Animal Biotechnology

ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20

Protective Effect of Adansonia digitata against Isoproterenol-Induced Myocardial Injury in Rats Mona A. M. Ghoneim, Amal I. Hassan, Manal G. Mahmoud & Mohsen S. Asker To cite this article: Mona A. M. Ghoneim, Amal I. Hassan, Manal G. Mahmoud & Mohsen S. Asker (2016) Protective Effect of Adansonia digitata against Isoproterenol-Induced Myocardial Injury in Rats, Animal Biotechnology, 27:2, 84-95, DOI: 10.1080/10495398.2015.1102147 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1102147

Published online: 25 Feb 2016.

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Date: 29 February 2016, At: 00:19

ANIMAL BIOTECHNOLOGY 2016, VOL. 27, NO. 2, 84–95 http://dx.doi.org/10.1080/10495398.2015.1102147

Protective Effect of Adansonia digitata against Isoproterenol-Induced Myocardial Injury in Rats Mona A. M. Ghoneima, Amal I. Hassana, Manal G. Mahmoudb and Mohsen S. Askerb Department of Radioisotopes, Nuclear Research Centre, Atomic Energy Authority, Giza, Egypt; bMicrobial Biotechnology Department, National Research Centre, Dokki, Cairo, Egypt

Animal Biotechnology 2016.27:84-95.

a

ABSTRACT

KEYWORDS

The baobab fruit (Adansonia digitata) was analyzed for proximate composition, amino acids, and minerals. The fruit pulp was found to be a good source of carbohydrates, proteins, phenols, and substantial quantities of K, Ca, and Mg. Amino acid analyses revealed high glutamic and aspartic acid, but the sulfur amino acids were the most limited. The present study was designed to investigate the role of Adansonia digitata (Baobab fruit pulp) against isoproterenol induced myocardial oxidative stress in experimental rats by demonstrating the changes in tissue cardiac markers, some antioxidant enzymes, interlukin-1 β (IL-1 β), monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), Collagen-1, galectin-3, and serum corticosterone. The activities of enzymatic antioxidant glutathione peroxidase (GPX) and non-enzymatic antioxidant reduced glutathione (GSH) in the heart tissue; additionally, histopathological examination of the heart was estimated. Male albino rats were randomly divided into four groups of ten animals each. Group I served as normal control animal. Group II animals received isoproterenol (ISP) (85 mg/kg body weight intraperitonealy (i.p.) to develop myocardial injury. Group III were myocardial oxidative animals treated with Baobab fruit pulp (200 µg/rats/day) for 4 weeks. Group IV received Baobab fruit pulp only. The data suggested an isoproterenol increase in levels of cardiac marker enzymes [creatine kinase MB (CK- MB), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST)], IL-1ß, MCP-1, MPO, Collagen, and galectin-3, with concomitant decrease in the activities GPX and GSH in heart tissue as well as corticosterone in serum. Baobab fruit pulp brings all the parameters to near normal level in ISP-induced myocardial infarction in rats. Histopathological examination of heart tissue of ISP-administered model rat showed infiltration of inflammatory cells and congestion in the blood vessels. However, treatment with Baobab fruit pulp (200 µg/rats/day) showed predominantly normal myocardial structure and no inflammatory cell infiltration. It has been concluded that Baobab fruit pulp has cardio protective effect against ISP-induced oxidative stress in rats.

Baobab fruit pulp; chemical composition; isoproterenol; oxidative stress

Abbreviations: Ca: calcium; CK-MB: creatine kinase MB; CVD: Cardiovascular disease; GPX: glutathione peroxidase; GSH: reduced glutathione; IHD: ischemic heart disease; IL-1 β: interlukin-1 β, ISP: isoproterenol; K: potassium; LDH: lactate dehydrogenase; Mg: magnesium; MCP-1: monocyte chemoattractant protein-1; MPO: myeloperoxidase; NaCl: sodium chloride; MI: myocardial injury; NaOH: sodium hydroxide; LV: left ventricle; RV: right ventricle; TTC: triphenyltetrazolium chloride

Introduction Ischemic heart disease is one of the most serious causes for the loss of human life, claiming 17 million lives per year (1). It has been shown that acute myocardial infarction can be produced in rats by injection of isoproterenol (2). The animals show signs of shock, develop congestive heart failure accompanied by dyspnea, tachycardia, and prostration. Isoproterenol [1-(3,4-dihydroxyphenyl)-2isopropylamino ethanol hydrochloride] (ISP), a synthetic

catecholamine and b-adrenergic agonist, causes severe oxidative stress in the myocardium, resulting in infarctlike necrosis of the heart muscle. It is also known to generate free radicals and to stimulate lipid peroxidation, which may be a causative factor in irreversible damage to the myocardial membrane (3). New therapies are needed to treat myocardial ischemia because current treatment has only a limited impact on survival and annual cost. The sequential histopathological changes that occur during the acute onset and repair of myocardial damage in isoproterenol-treated rats have been

CONTACT Amal I. Hassan [email protected] Egyptian Atomic Energy Authority, Department of Radioisotopes, Nuclear Research Centre, 9 Ahmed El-Zayat St. Dokki, P.O. 12311, Giza, Egypt. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt. © 2016 Taylor & Francis

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described (3). Cardiovascular disease, especially ischemic heart disease (IHD), remain the leading cause of death in industrialized countries and its incidence is also rising at an alarming rate in the developing world (4, 5). Many research studies have identified a protective role for a diet rich in fruits and vegetables against CVD (6). However, there is a growing interest in the use of alternative medicine for maintaining heart health and curing heart attack in high risk patients (7). Adansonia digitata L. (Bombacaceae family) is a native deciduous tree from the African savannas. Baobab fruit pulp has a particularly high antioxidant capability mainly because of its high natural vitamin C content, which is equivalent to 6 oranges per 100 g. Antioxidants protect the cells of organisms from damage by free radicals. Baobab fruit pulp is traditionally used against diarrhea, scurvy, cough, dysentery, smallpox, and measles. Several scientific studies have been performed such as on its antidiarrheic properties (8); demonstrating its anti-inflammatory, analgesic (pain killing), and antipyretic (temperature reducing) properties (9); and its effect against sickle cell anemia (10). Studies on the prebiotic effect of the fruit pulp were performed by the University of Piacenza (11). Baobab fruit pulp has a well-documented antioxidant capability (12–16). Antioxidants could help prevent oxidative stress related diseases such as cancer, aging, inflammation, and cardiovascular diseases as they may eliminate free radicals that contribute to these chronic diseases (15, 17). In the present study, we have investigated the role of Adansonia digitata against cardiac damage induced by ISP on the basis of biochemical assessment, confirmed by histopathological examination after determining the chemical composition of baobab fruit.

Materials and methods Dry Adansonia digitata fruit obtained from Dr. Amera Shawky, Department of Natural Resources (DNR), Institute of African Research and Studies, Cairo University. The dry pulp as mixed with 300 mL distilled water for 24 hours and the solution was filtration two times.

Chemical composition of Adansonia digitate The lipid, total carbohydrates, and reducing sugars were determined as described by the AOAC (18). Total phenols were determined using Folin-Ciocalteu reagent and Gallic acid as the standard (19). Total protein was determined by the Bradford method (20) using BSA as the standard. The total protein of the extract was expressed as mg of BSA equivalents 100 g of dry weight.

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Enzymatic hydrolysate Baobab fruit (150 g) was mixed with 825 mL of tap water and pasteurized at 85°C for 5 minutes (21). After the mixture cooled to 50°C, the pH was adjusted to pH 7.0 with 4 M NaOH. Flavourzyme (0.78 g) and Alcalase (0.78 g) were added, and the mixture was allowed to stand at 50°C for 5 hours. The pH was adjusted to pH 5.0 with HCl (4 M), and then 14.6 g of NaCl and 0.39 g of Flavourzyme were added. The hydrolysis continued at 50°C for a total of 24 hours. The enzymes were deactivated at 85°C for 5 minutes. After the mixture cooled to 50°C, the pH was adjusted to pH 6.5 with 4 M NaOH. After centrifugation the precipitate was washed with 300 mL of tap water and centrifuged again. The suspension of hydrolyzate sample in water was filtered and the hydrolyzed sample was subjected to analysis for free amino acids, and then freeze dried and stored at 10° C until further analysis. Analysis of amino acid Amino acid analysis was carried out using LC3000 Amino acid analyzer (Eppendorf, Biotronic, Maintal, Germany) equipped with a (75 � 6.0 mm) BTC guard column and BTC 2140 main column (145 � 3.2 mm) with the following conditions: Flow rate: 0.2 mL min 1, pressure of buffer from 0.0–50 bar, pressure of reagent from 0.0–150 bar and reaction temperature 123°C. Determination of metals The dry sample (1 g) was transferred to a 25-mL conical flask; 5 mL of concentrated H2SO4 was added, followed by 25 mL of concentrated HNO3 and 5 mL of concentrated HCl. The contents of the tube were heated at 200°C for 2 hours in a fuming hood, and then cooled to room temperature. Then, 20 mL of deionized water was added and the mixture was filtered using filter paper to complete the digestion of organic matter. Finally, the mixture was transferred to a 50-mL volumetric flask, filled to the mark, and allowed to settle for at least 15 hours. The resulted filtrate was analyzed by Atomic Absorption Spectrometer (AAS). The calculating of metal is based on the comparison of absorbance of samples against standard known concentration, and all results were converted to mg kg 1 (22). Experimental animals Forty adult female albino rats (180-200 g) were used. The animals were purchased from the animal house at the Institute of Ophthalmology, Academy of Science, Egypt.

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All rats had been led on standardized laboratory balanced diet and given water ad libitum. The animals were housed at a temperature of 20°C and were exposed to a 12 h light/ 12 h dark cycle. Animal experiments were according to guidelines of the institutional animal ethical committee. Animal maintenance and treatments were conducted in accordance with the National Institute of Health Guide for Animal, as approved by the Institutional Animal Care and Use Committee (IACUC, No. 10031). Chemicals Isoproterenol hydrochloride and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma Chemical Co., St. Louis, Mo, USA. All other chemicals used were of analytical grade.

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Induction of myocardial oxidative stress The myocardial oxidative stress was induced by intraperitoneally (IP) injection of isoproterenol hydrochloride (85 mg/kg body weight) dissolved in physiological saline, for two consecutive days at an interval of 24 hours (23). Experimental design The animals were randomly divided into four equal groups of 10 animals each. The first group (G I) was maintained as control, whereas Group II and III were treated with ISP (85 mg/kg bw) for 2 consecutive days (IP) to induce myocardial oxidative stress. Group III was treated with Baobab fruit pulp after 24 hours following the last injection of isoproterenol at a daily dose of (200 µg/rat/ day) IP for a period of 4 weeks. Group IV rats received Baobab fruit pulp only as in Group III for 4 weeks. Tissue sampling and biochemical analyses At the end of the trial, blood samples were collected from all groups from the retro-orbital venous plexus under anesthesia with diethyl ether. Subsequently, the hearts of the animals were excised. The blood samples collected were centrifuged at 3000 rpm for 10 min for the separation of sera. Corticosterone was estimated using RIA techniques according to (24). Also, serum IL-1 beta was measured by using ELISA (quantikine R &D system USA) according to the manufacturer’s instructions (25). Assaying of MPO activity was described by Mizutani et al. (26). MPO activity was assayed by measuring the change in optical density at 450 nm using tetramethylbenzidine, as substrate (1.5 mmol/L) and H2O2 (0.5 mmol/L). Results were expressed as MPO relative units/100 mg tissue. One unit of MPO activity was defined as the quantity of

enzyme degrading 1 mmol peroxide at 25°C, as well as tissues, CK-MB according to (27), LDH (28), and AST (29) were detected. Enzymatic antioxidant glutathione peroxidase (GPx) (30), and non-enzymatic antioxidant reduced glutathione (GSH) was assayed by the method of (31). Additionally, MCP-1, collagen-1, and galectin-3 were estimated by using gene expression real time PCR. General health condition was observed daily and development of MI was monitored by recording the change in the weight of left and right ventricles (LV & RV) throughout the experiment. The right and left ventricles were separated, blotted, and weighed, including the interventricular septum (32). Myocardial ischemia determination Frozen hearts were cut into 2-mm thick cross-sectional slices. These slices were stained in 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma Chemical Co., St. Louis, Mo, USA.) for 10 minutes at 37°C. After TTC staining, the slices were transferred to a formalin solution for ten minutes and then placed in phosphate buffer (pH 7.4). Heart slices were then placed between two sheets of glass and scanned into a computer and analyzed using infarct size planimetry software (Infarct Size, Vista Medical, Winnipeg, Canada). The percentage of the ischemic area was determined by using a computerized planimetry package (33). Preparation of tissue homogenate Heart tissue was removed immediately and washed with ice cold saline and homogenized in the appropriate buffer in a tissue homogenizer. Histopathological assessments Autopsy samples were taken from the heart of rats in different groups and fixed in 10% formol saline for 24 hours. Washing was performed with tap water, and then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven for 24 hours. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns thickness by slidge-microtome. The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin and eosin stain for routine examination, and then examination was performed through the light electric microscope (34). Statistical analysis Data were expressed as mean �SE. Differences among means were tested for statistical differences by one way

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analysis of variance (ANOVA),when differences were significant, Data were statistically analyzed using SPSS version 17.0 (SPSS, Cary, NC, USA). One way analysis of variance (ANOVA) was used to test the variations Duncan’s test used for multiple comparisons between groups.

Results

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Proximate composition of baobab fruit pulp The fruit pulp is an excellent source of carbohydrates (76.8%) and phenols (6.65%); whereas, the protein was (3.11%) and fat (0.25%). The proximate composition of fruit pulp was similar to that reported by Nour et al. (35). These values were expressed as percentages of dry matter (DM) of the fruit extract. It was determined before those polysaccharides were able to bind to phenolic compounds (36–38). They showed a statistically high linear relationship between the scavenging activities of the polysaccharides and their content of phenols. The results suggested that the polyphenols were completed to the polysaccharides (38). The amino acid contents of A. digitata after enzyme hydrolysis are shown in Table (2). The results reveal that enzyme-hydrolyzed A. digitata was principally made up 16 amino acids. The glutamic acid was the most abundant amino acid (125.63%), followed by aspartic acid (97.52%), leucine (72.52%), proline (63.62%), alanine (58.23%), and valine (55.83%). The amino acid profile agreed well with that reported for baobab from Burkino Faso (39). The mineral content of baobab fruit pulp was shown in Table (3), the fruit pulp is an excellent sources of potassium, calcium, and magnesium, but poor sources of iron, copper, and zinc. The fruit pulp mineral content is comparable to those reported for baobab fruit Sudan (33). The pulp showed exceptionally high calcium content; a similar finding was reported by Prentice et al. (40). The high calcium contents of the fruit pulp make the baobab fruit attractive as a natural source of calcium supplementation for pregnant and lactating women, as well as for children and the elderly. Effect of A. digitata on cardiac marker enzymes Figure 1a-c depict the levels of cardiac marker enzymes CK (MB), AST, and LDH in the cardiac tissues of normal and experimental groups, i.p. injection of isoproterenol caused a significant increase (P < 0.05) in the levels of CK (MB), AST, and LDH in GII compared to control (GI). Treating animals with A. digitata for 4 weeks significantly reduced the cardiac marker enzymes at near the control level.

Figure 1. (a, b, and c) The levels of cardiac marker enzymes (AST, CK-MB, and LDH) activities, respectively, in control and experimental groups.

Effect of A. digitata on cardiac antioxidants Figure 2a-b show the effect of A. digitata on GSH and GPX activities in different experimental groups There appears to be a significant (P < 0.05) decrease in the activities both of GSH and GPX in (GII) animals when compared to control (GI). Treatment of rats with A. digitata (G III) caused a significant increase (P < 0.05) in the activities both of GSH and GPx compared to isoproterenol group (GII) and approached near control level. Effect of A. digitata on cardiac collagen-1, galectin-3, and MCP-1 Results indicated that collagen-1, MCP, and Galectin-3 levels were significantly increased (P < 0.05) in heart

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Figure 4. The levels of cardiac MCP-1 in control and experimental groups.

Figure 2. (a and b) The levels of cardiac GSH and GPx activities, respectively, in control and experimental groups.

tissues of rats treated with isoproterenol (GII) when compared to the control (GI). While rats that were treated with isoproterenol followed by A. digitata (GIII) significantly decreased the levels of these parameters when compared with isoproterenol (GII) treated rats and preserved these levels to near normal (Fig. 3, Fig. 4, and Fig. 5), respectively. Effect of A. digitata on cardiac MPO and serum IL-1 β

Figure 5. groups.

The levels of galectin-3 in control and experimental

experimental rats, isoproterenol induced a significant increase (P < 0.05) in both MPO and IL-1 β levels in group GII rats as compared with group GI rats. A. digitata treatment significantly reduced both MPO and IL-1 β levels. Effect of A. digitata on corticosterone hormone

Figures 6 and 7 depict the levels of heart tissue MPO and proinflammatory cytokine IL-1 ß in serum of

A significant decrease (P < 0.05) in serum level of corticosterone hormone was observed in-group GII rats as compared with the group GI rats (Fig. 8). A. digitata

Figure 3. The levels of cardiac collagen-1 in control and experimental groups.

Figure 6. The levels of MPO in control and experimental groups.

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Figure 7. The levels of proinflammatory cytokine (IL-1ß) in control and experimental groups.

treatment significantly reduced the level of corticosterone similar to the control group (GI).

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Effect of A. digitata RV and LV weight Marked fibrosis was present 4 weeks after isoproterenol (G II) Fig. 9a-b. Compared with control rats, RV and LV weight in (G II) rats was doubled (P < 0.05). However, rats that were treated with isoproterenol, followed by A. digitata (GIII) significantly decreased; the RV and LV weight when compared with isoproterenol (GII) treated rats and preserved these levels to near normal. Effect of A. digitata on area of infarction Figure 10 shows the heart sections of normal and ISP injected rats stained with TTC. ISP injected rats showed an increased infarction area shown by yellowish color compared to the control rats. The percentage of mean infarct size was also calculated. ISP injected rats showed an increased area of infarction compared to control rats. Treatment with A. digitata for 4 weeks showed a significant (P < 0.05; 132.02%) decrease in infarct size as compared to ISP injected rats (17.8 � 0.07 and 41.3 � 2.11, respectively) (Fig. 10).

Figure 8. The levels of serum cortecosterone in control and experimental groups.

Figure 9. (a and b) Weight of right and left ventricles respectively in control and experimental groups.

Histopathological study The normal architecture of the cardiac cells was observed with no evidence of microscopic changes in the control and A. digitata treated groups only (Fig. 11A-B). Endocardium and pericardium were within normal limits. No inflammatory cell infiltration was observed. In isoproterenol-treated rats’ heart, histological changes such as a

Figure 10. Representative myocardial injury in each group carried out by TTC staining Values represent means �S.E. Bars with different letters are statistically significant (P < 0.05).

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Figure 11. (A) control heart (80 X) and (B) normal rats were administered A. digitata (80 X), (C and D) isoproterenol -treated rats (40 and 80 X, respectively), (E and F) isoproterenol (85 mg/kg body weight) followed by A. digitata (200 µg/rat/day) treated rats (40 and 80 X), respectively.

focal area in the degenerated myocardium showed myofibroblast proliferation with few inflammatory cell infiltration as well as congestion in the blood vessels of GII (Fig. 11C-D). In isoproterenol (85 mg/kg body weight) followed by A. digitata (200 µg/rat/day) treated rats, there was no appreciable change in the heart Table 1. Primer Collagen-1 Galectin-3 MCP-1 GAPDH

Primer sequences used for RT-PCR. Sequence FORWARD 5’- TGCCG TGAC CTCAAGATGTG-3’ REVERSE 5’- CACAA GCGTGCTGTAGGTGA -3’according to gene bank accession number NM_053304.1 Forward :50 -TGCCTCGCATGCTGATAACA-3’ Reverse: 50 -GGTTCAACCAGGACTTGTAT-3’ according to gene bank accession number NM_001197043. Forward :5’ TCT GGC ACC ACA CCT TCT ACA ATG 3’ Reverse: 5’ AGC ACA GCC TGG ATA GCA ACG 3’ according to gene bank accession number NM_031530.1 forward: 50 - CTCCCATTCTTCCACCTTTG-30 reverse: 50 - CTTGCTCTCAGTATCCTTGC-30 according to gene bank accession number XR_145951.1

(Fig. 11E-F). There was mild congestion in myocardial blood vessels (Table 4). Table 2. Amino acids contents of baobab fruit pulp. Amino Acid Aspartic acid Threonine Serine Glutamic acid Alanine Valine Methionine Isoleucine

%

Amino Acid

%

97.52 48.12 45.42 125.63 58.23 55.26 6.33 39.75

Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Proline Glysine

72.52 28.81 33.62 16.51 47.35 27.86 63.62 52.07

Table 3. Minerals content of baobab fruit pulp. Mineral

mg/g

Mineral

mg/g

Potassium Calcium Sodium Magnesium

1520 310 35 120

Phosphorus Iron Copper Zinc

215.0 12.0 1.8 2.3

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Table 4. Histopathological changes in the heart of male rats exposed to isoproterenol and the treatment effect of A. digitata, based on scoring severity of injury. Observation Focal area in the degenerated myocardium Congestion in the blood vessels Inflammatory cells

Control

Isoproterenol

Isoproterenol þA. digitata

þþþþ

þ

þþþþ

þþ

þþþþ

Normal ( ), minimal (þ), mild (þþ) severe (þþþþ).

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Discussion In the present study, we demonstrate that enzyme hydrolyzed A. digitata was principally made up 16 amino acids. The glutamic acid was the most abundant amino acid (125.63%), followed by aspartic acid (97.52%). Glutamic acid and its metabolites, glutamine, asparagin, and alanine, the ability of these amino acids to support a higher level of contractile function in ischemia or hypoxia also confirms an undoubtedly important role for glutamic acid and its precursors, arginine and ornithine, and also aspartic acid (41). It is apparent that the effect of amino acid supplementation on glycolysis is directly translated into improved regional function and reduced injury size (42). Sivakumar et al., (43) showed that aspartate and glutamate could reduce oxidative stress in MI induced rats. There was no increase in diastolic pressure in the presence of these amino acids, which is characteristic of the initial phase of reoxygenation (44). Moreover, the fruit pulp is an excellent source of potassium, calcium, and magnesium. Increasing consumption of potassium to 4.7 g per day predicts lower event rates for future cardiovascular disease, with estimated decreases of 6% to 11% in myocardial infarction (42). Oral magnesium acts as a natural calcium channel blocker, increases nitric oxide, improves endothelial dysfunction, and induces direct and indirect vasodilation (45). In the present study there was a significant increase in levels of cardiac marker enzymes (CK-MB, LDH, AST) in the heart tissue during isoproterenol (ISP) administration, which used as markers of myocardial injury. Isoproterenol administration produces free radicals (via a beta adrenoceptor mechanism) that affect the cell metabolism, such that toxic free radicals are formed producing myocardial cell necrosis (46). Generation of free radicals extensively damage the myocardium result in increased membrane permeability leads to leakage of CK-MB, LDH, and AST (47). When myocardial cells, containing AST, CK, and LDH, are damaged or destroyed due to deficient oxygen supply or glucose, the cell membrane becomes permeable or may rupture, which results in the leakage of enzymes. This accounts for the decreased activities of these enzymes in heart of rats with

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myocardial ischemia-induced by isoproterenol. This might be due to the damage caused to the sarcolemma by the β-agonist that has rendered it leaky (48). Treatment with A. digitata significantly inhibits the rise in level of cardiac marker enzymes in G III rats compared to GII rats that in consist with Song et al. (7). This might due to the scavenging ability (49) and antioxidant properties (50) of A. digitata polysaccharide. Also, A. digitata fruit has a well-documented antioxidant capability (15,16). From the obtained results shown in Fig. 2a-b, decreasing of GSH and GPX activities by isoproterenol may contribute to generating free radicals and stimulating lipid per oxidation, which may be a causative factor in irreversible damage to the myocardial membrane. GSH is a tripeptide that has a direct antioxidant function by reacting with superoxide radicals, peroxy radicals, and singlet oxygen followed by the formation of oxidized GSH and other disulfides. It plays an important role in the regulation of a variety of cell functions and in cell protection against free radical mediated injury (51). Thus, reduction in cellular GSH content could impair recovery after short period of ischemia. Depressed GSH levels may be associated with an enhanced protective mechanism to oxidative stress in MI. In this study, ISP administration was found to reduce the levels of GSH in cardiac tissue and the observation concurs with several earlier findings (52, 53). The activities of GSH and GPx antioxidant enzymes were declined in hearts ISP-administered. GPx offers protection to the cellular and subcellular membranes from the peroxidative damage by eliminating hydrogen peroxide and lipid peroxides. The lowered activities of GPx and GSH in heart in ISP-administered rats may due to the reduced availability of GSH. Decreased activities of these enzymes lead to the accumulation of these oxidants and make myocardial cell membranes more susceptible to oxidative damage. Inactivation of glutathione reductase (GR) in the heart leads to accumulation of oxidized glutathione (GSSG), which is an oxidized product of GSH. GSSG inactivates the enzymes containing SH-group and inhibits protein synthesis (54). Treatment of A. digitata significantly increases in the activities of GPx, and GSH when compared to individual treatment groups approach near normal level. A. digitata polysaccharide exhibits a free radical inhibitor or scavenger activity for DPPH and superoxide anion radical, as well as a primary antioxidant that reacts with free radicals, which may limit free radical damage occurring in the human body (38). A. digitata polysaccharide possesses the scavenger effect not only for radicals, but also for hydrogen peroxide (55, 56). Enhanced collagen turnover (breakdown and synthesis) occurs early after myocardial injury (MI) (57). Preservation of the extracellular

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matrix and collagen deposition at the site of myocyte necrosis are essential for structural integrity of the infarcted heart. Excessive collagen degradation and impaired fibrous tissue formation may reduce the tensile strength of the necrotic zone and lead to enhanced infarct expansion (58). In the current study, ISP induced a significant upregulation of collagen gene expression, whereas treatment with the A. digitata approach was used for the controls. It was noticed earlier that the neutrophils, a major source of free radicals, characteristically invaded the myocardial tissue during ischemia (59). The observations of this study showing that A. digitata treatment blocked the elevation of MPO activity, which indicated that A. digitata suppressed neutrophil infiltration into the injured myocardium. The inhibition of neutrophils infiltration and its function resulted in reduced generation of oxygen free radicals, and during ischemia, may contribute to the protective action of A. digitata against myocardial ischemia. As prominent and early mediators of inflammation, proinflammatory cytokines critically regulate the response to cardiac injury. Interleukin (IL)-1 β signaling mediates chemokine synthesis in the infarcted myocardium and stimulates infiltration of the injured with leukocytes (60). In the present study, there was a significant increase in IL-1 β in the animals exposed to ISP, whereas A. digitata following an ISP modulate increased and approached normal values. Inflammation is a common underlying cause of many diseases, infectious and otherwise, and can occur in many organs and tissues, although a controlled acute inflammatory reaction is a normal part of our innate immune response to infection and injury. Plant extracts can be evaluated for inflammatory properties in such a system and direct antiviral effects can also be tested against the same viruses (61). Also, the study showed a strong increase in MCP-1 levels in the ISP group as compared to the controls. Similar to Kralisch et al. (60) have shown that ISP increases MCP-1 mRNA levels in fibroblasts. A key role is played by MCP-1, which controls not only the accumulation of macrophages, but also a fibroblast proliferation, the induction of TGF-β1, and the appearance of fibrosis (62, 63). Galectin-3 is a member of the galectin family, which consists of animal lectins that bind β-galactosides. Recently, a role for galectin-3 in the pathophysiology of heart failure has been suggested. It was observed that galectin-3 is specifically upregulated in decompensated heart failure compared with compensated heart failure in animal models of heart failure (64). In the present study, ISP induced a significantly increased of galectin-3. The increased expression levels of galectin-3 are associated with the tendency to develop decompensated heart failure, and in clinical cohorts, increased plasma galectin-3 levels

are linked with worse prognoses (64). Therefore, galectin-3 may be advocated as a novel biomarker, but it may also be in the pathophysiologic circle of heart failure (“culprit biomarker”), and therefore it may also be a target for intervention (65). A. digitata (GIII) significantly decreased galectin-3 and approached normal values. The role of the right ventricle (RV) in cardiac function and disease has often been neglected as more attention is given to the function of the left ventricle (LV). However, it is well recognized that there is a relationship between the functioning of the two ventricles. Thus, impairment of the RV may impede LV function (or vice versa) (66). In the present study, A. digitata (GIII) significantly decreased the RV and LV weight when compared to ISP (GII) treated rats and preserved these levels to near normal. Chowdhury et al. (67). demonstrated that ISP- induced cardiac hypertrophy affects the molecular level, affecting the cardiac protein expression profiles in rats. Left ventricular function in vivo was markedly impaired after 1 week of treatment with ISP; the impaired cell was shortened both under basal conditions and in response to beta-adrenergic stimulation, indicating a functional defect intrinsic to the cardiac muscle. Previous studies have shown that ISP mediated oxidative stress could progress to myocardial necrosis, which leads to cardiac dysfunction characterized by increased end-diastolic volume and pressure and left ventricular wall thickness (68). These changes are significantly prevented by antioxidants. The increased heart weight has been attributed to the fact that the poor metabolic product of the extract resulted in the poor oxygen carriage. The heart needs to pump more blood leading to increased metabolic activities of the heart, which resulted in enlargement and increase in the heart weight (69). Patel et al. (52) have reported that the observed increase in the heart weight in ISPinduced rats might be due to the increased water content, edematous intramuscular space, and extensive necrosis of cardiac muscle fibers followed by the invasion of damaged tissues by the inflammatory cells. In the present study, the favorable modulation of right and left ventricular weight by A. digitata treatment also supports the previous observations, which may modify the course of action and help in retaining the function of heart to normal level. The progression of LV dysfunction and heart failure in animal models (rats and mice) is similar to that observed in humans who sustain a myocardial oxidative, survive and then subsequently develop heart failure (70). The LV fibrosis in the rats (GII) was associated with higher mRNA of collagen-I. Moreover, Stefanon et al. (70) assayed factors involved in collagen turnover in both ventricles following MI in rats presenting signs of heart failure. The animals in the GII

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presented different patterns of remodeling in both ventricles and fibrosis and seemed to be a consequence of collagen production in LV (71). Xu et al. (72) reported that collagen protein expressions in ischemic myocardium of rats with acute myocardial infarction (AMI). Examination of viable myocardium in both humans and animals subsequent to myocardial infarction has shown an increase in interstitial collagen (73). The total content of non-collagenous and collagenous proteins was elevated in the viable heart with large infarct and the synthesis of collagenous proteins was greater than that of noncollagenous proteins in hearts subsequent to myocardial infarction in rats (74). Furthermore, myocardial fibrosis is considered to be intimately associated with an abnormal increase in myocardial collagen concentration and is believed to result in diastolic dysfunction (75). The histopathological examination of myocardium in ISP control animals showed the presence of focal myonecrosis with myophagocytosis and lymphocytic infiltration in sub-endocardial region indicative of infarctlike lesions similar to a previous study (68). Treatment with A. digitata extract preserved the normal histoarchitecture of the myocardium as evidenced by reducing myonecrosis and lesser infiltration of inflammatory cells. Taken together, the biochemical and histopathological results of the present study demonstrate the cardioprotective effects of A. digitata.

Conclusion In conclusion, our study reveals that treatment with A. digitata exerts significant cardioprotective effects against ISP-induced myocardial injury in rats. This myocardial protective effect could be associated with the enhancement of antioxidant defense system through the activation of GSH and GPx in heart tissue. Also, A. digitata treatment proved to be effective in reducing the extent of myocardial damage by blocking the increase of cardiac marker enzymes. There was also a trend toward reduced LV fibrosis and collagen-1 with treatment.

Competing interests The authors have declared that no competing interests exist.

Acknowledgment The authors are grateful to Dr. Adel M. Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, Cairo University for his professional help in carrying out the histopathological examination.

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