Study the effects of natural honey on cardiac arrhythmias and infarct size in ischemic-reperfused isolated rat heart

Abstract

Natural honey has been used as an effective medicinal compound for many years and utilized traditionally for treatment of different diseases. So far, majority of studies have focused on the antibacterial, antifungal, antiviral, pain relief and wound healing properties of natural honey, however, its efficacy on cardiovascular diseases such as arrhythmias and myocardial infarction was not studied. So, in the present study, potential cardioprotective effects of natural honey against ischemia- reperfusion (I/R)-induced cardiac arrhythmias and myocardial infarction were investigated in isolated rat heart.

Materials and methods

Male SD rats (270-320g) were divided into 5 groups (n=8 in each group) randomly and pretreated with intraperitoneal (i.p) injection of 300 IU heparin then anaesthetized with sodium pentobarbital (50-60 mg/kg, i.p). As soon as deep anesthesia was achieved, the hearts were removed and quickly mounted on a Langendorff apparatus and then perfused by a carbogenated Krebs-Henseleit (K/H) solution under constant pressure at 37oC. Normal K/H was perfused during stabilization and 30 min regional ischemia followed by 120 min reperfusion in control group while in the test groups, during I/R, isolated hearts were perfused with 0.125, 0.25, 0.5 and 1% of honey-enriched K/H solution, respectively. To investigate the potential cardioprotective mechanisms of honey, K/H solution enriched by pure fructose or glucose (which present in 1% honey) also perfused in the individual test groups. An epicardial ECG was continuously recorded by physiograph throughout the experiment. Cardiac arrhythmias which examined during 30 min ischemia and first 30 min of the reperfusion, included total number of ventricular ectopic beats (VEBs), number, duration and incidence of ventricular tachycardia (VT), duration and incidence of reversible ventricular fibrillation (Rev VF), incidence of irreversible ventricular fibrillation (Irrev VF) and incidence of Total VF (sum of incidences of Rev VF and Irrev VF). To determine myocardial infarct size, at the end of reperfusion, 0.25% Evans Blue solution was infused to stain the non-ischemic area. Then the heart was cut into slices and incubated by 1% Triphenyltetrazolium chloride (TTC) solution and fixed by 10% formalin. The area of infarcted tissue and area at risk were determined by computerized planimetry.

Results

During ischemia, perfusion of 1% of honey-enriched K/H solution had statistically significant reduction effect on duration and incidence of VT (p<0.05 and p<0.01, respectively) compared to the control group. Honey (0.25 and 0.5%) decreased the number of VEBs and number and duration of VT (p<0.05). Additionally, the time spent in Rev VF and incidences of Rev VF and Total VF were significantly reduced by 0.25, 0.5 and 1% honey (p<0.05 for all). Application of fructose or glucose-enriched K/H solution produced marked and significant reduction in the number, duration and incidence of VT (p<0.01 for all). At the reperfusion phase, honey (1%) significantly decreased duration of Rev VF (p<0.001) and incidences of Rev VF and Total VF (p<0.05). Moreover, administration of 0.5% honey markedly reduced the number of VEBs and VT (p<0.01and p<0.05, respectively). The time spent in Rev VF and incidences of Rev VF and Total VF were significantly lowered by the same concentration. Furthermore, honey (0.25%) showed clear reduction in the number, duration (p<0.01 for both) and incidence of VT (p<0.05). Duration of Rev VF and incidences of both Rev VF and Total VF were also reduced by 0.25% honey (p<0.001 for duration and p<0.01 for incidences). Perfusion of the lowest concentration (0.125%) produced significant reduction in the number of VEBs, number and duration of VT and duration of Rev VF (p<0.05). Both fructose and glucose administered groups markedly reduced the number and duration of VT and the number of VEBs (p<0.05). Additionally, duration of Rev VF and incidences of Rev VF and Total VF were only decreased by fructose (p<0.05). Moreover, perfusion of honey (0.125, 0.25 and 0.5%) resulted in marked reduction of infarct size versus the control group (p<0.001, p<0.01 and p<0.05, respectively). Similarly, fructose and glucose significantly lowered the extent of myocardial infarct size (p<0.001 for both).

Discussion and conclusion

During both ischemia and reperfusion phases, perfusion of 0.25% honey for the whole period of I/R, significantly reduced the number, duration and incidence of VT. Additionally, the time to spend in Rev VF and incidences of Rev VF and Total VF were significantly reduced by 0.25, 0.5 and 1% of honey. Although 0.125% honey showed significant anti-arrhythmic effects on the number and duration of reperfusion-induced arrhythmias, but this concentration had no significant effect on ischemia-induced arrhythmias versus the control group. Overall, it seems that anti-arrhythmic effects of honey during the reperfusion were grater than ischemic period. In addition, the results showed that honey (0.25 and 0.5%) were optimum concentrations to protect ischemic reperfused rat hearts against arrhythmias in this model of study. However, honey (1%) had some non-significant pro-arrhythmic actions at the reperfusion phase. At the same time, the lowest concentration of honey was not efficient enough to show significant anti-arrhythmic effects. In our study, fructose and glucose desirably produced anti-arrhythmic effects on both ischemia and reperfusion phase arrhythmias. As a result, it could be suggested that fructose and glucose may play pivotal role in the anti-arrhythmic effects of natural honey. On the other hand, under conditions of I/R, the myocardium may be subjected to oxidative damage by free oxygen radicals. Therefore, it can be suggested that protective effects of natural honey against I/R-induced arrhythmias may also be related to its both nonenzymatic antioxidants (such as glutathione and ascorbic acid) and enzymatic antioxidants (such as superoxide dismutase and catalase). In addition, there are some findings that mention to probable anti-arrhythmic effects of the mineral elements such as zinc, magnesium, sodium and chlorine, which are found in natural honey in a relatively high level. Altogether, the used concentrations of honey significantly lowered myocardial infarct size in the ischemic reperfused rat heart (except for 1% honey). At the same time, fructose and glucose diminished infarct size markedly. The results showed that the anti-infarct properties of honey were reversely concentration dependent (equation: y=-9.02x+50.9, r2=0.992) and 0.125 and 0.25% solutions of honey were optimum concentrations to protect ischemic reperfused rat heart against infarction. Similar to the anti-arrhythmic effects, fructose and glucose may probably have important role in the efficacy of natural honey on myocardial infarction due to provide rich energy source. Since effectiveness of honey against I/R-induced injuries are not clear, there are no exact cardioprotective mechanisms in relation with the natural honey. However, other potential mechanisms of honey to decrease myocardial infarct size may include antioxidant and free radical scavenging activity, anti-inflammatory effects, liberation of inflammatory cytokines, decrease in the measure of necrotized tissue and anti-arrhythmic effects. In summary, for the first time we found that natural honey produced significant anti-arrhythmic and anti-infarct effects when it was used for the whole period of I/R. Also, we suggested that fructose and glucose, as the most constituents of honey, may play important role in the both anti-arrhythmic and anti-infarct properties of honey. Finally, it seems that natural honey should be considered as a potent cardioprotective natural product.