Abstract
Omega-6 polyunsaturated fatty acids (n-6 PUFA) are well known for their role in cardiovascular disease (CVD). We proposed that Evening prime rose oil (EPO) can improve the outcome of a heart with myocardial infarction (MI) in the presence of diet-induced hyperaggregability. This study was designed to examine its cholesterol lowering, antithrombotic and anti-inflammatory effects. High fat diet was administered for 4 weeks then MI was induced by isoproterenol (85 mg/kg/s.c./24 h). Treatment with EPO (5 or 10 gm/kg/day) for 6 weeks improved the electrocardiographic pattern, serum lipid profile, cardiac biomarkers as well as Platelet aggregation percent. We reported decreased serum level of TNF-α, IL-6 and COX-2 with attenuation of TNF-α and TGF-β in the cardiac homogenate. Moreover, histopathology revealed marked amelioration. Finally, we provide evidence that EPO improve cardiac recovery in hypercholesterolemic myocardial infarct rats. These effects are attributed to direct hypocholesterolemic effect and indirect effect on the synthesis of eicosanoids (prostaglandins, cytokines).
Keywords: Hypercholesterolemia, high fat diet, myocardial infarction, evening primrose oil
Introduction
The relation of dietary fat to risk of cardiovascular disease (CVD) has been studied extensively using different approaches. Elevated levels of serum total or low-density lipoprotein cholesterol have been related to increased risk of CVD [1]. Changes in plasma lipoproteins affecting platelet function have been found in hyperlipidemia [2]. High LDL-C levels could trigger spontaneous platelet aggregation [3] via intraplatelet calcium mobilization [4] and increasing platelet function and sensitivity [5,6]. The increase in transfatty acids in our diet interfere with the desaturation and elongation of omega-6 fatty acids (n-6 PUFA) [7]. n-6 PUFAs have a critical role in CVD risk reduction mainly by lowering LDL-C levels [8]. In addition to blood pressure, inflammatory markers, and hemostatic parameters reduction [9]. About 10% of total energy from n-6 PUFA appears justified for the prevention of ischemic heart disease according to the American heart association recommendation [10-12] mainly through reducing inflammatory states and preventing atherogenesis, even in a pro-inflammatory condition [13-15]. Cyclooxygenase (COX) is considered as one of the major enzyme families that catalyze the rate-limiting step in the formation of prostaglandins (PGs) and (TXA2) [16]. COX-2 is an inducible form that is expressed during inflammation as a result of stimulation by cytokines, nitric oxide and growth factors [17,18]. A dietary supplement that proved useful in cardiovascular protection is EPO. EPO supplies (LA) and γ-linolenic acid (GLA), a product that increases the production of series 1 prostaglandins. These in turn have antithrombotic activity, enhances smooth muscle relaxation and vasodilation. In accordance, EPO reduces platelet hyperaggregability in rabbits fed an atherogenic diet [19]. Based on above information, we proposed that EPO can improve the outcome of a heart with myocardial infarction in the presence of diet-induced hyperaggregability. Therefore, this study was designed to examine the cholesterol lowering, antithrombotic and anti-inflammatory effects of EPO in the current model of combined hypercholesterolemic myocardial infarct rats.
Materials and methods
Animals
All animal care and experimental procedures were approved by the Institutional Laboratory Animal Care and Use Committee in The Faculty of medicine, Suez Canal University. Sixty adult male Wistar rats (with a weight range of 280-300 g) were purchased from the Egyptian Organization for Biological Products and Vaccines (Cairo, Egypt). Before experiments, animals were allowed to adapt to the new environment for one week. Rats were housed in groups of four in well-ventilated clean stainless steel cages under controlled environmental conditions (21-25°C) and normal dark/light cycle). Food and water were provided ad libitum.
Drugs and chemicals
Cholesterol was obtained from GFS chemicals & reagents (Texas, USA). Bile salts were purchased from SAS Chemicals Co. (Mumbai, India). Pork fat was purchased from the market and used to prepare the high fat diet (HFD). Isoproterenol hydrochloride was purchased from Sigma-Aldrich (MO, USA) and was freshly prepared by dissolution in sterile saline. EPO containing 9% GLA was purchased from Now Foods Co. (Illinois, USA). All the other chemicals were of analytical grade from commercial suppliers.
Experimental design
Rats were randomly divided into the following groups; 10 rats each.
Group I: served as a normal group and was maintained on normal palatable diet (NPD) throughout the experiment (10 weeks).
The remaining four groups were maintained on a HFD containing 87.7% standard diet (w/w), 10% pork fat (w/w), 2% cholesterol (w/w) and 0.3% bile salt (w/w) throughout the experiment [11].
Group II (HFD control group): rats were injected with two successive doses of saline (vehicle of isoproterenol, 2 ml/kg/day, s.c.) at the end of week 4.
Group III (HFD-isoproterenol group): rats were injected with two successive doses of isoproterenol (85 mg/kg/day, s.c.) at the end of week 4 for induction of acute myocardial infarction (AMI) [20].
Group IV and V: rats were injected with isoproterenol at the same above-mentioned schedule at week 4. At this time, rats of group IV and V started a therapeutic regimen with EPO (5 or 10 gm/kg/day), respectively that continued for six weeks. In general, EPO was administered daily by gastric gavage for six weeks.
Another group of rats (NPD-isoproterenol group) were fed NPD for four weeks followed by 2 injections of isoproterenol (85 mg/kg/day) followed by six weeks of NPD feeding; this group was used to determine the difference in sensitivity to isoproterenol between rats fed with a NPD and those fed a HFD (group III). However, this group was not demonstrated in illustrations to focus on the effect of EPO on the measured parameters compared to HFD-fed rats.
ECG monitoring
At the end of week 4 and 10, all rats were anesthetized using thiopental sodium (50 mg/kg i.p.) [21]. Then, ECG was recorded using research Biopac data acquisition device mp150. The electrodes were placed subcutaneously in the gently extended limbs of the supine animal. ECG amplifier was adjusted to channel (I). Data were obtained from lead II. V+ needle was inserted to the left leg of the rat and V- to the right arm and ground to the right leg of the rat. For each ECG tracing ST segment displacement was recorded and pathological Q wave was monitored [22].
Blood sampling
After ECG recording, the body weight of rats was registered. Then, blood samples were collected by cardiac puncture. Half of this amount was used for platelet aggregation assay and the other half was processed by centrifugation at 2000 × g for 15 min within 30 min of collection. After that, serum samples were separated, collected in clean tubes and stored at -80°C until used for different assays. A midline incision were made and the hearts were removed, trimmed of connecting tissue and rinsed with ice-cold phosphate buffered saline. The heart as well as the left ventricles were weighed to calculate the ratio of the heart weight to left ventricular weight to the body weight. These ratios were determined as indices for cardiac hypertrophy. Ventricular myocardium was divided into two parts. The first part (0.2 g) was kept at -80°C), whereas, the remaining parts were fixed using 10% phosphate-buffered paraformaldehyde solution (PH1/4 7.4) for 18 h and then embedded with paraffin. After that, all tissues were re-sectioned at 4 mm thickness at the cardiac apex and left to dry over night at 37°C. Sections were then subjected to deparaffinization, rehydration and prepared for histopathological staining.
Determination of serum cardiac biomarkers
Serum lactate dehydrogenase activity was estimated using an enzymatic kit [23]. Creatine phosphokinase activity was estimated according to the method developed previously [24]. All the previous methods were performed using an ultraviolet- visible spectrophotometer (UV-160 1PC Schimadzu, Japan). The activity of serum creatine kinase-MB isoenzyme was measured using immune- inhibition method [25]. All the previous methods were performed using commercial kits purchased from Stanbios (Texas, USA). All procedures followed the manufacturer’s instructions.
Platelet aggregation assay
Half the amount of blood was collected from anesthetized rats by cardiac puncture was processed into 3.8% sodium citrate solution (9:1 V/V). To prepare platelet-rich plasma (PRP), samples were centrifuged immediately at 160 × g for 15 min at room temperature, then PRP was transferred into plastic tubes and the remaining blood was centrifuged at 3000 × g for 10 min to obtain platelet-poor-plasma (PPP). Platelet count in PRP was adjusted to (5 × 108/ml) with PPP. Platelet aggregation was measured by addition of 5 μg/ml collagen (Chrono-Log corp.) using a dual channel aggregometer (Clot 2, SEAC- Radium Company, Italy). Results were expressed as a percentage of aggregation, extent of aggregation was estimated by change in light transmission [12].
Measurement of serum lipid profile
Serum triglycerides (TGs), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were assayed colorimetrically using commercially available kits (Biodignostic®, Cairo, Egypt). The absorbance was measured using a UV-visible spectrophotometer (UV-1601-PC, Shimadzu, Japan).
Determination of serum levels of TNF-α, TGF-β, IL-2 and COX-2 using ELISA kits
Serum levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) were determined, as well as, serum levels of interlukien-2 (IL-2) and cyclooxygenase enzyme-2 (COX-2) were assessed. Rat ELISA kits (Ray Biotech Inc., Norcross, USA) were used according to the instructions of the manufacturer using an automated ELISA reader (Europe S.A. Belgium).
Determination of cardiac levels of TNF-α and TGF-β using ELISA kits
Each frozen cardiac sample (0.2 g) were homogenized using a teflon homogenizer (Glascol homogenizer system, Vernon hills, USA). Homogenization was carried out in 1 ml phosphate-buffered saline solution that contains a protease inhibitor and centrifuged at 3000 g for 15 min at 4 C. The supernatant was collected and divided into 2 dry tubes. ELISA kits for tumor necrosis factor-α (TNF-α) (Ray Biotech Inc., Norcross, USA), transforming growth factor-β (TGF-β) (Boster Biological technology, Wuhan, China) were used for determination of tissue level of these markers. The assays were recarried out following the instructions of the manufacturer using an automated ELISA reader (Europe S.A., Belgium).
Histopathological examination of the heart
The prepared sections were assessed for myocardial morphology by hematoxylin and eosin staining was done under a light microscope (Olympus, CX21, Japan). Also, the separate set of paraffin-embedded tissue sections at 4 μm were stained using Masson’s trichrome (Sigma-Aldrich Co., St. Louis, MO, USA). Tissues stained with Masson’s Trichome were quantified sterologically on ten regularly spaced sections covering the entire surface of the heart. Each section was first viewed at low power (× 10 magnification) whereas; the scoring was performed at high power (× 40 magnification). Myocardial fibrosis and necrosis was evaluated in each section of the heart tissue using a morphometric point counting procedure [26]. Two pathologists grade the histopathological changes as 1, 2, 3, 4, for low, moderate, high and intensive pathological changes, respectively.
Statistical analysis
All results are expressed as mean ± standard error of the mean. Results were assessed by one-way repeated measures analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. The difference between the HFD-isoproterenol rats and NPD-isoproterenol rats was determined using unpaired student t test. Data were analyzed using The Statistical Package for the Social Sciences, version 17 (SPSS Software, SPSS Inc., Chicago, USA). Differences between means were considered to be statistically significant when P < 0.05.
Results
Effect of treatment with EPO on percent survival and ECG pattern
In the present study, changes in ST segment elevation and % abnormal Q waves as well as % survival are shown in Table 1. NPD and HFD groups showed normal ECG pattern whereas HFD + isoproterenol treated group showed an elevated ST segment as well as significant increase in the % abnormal Q waves when compared to either NPD or HFD groups (P < 0.05). Oral treatment with EPO (5 and 10 gm/kg/rat) in HFD + isoproterenol treated rats induced a significant decrease in ST segment as well % Q waves when compared to HFD + isoproterenol treated group (P < 0.05). The percentage of survival decreased significantly in the HFD + isoproterenol group when compared to the NPD or HFD groups (P < 0.05). Even when the dose of EPO was increased to 10 gm/kg the percentage of survival was decreased significantly when compared to the NPD and HFD groups (P < 0.05).
Table 1.
Effect of treatment with EPO (5 or 10 gm/kg) for six weeks on % survival and ECG findings in HFD-myocardial infarct rats
| Groups | Percentage survival | ST segment elevation (mm) | % Pathological Q waves |
|---|---|---|---|
| NPD | 100 | 0 | 0 |
| HFD | 100 | 0 | 0 |
| HFD + Isoproterenol | 50*,† | 3.2 ± 0.31*,† | 92.4 ± 5.1*,† |
| HFD + Isoproterenol + EPO (5 gm/kg) | 50*,† | 1.8 ± 0.15*,†,‡ | 45 ± 2.9*,†,‡ |
| HFD + Isoproterenol + EPO (10 gm/kg) | 40*,† | 1.1 ± 0.08*,†,‡ | 26 ± 1.8*,†,‡,$ |
Rats were fed with a HFD for four weeks and then injected with two doses of isoproterenol (85 mg/kg/24 h, s.c.) to induce acute myocardial infarction. Rats were treated with EPO (5 or 10 gm/kg/rat/day) for another six weeks. NPD: normal palatable diet, HFD: high-fat diet, ECG: electrocardiography, ST segment elevation is represented by the number of small squares shifted from the base line. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons.
Compared to NPD group at P < 0.05.
Compared to HFD group at P < 0.05.
Compared to HFD + isoproterenol group at P < 0.05.
Compared to HFD + isoproterenol + EPO (5 gm/kg) group at P < 0.05, n = 6-10.
Effect of treatment with EPO (5 or 10 gm/kg/rat) on LV weight, Ht weight and body weight
In the current study, treatment with EPO (5 or 10 gm/kg/rat) for 6 weeks significantly ameliorated the LV to BWt ratio compared to HFD + isoproterenol group. Further, EPO (10 gm/kg/rat) decreased the Ht W to BWt ratio compared to HFD + isoproterenol group. Remarkably, the effect of the high dose of EPO (10 gm/kg) on the LV to Bwt ratio and Ht Wt to Bwt ratio was different from that produced by the low dose (5 gm/kg/rat) (P < 0.05, Table 2).
Table 2.
Effect of treatment with EPO (5 or 10 gm/kg) for six weeks on body weight and heart weight ratios in HFD myocardial infarct rats
| Groups | Baseline Bwt (g) | Final Bwt (g) | LV wt to Ht W Ratio (g/g) | LV wt to Bwt ratio (g/g xE-03) |
|---|---|---|---|---|
| NPD | 230.2 ± 4.2 | 288.4 ± 1.8 | 0.315 ± 0.001 | 3.05 ± 0.17 |
| HFD | 236.2 ± 4.6 | 364.43 ± 5.2* | 0.341 ± 0.04 | 3.45 ± 0.19 |
| HFD + Isoproterenol | 212.3 ± 4.8 | 359.4 ± 4.3* | 0.403 ± 0.01*,† | 3.98 ± 0.20* |
| HFD + Isoproterenol + EPO (5 gm/kg) | 227.1 ± 5.4 | 320.3 ± 1.9†,‡ | 0.352 ± 0.02 | 3.51 ± 0.22 |
| HFD + Isoproterenol + EPO (10 gm/kg) | 239.7 ± 4.4 | 324.5 ± 2.6†,‡ | 0.345 ± 0.006 | 3.16 ± 0.17† |
Rats were fed with a HFD for four weeks and then injected with two doses of isoproterenol (85 mg/kg/24 h, s.c.) to induce acute myocardial infarction. Rats were treated with EPO (5 or 10 gm/kg/rat/day) for another six weeks. NPD: normal palatable diet, HFD: high-fat diet, Bwt: body weight, LV: left ventricle, Ht wt: heart weight. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons.
Compared to NPD group at P < 0.05.
Compared to HFD group at P < 0.05.
Compared to HFD + isoproterenol group at P < 0.05, n = 6-10.
Effect of treatment with EPO (5 or 10 gm/kg/rat) for six weeks on serum lipid profile in HFD myocardial infarct rats
It is clear that there is a significant increase in total cholesterol level when comparing NPD group with the other 4 groups (P < 0.05, Table 3). Remarkably, total cholesterol and triglycerides level increased in the HFD and the HFD + Isoproterenol in comparison to the NPD (P < 0.05, Table 3). The effect of the treatment with EPO (5 or 10 gm/kg) on total cholesterol and triglycerides level was significant on comparison with either the HFD or HFD + isoproterenol group (P < 0.05, Table 3). There is also a significant increase in LDL-C when comparing HFD or HFD + isoproterenol group or even the 5 gm/kg EPO treated group with NPD group. Fortunately, there is a significant decrease in the LDL-C level in the 10 gm/kg EPO group when comparing with HFD and HFD + isoproterenol groups (P < 0.05, Table 3). In the current study, HDL-C level was decreased significantly when comparing the NPD group to the remaining four groups (P < 0.05, Table 3).
Table 3.
Effect of treatment with EPO (5 or 10 gm) for six weeks on serum lipid profile in HFD myocardial infarct rats
| Groups | Total cholesterol (mg/dl) | Triglycerides (mg/dl) | LDL-C (mg/dl) | HDL-C (mg/dl) |
|---|---|---|---|---|
| NPD | 213.2 ± 17.2 | 21.73 ± 1.8 | 121.2 ± 9.2 | 98.3 ± 8.7 |
| HFD | 365.2 ± 22.6* | 64.43 ± 5.2* | 239.5 ± 18.4* | 61.4 ± 5.9* |
| HFD + Isoproterenol | 358.3 ± 26.8* | 59.4 ± 4.3* | 247.1 ± 17.6* | 54.3 ± 4.2* |
| HFD + Isoproterenol + EPO (5 gm/kg) | 263.1 ± 21.4†,‡ | 27.3 ± 1.9†,‡ | 232.9 ± 19.2* | 67.1 ± 4.8* |
| HFD + Isoproterenol + EPO (10 gm/kg) | 273.7 ± 20.4†,‡ | 24.5 ± 2.6†,‡ | 189.5 ± 21.6†,‡ | 62.6 ± 5.7* |
Rats were fed with a HFD for four weeks and then injected with two doses of isoproterenol (85 mg/kg/24 h, s.c.) to induce acute myocardial infarction. Rats were treated with EPO (5 or 10 gm/kg/day) for another six weeks. NPD: normal palatable diet, HFD: high-fat diet, LDL-C: low density lipoprotein, HDL-C: high density lipoprotein. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons.
Compared to NPD group at P < 0.05.
Compared to HFD group at P < 0.05.
Compared to HFD + isoproterenol group at P < 0.05, n = 6-10.
Effect of treatment with EPO (5 or 10 gm/kg) for six weeks on cardiac enzymes in HFD myocardial infarct rats
Increased levels of cardiac markers such as LDH, CK, CK-MB as well as cardiac index were observed in the HFD + isoproterenol as well as the 5 gm EPO treated groups on comparing with either NPD or HFD groups. The HFD + isoproterenol + EPO (5 gm/kg) group showed significant amelioration in the levels of LDH as well as CK in comparison with HFD + isoproterenol group. A remarkable decrease in all cardiac markers were observed in the HFD + isoproterenol + 10 gm EPO in comparison to either HFD or HFD + Isoproterenol group. Moreover, High dose treated EPO group showed a significant decrease in all LDH, CK-MB and cardiac index on comparison with low dose treated EPO group (P < 0.05, Table 4).
Table 4.
Effect of treatment with EPO (5 or 10 gm/kg) for six weeks on serum LDH activity, CK activity and CK-MB activity in HFD myocardial infarct rats
| Groups | LDH (U/L) | CK (U/L) | CK-MB (U/L) | Cardiac CK-MB index (%) |
|---|---|---|---|---|
| NPD | 582.1 ± 45.3 | 276.5 ± 19.2 | 54.2 ± 3.2 | 19.5 ± 1.6 |
| HFD | 573.2 ± 55.2 | 255.3 ± 21.3 | 48.3 ± 3.7 | 18.9 ± 1.4 |
| HFD + Isoproterenol | 692.7 ± 41.3*,† | 487.5 ± 38.5*,† | 178.9 ± 25.5*,† | 36.7 ± 4.3*,† |
| HFD + Isoproterenol + EPO (5 gm/kg) | 643.3 ± 58.2*,†,‡ | 396.5 ± 34.2*,†,‡ | 162.3 ± 17.2*,† | 40.9 ± 4.1*,† |
| HFD + Isoproterenol + EPO (10 gm/kg) | 611.6 ± 53.1†,‡,$ | 355.4 ± 31.7*,†,‡ | 103.7 ± 9.4*,†,‡,$ | 29.4 ± 3.2*,†,‡,$ |
Rats were fed with a HFD for six weeks and then treated with two doses of isoproterenol (85 mg/kg/24 h, s.c.) to induce acute myocardial infarction. NPD: normal palatable diet, HFD: high fat diet, LDH: lactate dehydrogenase, CK: creatinine kinase, CK-MB: creatinine kinase isoenzyme MB. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons.
Compared to NPD group at P < 0.05.
Compared to HFD group at P < 0.05.
Compared to HFD + isoproterenol group at P < 0.05.
Compared to HFD + isoproterenol + EPO (5 gm/kg/rat) group at P < 0.05, n = 6-10.
Effect of treatment with EPO (5 or 10 gm/kg) for six weeks on the percentage of platelet aggregation
It was evident that the platelet aggregation percentage increased significantly on comparing the HFD + isoproterenol group as well HFD group with the NPD. But the 2 doses of EPO treatment revealed a significant decrease in the platelet aggregation percentage when compared to either HFD or HFD + isoproterenol groups (P < 0.05, Figure 1).
Figure 1.
Platelet aggregation in the experimental groups. NPD: normal palatable diet, HFD: high-fat diet, EPO: evening primrose oil. Rats were injected twice with isoproterenol (85 mg/kg, s.c.) to induce acute myocardial infarction. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons. *Compared to NPD group at P < 0.05. †Compared to HFD group at P < 0.05. ‡Compared to HFD + isoproterenol group at P < 0.05. $Compared to HFD + isoproterenol + EPO (5 gm/kg) group at P < 0.05, n = 4-10.
Effect of treatment with EPO (5 or 10 gm/kg/rat) for six weeks on serum level of TGF-β1, TNF-α, IL-6 and COX-2 and cardiac tissue level of TGF-β1 and TNF-α in the experimental groups
The serum level of TNF-α, IL-6, TGF-β1 and COX-2 were increased significantly in the HFD + isoproterenol group in comparison to the NPD and the HFD. Also, serum level of TNF-α, IL-6 and COX-2 were decreased significantly in the HFD + isoproterenol + EPO 5 gm/kg and 10 gm/kg groups in comparison to the HFD + isoproterenol group. Moreover, serum level of TNF-α was decreased significantly in the HFD + isoproterenol + EPO 10 gm/kg group in comparison to the HFD + isoproterenol + EPO 5 gm/kg group (P < 0.05, Figure 2). In addition, EPO (5 or 10) decreased the level of TNF-α in the cardiac homogenate significantly when comparing with the HFD + isoproterenol group (P < 0.05, Figure 3).
Figure 2.
Serum level of TGF-β1 (A), TNF-α (B), IL-6 (C), and COX-2 (D) in the experimental groups. NPD: normal palatable diet, HFD: high-fat diet, EPO: evening primrose oil. Rats were injected twice with isoproterenol (85 mg/kg, s.c.) to induce acute myocardial infarction. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons. *Compared to NPD group at P < 0.05. †Compared to HFD group at P < 0.05. ‡Compared to HFD + isoproterenol group at P < 0.05. $Compared to HFD + isoproterenol + EPO (5 gm/kg) group at P < 0.05, n = 4-10.
Figure 3.
The level of TGF-β1 (A), TNF-α (B) in the cardiac homogenate of the experimental groups. NPD: normal palatable diet, HFD: high-fat diet, EPO: evening primrose oil. Rats were injected twice with isoproterenol (85 mg/kg, s.c.) to induce acute myocardial infarction. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons. *Compared to NPD group at P < 0.05. †Compared to HFD group at P < 0.05. ‡Compared to HFD + isoproterenol group at P < 0.05. $Compared to HFD + isoproterenol + EPO (5 gm/kg) group at P < 0.05, n = 4-10.
Effect of treatment with EPO on Platelet aggregation % when correlated to LDL-C, IL-6 and COX-2 respectively
Person correlation coefficient derived from the linear regression equation described a strong correlation between Platelet aggregation % and LDL-c. Moreover, there is a positive correlation between platelet aggregation % and IL-6 as well as Cox-2 serum levels (Figure 4).
Figure 4.
Linear regression lines correlate Platelet aggregation % and LDL-c (A), IL-6 concentration (B), COX-2 concentration (C) in the experimental groups. The correlation coefficient (r2) indicates positive correlation between platelet aggregation % and the different parameters.
Histopathological examination of the cardiac tissues
The myocardial fibers were arranged regularly with clear striations with no evident inflammation, degeneration, necrosis or even fibrosis in both NPD and HFD in contrast to isoproterenol + HFD group (Figure 5). The 2 doses of EPO (5 and 10 gm/kg/rat) significantly ameliorated the widespread subendocardial necrosis, hypertrophia and fibroplastic hyperplasia which appeared apparent in the HFD + isoproterenol group (Figure 5). A significant difference in the mean fibrosis grade was observed on comparing the isoproterenol treated groups with either NPD or HFD. Whereas the two doses of EPO significantly decreased the mean fibrosis grade (P < 0.05, Table 5).
Figure 5.
Histopathologic examination of the heart muscles from the experimental groups. NPD: normal palatable diet, HFD: high-fat diet, EPO: evening primrose oil. The left side represents photomicrographs of the cardiac muscles stained with hematoxylin & eosin (× 40). The right side represents photomicrographs of the cardiac muscles stained with Masson’s trichrome stain (× 40). Heart tissue from the saline treated rats showed normal appearance of the cardiac tissue whereas rats treated with the 2 doses of isoproterenol (85 mg/kg/s.c) showed intensive fibrosis. Treatment with EPO alleviated the extent of fibrosis.
Table 5.
Fibrosis grades in the cardiac tissues from the different experimental groups
| Grades | Mean fibrosis grade | |||||
|---|---|---|---|---|---|---|
|
|
||||||
| Groups | 0 | 1 | 2 | 3 | 4 | |
| NPD | 10 | - | - | - | - | 1 ± 0 |
| HFD | 7 | 2 | 1 | - | - | 0.4 ± 0.22 |
| HFD + isoproterenol | - | - | - | 2 | 3 | 3.6 ± 0.22 *,† |
| HFD + isoproterenol + EPO (5 gm/kg) | - | - | - | 5 | - | 3 ± 0 *,†,‡ |
| HFD + isoproterenol + EPO (10 gm/kg) | - | - | 3 | 1 | - | 2.25 ± .19 *,†,‡ |
Rats were fed with a HFD for four weeks and then injected with two doses of isoproterenol (85 mg/kg/24 h, s.c.) to induce acute myocardial infarction. Rats were treated with EPO (5 or 10 gm/kg/rat/day) for another six weeks. NPD: normal palatable diet, HFD: high-fat diet. The histopathological changes were assigned as 1, 2, 3, and 4 for low, moderate, high, and intensive pathological changes, respectively. Mean values are expressed as mean ± S.E.M. *Compared to saline group at P < 0.05. †Compared to HFD group at P < 0.05. ‡Compared to HFD-isoproterenol group at P < 0.05. $Compared to EPO (5 g/rat) group at P < 0.05, n = 6-10. Values are expressed as mean ± S.E.M. and analyzed using one-way ANOVA followed by the Bonferroni’s test for multiple comparisons.
Compared to NPD group at P < 0.05.
Compared to HFD group at P < 0.05.
Compared to HFD + isoproterenol group at P < 0.05, n = 6-10.
Discussion
In this study, our combined hypercholesterolemic myocardial infarct rat model was treated with EPO (5 or 10 gm/kg/rat). Hence, the rats were injected with isoproterenol on two subsequent days to induce myocardial ischemia with a concomitant HFD to ensure the presence of thrombotic, hyperlipidemic as well as hyperaggregable condition all through the study.
EPO is rich in n-6 essential fatty acids, including (LA) and (GLA) in high levels [27,28]. GLA represents 7-14% of total fatty acids [29] and needed to increase the tissue content of GLA [30]. So, we selected EPO as a natural source to increase the GLA content as well as LA in the tissue. Isoproterenol induced significant ECG changes in HFD group when compared to either the NPD or HFD rats; this may be attributed to the reduced mechanical capacity of the ventricles. The ST-segment elevation is the most sensitive parameter for MI as it reflects myocardial necrosis as well, the parallel loss of the cell membrane integrity in the injured myocardium [31]. Q wave reflects the extent of myocardial damage present in the isoproterenol treated group. The generation of highly cytotoxic free radicals with the subsequent disturbance in the physiological balance between production of free radicals and the antioxidant defense mechanism [32,33] have been implicated as an important possibility in the loss of myocardial membrane integrity as well as function. Administration of isoproterenol generates the free radicals in the myocardium through oxidative stress with subsequent loss of cell membranes leading to myocardial necrosis [34].
Oral treatment with EPO (5 or 10 gm/kg/rat) significantly improved the pathological alterations. EPO may counteract the postulated oxidative stress hypothesis by the fact that GLA is rapidly elongated to DGLA then converted to arachidonic acid (AA) very slowly, approximately 0.2% [35-37]. Moreover, DGLA in the macrophages converted into PGE1 by the COX activity [38] this reduces the oxidative stress [30]. Excess DGLA inhibits the synthesis of AAderived eicosanoids [38] so it may reduce the inflammatory condition within the ischemic heart.
Treatment with EPO (5 or 10 gm/kg/rat) significantly decreased the serum levels of cardiac markers revealing an ameliorating effect on the myocardium. Isoproterenol is cardiotoxic agent due to its ability to destroy myocardial cells [39]. This amelioration may be due to restricting the leakage of these enzymes from the myocardium. EPO resulted in the accumulation of DGLA in tissue phospholipids [38,40,41]. GLA enhances the integrity and fluidity of the membranes [41]. Collectively, this reflects suppression of membrane damage. The previous reported amelioration in cardiovascular mechanical and biochemical parameters in our study came on line with the results of some studies [42-44].
In our study, feeding with HFD led to significant mixed hyperlipemia: hypercholesterolemia and hypertriglyceridemia. The total cholesterol level showed a significant reduction and so the triglycerides did after the treatment with both doses of EPO. We found that LDL-C level was decreased significantly in EPO 10 gm/kg/rat treated group. These findings were similar to other studies [45-49]. This may be explained by the findings that PUFAs are potent inhibitors of 3-hydr-oxy 3-methyl glutaryl coenzyme A reductase (HMG-C-oA reductase) enzyme [46,50] which is the rate limiting step in the mevalonate pathway for cholesterol biosynthesis [51]. Its action mimics those of statins and this could be one mechanism by which they lower cholesterol levels.
Platelet aggregation is one of the main determining factors in MI prognosis especially in concomitance with HFD. Our findings showed that platelet hyperactivity caused by feeding with a HFD was reduced when EPO was added to the diet. Platelet hyperaggregability in hyperlipemic states has been documented in several studies [52,53] by increased thromboxane synthesis [52], increased the production of PGE-1 or increased oxidized LDL [2,53]; the latter mechanism may be applied to our study as LDL levels were elevated as shown previously. So, EPO may inhibit platelet hyperaggregability by reducing hyperlipemia [19,54]. To test the influence of EPO consumption on inflammation, we measured local and plasma levels of TNF-α and IL-6. TNF-α plays a key role in activation of other inflammatory cytokines. TGF-β as anti-inflammatory marker was also monitored. We found that the local and plasma levels of TNF-α and IL-6 were decreased significantly in the hypercholesterolemic infracted rats treated with EPO 5 gm/kg/rat and 10 gm/kg/rat groups in comparison to the diseased group. Our findings may be supported by many studies [14,55,56].
Fortunately, we can explain the ameliorating effect of EPO. High cholesterol level implies proinflammatory actions and HFD directly activate leukocytes and macrophages to produce pro-inflammatory cytokines: IL-6 and TNF-α [50], moreover, local activation of neutrophils in the ischemic myocardium [14], So, EPO hypocholesterolemic effect as reported previously may be the corner stone in explaining this amelioration in the inflammatory markers level. Mimicking statins in inhibition of IL-6 and TNF-α production through down-regulation of nuclear factor-κB and activation of peroxisome proliferator activated receptor-γ [57] may be the claimed mechanism but our study design did not allow us to test or confirm this mechanism.
We reported a significant reduction in the serum COX-2 levels in EPO treated groups and this may enhance EPO anti-inflammatory effect. COX-2 is undetectable in most normal tissues becoming abundant in activated macrophages and other cells at sites of inflammation [58-60]. COX-2 oxygenate DGLA (ω-6) to give the series-1 prostanoids, which are less inflammatory [30]. DGLA is competitive inhibitors with AA for the COX pathways. This inhibition is a major mode of action in the way that dietary sources of DGLA (e.g. EPO) reduce inflammation [61].
Histopathological examination revealed higher degree of fibrosis in isoproterenol treated rats with evident amelioration in EPO treated groups. This amelioration could be attributed to the reported attenuation in the inflammatory markers and platelet aggregation % in this combined model. Besides, the increase in DGLA exerts an anti-inflammatory effect [35] and hence, cardiac inflammation and consequent fibrosis showed this significant amelioration with evident cardiac remodeling and decreased extent of fibrosis.
We found that there is a strong positive correlation between platelet aggregation % and LDL-C. Also, there is a weak positive correlation between platelet aggregation % and IL-6 and COX-2 respectively. This paid our attention to the link between platelet aggregation % and these cholesterol as well as inflammation determining factors. So, EPO may ameliorate the condition of the ischemic myocardium with HFD mainly via interruption of this link; modulation of hyperaggregability. Thus, modulation of platelet aggregation % using EPO may potentiate the effect of other medications commercially used in MI subjects with hypercholes-terolemia.
Overall, we can say that our results agrees in conclusion with aggregate data from randomized trials, case-control, cohort studies, and long-term animal feeding experiments indicate that the consumption of omega-6; reduces the risk of CVD [59,62], protects against CVD and lowers incidence of myocardial infarction or CHD death [14].
Conclusion
Finally, we concluded that EPO has a potent anti-inflammatory, anti-aggregatory and hypocholesterolemic effect. These effects can ameliorate MI even in a state of hyperaggregability produced by concomitant HFD. These therapeutic effects of EPO in general are attributed to direct hypocholesterolemic effect and the indirect effect on the synthesis of eicosanoids (prostaglandins, cytokines, cytokine mediators). Further studies are still needed to justify the proper EPO dosage and timing of administration as well as its interaction with other drugs used in hypercholesterolemic MI subjects.
Acknowledgements
The authors wish to acknowledge the generous gift of Evening Primerose Oil from Al-Hekma pharmaceutical Co. (6th of October City, Egypt).
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