demonstrated an inverse correlation between flavonoids intake and heart disorders incidence. [19][20][21] Therefore, flavonoids have the most potential to be developed as safe therapeutics for cardiovascular disease. Isorhamnetin, a naturally distributed flavonoid in the sea buckthorn, Oenanthe javanica and Ginkgo biloba L., 22 has been confirmed that it can attenuate atherosclerosis, protect cardiomyocytes against oxidation 23,24 or anoxia/reoxygenation, 25 ameliorate cardiac hypertrophy 26 and rescue rat ventricular myocytes from I/R injury in vitro via repressing apoptosis. 27 These findings highlight the importance of antioxidant and anti-apoptotic properties of isorhamnetin on the therapy of cardiovascular diseases. However, whether isorhamnetin has the protective effect against myocardial I/R injury via the inhibition of oxidative stress and apoptosis have yet to be fully uncovered.
With this in mind, in this study, we intend to examine the effect of isorhamnetin treatment on myocardial I/R injury in isolated rat heart and investigated the underlying mechanism underpinning this effect.

| Animals and ethics statement
Adult male Sprague-Dawley (SD) rats weighing 250 ± 20 g were purchased from Animal Experimental Center of Central South University and were housed under laboratory pathogen-free conditions with a 12:12-hour light-dark cycle at 25 ± 2°C and 50% ± 15% humidity.
The animals had free access to a regular pellet diet and tap water. All animal experimental procedures were conducted in adherence with the guidelines approved by the Animal Care and Use Committee of the Second Xiangya Hospital, Central South University in Changsha, China.

| Induction of I/R and experimental design
The establishment of I/R model was performed as previously described by Jiang et al, with some modifications. 28 All rats were allowed to acclimatize the experimental environment for one week and then anaesthetized with pentobarbital sodium (100 mg/kg) via intraperitoneal injection (i.p.), followed by i.p. injection of 250 U/ kg heparin to avoid coagulation. After that, the hearts were quickly

| Measurement of myocardial infarct size and cardiac marker enzymes
The myocardial infarct size was measured by TTC staining method as previously described. 29 After 45 minutes of reperfusion, the hearts from three mice in each group was rapidly excised and frozen at −70°C until being sliced transversally into 2 mm thick axial sections.
These sections were soaked in 1% TTC (pH 7.4) away from light for 20 minutes at 37°C and then subsequently fixed in 4% paraformaldehyde for 24 hours. TTC-positive staining area in deep red colour represents the viable myocardial tissue, while the pale white section means nonviable myocardium. The percentage of infarct area size at risk was assessed digitally using Image J software as previously described. 30 After the experiment, the levels of LDH and CK in the perfusate from the other left mice (n = 7) in each group were spectrophotometrically measured to evaluate the degree of cardiac cell death using respective cytotoxicity detection commercial kit in accordance with the manufacturer's protocols.

| Histopathology analysis
For histological analysis, a portion cardiac tissues from each group (n = 7) were fixed in 4% paraformaldehyde solution at 4°C overnight for embedding in paraffin. The paraffin-embedded sections (5 μm) were stained with haematoxylin and eosin (H&E), followed by histopathological examination under a light microscope at 400× magnification. 31

| Measurement of myocardial apoptosis
At the end of experiment, a portion of myocardial tissue from various groups (n = 7) was fixed with 4% formalin, embedded in paraffin, and then sectioned at 5 μm thickness, as described previously. 31 For apoptosis determination, a terminal deoxynu-cleotidyl transferase dUTP nick end labelling assay (TUNEL) assay was performed using an In Situ Cell Death Detection kit according to the manufacturer's recommendations. After TUNEL staining, the stained specimen was then added with haematoxylin to stain myocardial cell nuclei,

| Assessment of oxidative stress
A portion of heart tissues in different group (n = 7) were lysed and homogenized with the appropriate phosphate buffer using a microcentrifuge tube homogenizer, followed by centrifugation (5000 rpm, 15 minutes) to yield their individual supernatants. The MDA content and the activity of CAT, GSH-Px and SOD were measured in cardiac homogenates supernatants using respective specific assay kits according to the manufacturer's protocol.

| Western blot analysis
The expression levels of Bax, Bcl-2 and cleaved-caspase-3 protein extracted from myocardial tissues were measured using Western F I G U R E 1 Experimental protocol for myocardial ischaemia/reperfusion (I/R) stimulation and isorhamnetin addition in perfused rat heart blot, as documented by Yao et al 32 Briefly, after reperfusion, the same regions of myocardial tissue from each group (n = 7) were crushed and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and then homogenized under 4°C. The resulting samples were kept on ice for 20 minutes, after which the lysate was subject to centrifugation (12,000 g for 10 minutes) to yield the total proteins. The content of this total protein was quantified using the BCA protein assay kit. Each sample with a total of 30 μg proteins was separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes, followed by blocking with 5% non-fat milk at 30°C for 30 minutes. Thereafter, the mem-  The CK levels in coronary effluent were measured by commercial assay kits. The data were expressed as mean ± S.D (n = 7). # P < .05 and ## P < .01 compared with the I/R group; *P < .05 and **P < .01 compared with the sham group

| Statistical analysis
All data are expressed as the mean ± standard deviation (SD) and evaluated by a one-way or two-way mixed analysis of variance

| Isorhamnetin reduced myocardial structure injury, myocardial infarct size and myocardial enzyme activities in isolated rat hearts subjected to I/R
The myocardium histopathological change in different groups was examined by H&E staining (Figure 2A). The myocardial cells from sham operation rats showed well-arranged cell architecture and intact muscle fibres, while I/R injury lead to aggravated myocardium damage, including irregularly arranged muscle fibres, widespread necrosis, cell oedema and the blurred boundaries between cells.
However, treatment with isorhamnetin at 5, 10 and 20 μg/mL significantly reversed I/R-induced pathological changes in isolated rat hearts tissues when compared with I/R group. The arrangement of myocardial fibres was relatively neat and tight, and the necrosis extent of cardiomyocytes was significantly reduced.
In consistent with the myocardial morphological alteration, I/R operation caused a significant increase of infarction size manifested by the more pale white areas as compared with the sham group.
Digital computation of the TTC staining displayed that isorhamnetin treatment (5, 10 and 20 μg/mL) markedly reduced the infarction area of the isolated rat hearts in a dose-dependent manner ( Figure 2B).
After 20 minutes of ischaemia followed by 45 minutes of reperfusion, the coronary effluent samples were collected to measure the release of LDH and CK as degree of myocardial injury ( Figure 2C,D). The serum CK and LDH level in the I/R group was notably higher than those in sham control (P < .01). However, sorhamnetin treatment (5, 10 and 20 μg/mL) resulted in a visible reduction in these two myocardial enzyme activities with relative to those of I/R groups (P < .05 or P < .01).

| Isorhamnetin improved cardiac function in isolated rat hearts subjected to I/R
To verify whether isorhamnetin has a protective effect on IR rats, haemodynamic parameters (LVDP, ±dp/dtmax, HR and CF) were continuously recorded during the experiments to evaluate the cardiac function using a computer-based data acquisition system. As illustrated in Table 1, I/R treatment resulted in a remarkable reduction in the levels of LVDP, ±dp/dtmax and CF in isolated rat heart following reperfusion at 15, 30 and 45 minutes when compared to those in the shaml group (P < .01). There is not any statistical difference for HR between sham and I/R groups (P > .05). However, these haemodynamic function indexes were totally reversed by isorhamnetin treatment at three doses (5, 10 and 20 μg/mL) as comparison with I/R group (P < .05 or P < .01).

| Isorhamnetin attenuated myocardial apoptosis in isolated rat hearts subjected to I/R
Next, the influence of isorhamnetin on myocardial I/R-induced cardiomyocyte apoptosis was confirmed by TUNEL staining. Cells with brown granules in the nucleus were considered as apoptotic cells.
Under optical microscopy, this staining showed the more appearance of TUNEL-positive cells with dark brown cell nuclei in the I/R group than that in the sham group; whereas this increase was changed to the opposite by isorhamnetin in a dose-dependent way ( Figure 3A).

| Isorhamnetin regulated apoptotic protein expression in isolated rat hearts subjected to I/R
To confirm the involvement of isorhamnetin on reducing cardiomyocytes apoptosis, the expression of the apoptotic regulatory protein

| Isorhamnetin attenuated oxidative stress in isolated rat hearts subjected to I/R
Oxidative stress is one of the important factors in myocardial I/R injury. We further opted to measure the level of oxidative stress in isolated rat hearts subjected to I/R injury ( Figure  This result suggested that isorhamnetin plays a cardioprotective role by regulating oxidative stress.

| D ISCUSS I ON
The present study is the first, to the best of our knowledge, to ex- CK and LDH are two classical cytosolic enzymes, serving as biomarkers for the diagnosis of myocardial damage. 35 In the normal physiological state, they constitutively retain in endochylema of cardiomyocytes. When the cell membrane becomes permeable or destroyed during myocardial IR injury, CK and LDH can easily transit through cytoplasmic membrane and released into the blood.
Thus, their activity indirectly represents the extent of myocardial damage. 36 Next, we measured the release of LDH and CK to evaluate the degree of myocardial injury. Compared with sham-operated hearts, LDH and CK levels significantly increased in serum of different groups in response to I/R treatment. This increase was effectively suppressed in isolated rat hearts suffering isorhamnetin treatment in a dose-dependent manner. In line with this result, decreased LDH and CK levels were adopted as the important benchmarks for evaluating the cardioprotective effect of potential drug in many literatures. 30,36 As we know, for ischaemic heart diseases, reperfusion of the ischaemic myocardium may lead to cardiac dysfunction and eventually cardiomyocyte apoptosis. 37 Myocardial I/R injury is a complicated pathophysiological process in which oxidative stress serve crucial roles, thus oxidative stress inhibition is recognized as an important approach to treat I/R-induced cardiac injury. 45,46 Wang et al reported that xanthones from Gentianella acuta alleviate myocardial I/R injury in isolated rat heart via attenuation of oxidative stress. 39 It was also reported that attenuation of oxidative stress contributed to the cytoprotective effect of gypenosides against myocardial I/R injury. 47

| CON CLUS IONS
In conclusion, our findings provided the first evidence that isorhamnetin could alleviate myocardial I/R injury by suppressing apoptosis via attenuating apoptosis and oxidative stress. Therefore, it is suggested that isorhamnetin can be developed as a promising therapeutic agents for the treatment of myocardial I/R cardiovascular diseases. However, Langendorff isolated heart model, as an ex vivo model, has its limitations. For example, the isolated and perfused heart is free of neurohumoral control, although the preparation is simple and reproducible, and enables the study of the heart without intervention by other organ systems. Next, further studies are still needed to investigate the precise mechanism by which isorhamnetin protects the heart against myocardial I/R injury in vivo and confirm whether isorhamnetin can be used in a clinical setting.

CO N FLI C T O F I NTE R E S T S
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N
Yan Xu, Chun Tang, Shengyu Tan, Juan Duan and Yu Yang were involved in conception, design, statistical analysis and drafting of the manuscript. Shengyu Tan and Hongmei Tian contributed in data collection. All authors approved the final version for submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.