EFFECT OF HONEYBEE VENOM (APIS MELLIFERA) ON RESPIRATORY FUNCTIONS OF HYPERCHOLESTEROLEMIC MALE ALBINO RATS

Assiut University web-site: www.aun.edu.eg

EFFECT OF HONEYBEE VENOM (APIS MELLIFERA) ON RESPIRATORY FUNCTIONS OF HYPERCHOLESTEROLEMIC MALE ALBINO RATS

AZZA M. MAREI

Department of Zoology, Faculty of Science, Benha University, Egypt.

Received: 10 December 2020;     Accepted: 20 January 2021

INTRODUCTION

Hyperlipidemia suggests abnormally increased levels of lipids or lipoproteins in the blood due to abnormal fat metabolism or function, and it is induced by dietary disorders, obesity, genetic illnesses such as familial hypercholesterolemia (FH) or different ailments such as diabetes (Yao et al., 2020) which caused a reduction in O₂ transport due to increasing the cholesterol content in RBCs membrane  delay  diffusion

Corresponding author: AZZA.M. MAREI

E-mail address: azza.marei@fsc.bu.edu.eg

Present address: Department of Zoology, Faculty of Science, Benha University, Egypt.

of O₂ in and out of RBCs (Buchwald et al., 2000) and caused respiratory alkalosis and decrease O2 saturation (Abd-El-Maksoud, 2009). Nowadays many types of research were directed to natural therapy to avoid the side effects of drugs used as anti-hypercholesterolemia. Honey bee venom (Apis mellifera) is a bitter, colorless liquid, and its active portion contains many peptides (adolapin, apamin, melittin, and mast cell degranulating peptide), enzymes (phosphatase, hyaluronidase, α-glucosidase, phospholipase B, and phospholipase A2) and low molecular weight components. It also had a non-peptide fraction (histamine, dopamine, and nor-epinephrine) (Raghuraman and Chattopadhyay, 2007). BV has various polypeptides, the primary one is that melittin and is also the major component of BV and it has many favorable biological effects and low toxicity. BV has been used as a drug because of its beneficial effects on many diseases (Abdela and Jilo, 2016; Sforcin et al., 2017). Studies on the use of BV to treat patients with various nervous system degenerative disorders such as multiple sclerosis (MS) have been published, Alzheimer’s disease, and Parkinson's disease (Kim et al., 2011 and Lee et al., 2012). In cell and animal studies BV has also been found to work against different types of cancers (Orslic, 2012). However, no differences between the cancer incidences in normal humans and beekeepers have been reported (Mcdonald et al., 1979). reactive oxygen species (ROS), free radicals, and reactive nitrogen species are the most recognized cellular oxidants (Asmat et al., 2016) and BV is believed to be counteracting oxidant activity (Roy et al., 2015). BV also had decreased effects on elevated blood glucose and dyslipidemia (Gawad, Fikry, Amin, Elmahdi, and Elaziz, 2016). The present work aimed to evaluate the potential effects of honeybee venom (BV) on the hematology and respiratory functions of hypercholesterolemic male albino rats.

MATERIALS AND METHODS

1. Experimental animals

Forty-two male albino rats (Rattus norvegicus) weighing 100-150g were collected from Helwan Farm of the Egyptian Organization for Vaccine and Biological Preparations. Rats were caged at 25±2 ° C, 12 hours light / dark cycle and given food and water ad libitum for 10 days before the start of the experiment in the laboratory.

2. Cholesterol

It was purchased from the Middle East Company for Medical and Scientific Apparatus Laboratory Equipment and Chemicals, Cairo, Egypt, it is white crystalline powder, dissolved in coconut oil

-Chemical name: (3β)-cholest-5-en-3-ol

-Empirical formula: C₂₇H₄₆O

-Molecular weight: 386.65 g/mol

-Density: 1.052 g /cm³

-Structure formula:

3. Coconut oil

 It was purchased from a pyramid company for new industry, used for dissolving of cholesterol.

4. Honeybee venom

Lamarck's honeybee or the Egyptian honeybee, Apis mellifera lamarckii.BV was purchased from the Faculty of Agricultural Environmental Sciences, Benha University. The venom was collected from healthy colonies of local A. mellifera lamarckii. The collection was made following the standard electroshock method (Pence, 1981), when the wires at the top of the hive were electrified and a very mild shock was applied to the bees, they covered the surface of the wired glass plate and stung the surface of the glass plate in response to the electrical stimulation. Secreted venom from bee sting dried rapidly when exposed to the air. Dried venom was scraped off with a sharp scalpel and transferred to the laboratory and was stored at a temperature of −20 ◦C until further analysis. Extraction was made for 15–20 min on each colony and was repeated twice every 2 weeks.

5. Induction of Hypercholesterolemia

Cholesterol was dissolved in coconut oil and was given to the rats by oral gavage at a daily oral dose of (450 mg/kg b.wt) dissolved in 0.5 ml coconut as described byNwichi et al. (2012),the presence of hypercholesterolemia in the induced rat model was documented by evaluating the lipid profile at the end of induction period (6 weeks) in the cholesterol group as compared to negative control and coconut oil groups.

6. Experimental groups and design of the work:

Animals were divided randomly into six groups (7 animals each), fed on a standard diet, and supplied with water adlibitum.

6.1. First group: (control group). Normal untreated rats.

6.2. Second group: (coconut oil group). Ratsadministered a daily oral dose of coconut oil (0.5ml) for 6 weeks.

6.3. Third group: (cholesterol group). Ratsadministered a daily oral dose of cholesterol (450 mg/kg b.wt) dissolved in 0.5 ml coconut oil for 6 weeks.

6.4. Fourth group: (Bee venom group). Bee venom was freshly prepared by dissolving in distilled water just before treatment and was injected intraperitoneally (IP) at a concentration of 0.5 mg/kg for 6 weeks (Mousavi et al., 2012).

6.5. Fifth group: (prophylactic group). Rats injected intraperitoneal (IP) with bee venom 0.5 mg/kg for 6 weeks then administered cholesterol for another 6 weeks (450 mg/kg b.w dissolved in 0.5 ml coconut oil).

6.6. Sixth group: (therapeutic group). Rats administered a daily oral dose of cholesterol (450 mg/kg b.wt dissolved in 0.5 ml coconut oil) for six weeks then injected intraperitoneal (IP) with bee venom 0.5 mg/kg for another six weeks.

7. Blood Sampling:

At the end of the experimental period, rats were fasted overnight then anesthetized by ether inhalation (Sinet et al., 1984). Rats were dissected to expose the dorsal aorta of each control and treated animals as previously described by (Eissa et al., 1988; El-Shafey, 1990 and El-Shafey and Selim, 2002) and the arterial blood samples of the rats were collected into 1.0 ml syringes containing heparin (500 IU/ml). Heparin is used because of its limited effect on the biochemical composition of blood (Muller-Plathe and Schebusch, 1991).

8. Hematological analysis:

Determination of hemoglobin content, hematocrit value, andred blood cells "RBCs" count by Automated Hematology Analyzer (Diff3) Mek-6410/Mek-6420.

9. Determination of Respiratory Functions of Blood

9.1. Blood gases and acid-base balance

Determination of blood gases (oxygen and carbon dioxide partial pressures; PO₂ & PCO₂in mmHg), % blood O₂ saturation, and blood acid-base status parameters (pH value, HCO₃) were carried out using MEDICA, REF7001, Analyzer, English. Oxygen carrying capacity was calculated as follows: -

One gm of Hb can hold about 1.34 ml of oxygen at 100% saturation, thus Oxygen-Carrying capacity = 1.34 X g/ L of Hb.

9.2. Blood oxygen equilibrium curve “OEC”

The best way to measure the oxygen affinity is to draw an OEC by plotting the percentage of O₂ saturation of hemoglobin at a different partial oxygen pressure using Sigma Plot (version 10) program produced by Systat Software, Chicago, USA and determination of the blood oxygen equilibrium curve “OEC” was performed as previously described by Eissa et al. (1988) and El-Shafey (1998)

10. Statistical analysis

The values of measured and calculated parameters were expressed as the mean of 7 individual values ± standard deviation “SD”. Statistical analysis was carried out using a one-way analysis of variance (ANOVA) followed by Duncan´s test by using SPSS (version 20) program produced by IBM Software, Inc. Chicago, USA(George and William, 1980). In the same raw, similar letters mean a non-significant difference at P< 0.05 and different letters mean a significant difference at P< 0.05.

RESULTS

1. Hemoglobin content

By analysis of variance, hemoglobin content showed significant declines in Hb content in cholesterol treated groups as compared to the control group and other treated groups (Table 1). BV treatment caused a significant increase in Hb content. Treatment with BV before or after cholesterol help to return Hb content toward control value.

2. Hematocrit value

Hematocrit values (Table 1) showed a significant difference between control and treated groups by analysis of the variance. Rats treated with cholesterol showed significant decreases in Hct value as compared to all groups. BV and BV then cholesterol treatment (protective) induced significant increases in Hct value as compared to control and other treated groups.

3. Red blood cells (RBCs) count 

Data in table (1) showed a significant difference in RBCs count between control and all treated groups (ANOVA). After cholesterol treatment RBCs count showed significant decreases as compared to the control group and all other treated groups. Bee venom and BV then cholesterol treatments caused significant increases in RBCs count as compared to control and other treated groups.

4. Respiratory functions of blood

Data in table (2) showed a significant increase in partial pressure of oxygen (PO₂), oxygen saturation, oxygen carrying capacity in the bee venom group in comparison to all other groups. There was no significant difference between BV then cholesterol group, coconut, and control groups in partial pressure of oxygen, oxygen saturation, oxygen carrying capacity. There was a significant decrease in the cholesterol treated group in partial pressure of oxygen, oxygen-carrying capacity, oxygen saturation, and partial pressure of carbon dioxide (PCO₂). There was no significant difference between BV and BV than cholesterol, coconut, and control groups in partial pressure of carbon dioxide (PCO₂).

5. Blood acid-base status parameters

Table (3) illustrated the changes in blood pH, bicarbonate ‟HCO₃”. Treatment with cholesterol and cholesterol then bee venom showed significant increases in blood pH and HCO₃   compared to the control group and other treated groups. There were non-significant differences between control, coconut oil, and BV in blood pH, bicarbonate ‟HCO₃”. There were non-significant differences between BV and BV then cholesterol groups.

-Blood oxygen equilibrium curve ‟OEC” Fig (1) showed the oxygen equilibrium curves (OEC) of all groups. The OEC of cholesterol and cholesterol before BV treated groups showed a left shift in comparison with that of control and other treated groups. Blood oxygen half-saturation pressure (P₅₀ value) found to be (28.52±1.3), (29.32±0.82), (24.10±1.76), (30.23±1.51), (29.32±0.95) and (25.51±2.31) for control, coconut oil, cholesterol, BV, BV then cholesterol and cholesterol BV treated groups respectively (Table 4). ANOVA showed significant decreases in cholesterol and cholesterol then RJ treated groups in comparison with those of control and other treated groups, while there were non-significant differences between all other treated

Table 1: Hb content, Hct value, RBCs count of hypercholesterolemic male albino rats before or after bee venom treatments

Table 2: Partial pressure of oxygen “PO₂”, O₂ saturation “%O₂ sat.”, oxygen-carrying capacity and partial pressure of carbon dioxide “PCO₂” of hypercholesterolemic male albino rats before and after bee venom treatments.

Table 3: PH, bicarbonate concentration (HCO₃) of hypercholesterolemic male albino rats before or after bee venom treatments.

Table 4: Blood oxygen half-saturation pressure “P₅₀” of hypercholesterolemic male albino rats before or after bee venom treatments

Fig.1. Blood oxygen equilibrium curves of hypercholesterolemic male albino rats before or after bee venom treatments.

DISCUSSION

Bee venom can have therapeutic, defensive, and hypolipidemic effects by increasing lipid consumption in adipose tissue and triglyceride hydrolysis (Hassan et al., 2019). Treatment with cholesterol, induced decreases in levels of Hb, Hct, and RBCs count. These results are in agreement with those of (Prasad, 2010) who reported that hypercholesterolemia caused reductions in the hematocrit and hemoglobin values. These reductions may be due to a decrease in RBC count which results from a decrease in the formation of RBCs, damage of erythrocyte membrane, generate micro-nucleated erythrocyte, and alteration in blood viscosity and increase in the fluid volume (Abd Elhalim and Alhadlaq, 2008).

Bee venom induced significant increases in Hb, Hct, RBCs count, PO₂, O₂ saturation, and O₂ carrying capacity this may be due to BV improves erythropoiesis and increases the Hb content, RBCs and Hct which improve O₂ transport by increasing coronary and peripheral circulation and improves circulation of blood in the micro blood vessels and these results are in line with (Mohammed and Hassan, 2019) and (Son et al., 2007). Using BV before cholesterol has a preventive role in the protection of the body from the high level of cholesterol and improvement renewal of RBCs (Salman et al., 2015).

Cholesterol treated group showed a decrease in O₂ saturation, PO₂ and O₂ carrying capacity this may be due to increased RBC membrane cholesterol in hypercholesterolemia appears to decrease the transmembrane O₂ diffusion rate.  (Buchwald et al., 2000 and Awwad, 2008) and also, this group showed respiratory alkalosis which indicated by a significant decrease in PaCO₂, increase in pH, and compensatory decreases in blood HCO₃ concentration (Johnson, 2015). The high RBC membrane cholesterol content seemed to impair O₂ diffusion into the RBCs, thereby maintaining higher PO₂ plasma levels in the cholesterol group (Buchwald et al., 2000). This data agreed with Menchaca et al. (1998) who compared arterial blood samples from cholesterol-supplemented rabbits and from non-supplemented control and reported that high cholesterol concentrations reduce blood O₂ transport and reported finally that increased RBC membrane cholesterol in hypercholesterolemic rabbits decreased the trans-membrane O₂ diffusion rate. The oxygen dissociation curve (ODC) describes the dependency of the oxygen saturation on the oxygen partial pressure (PO₂) and with its sigmoid shape, the curve is subjected to right or left shifts, thereby changing hemoglobin-O₂ affinity (Woyke et al., 2020). A shift to the left implies an increased oxygen affinity and, hence, tighter binding due to the higher oxygen saturation to the PO₂. On the other hand, a shift to the right corresponds to a decreased oxygen affinity and easier release of oxygen to the tissues. It is well known that the ODC shifts in response to changes in pH, PCO₂ and 2,3 diphosphoglycerate (Hamilton et al., 2004). The oxygen equilibrium curves (OEC) of all treated groups showed that there was a non-significant change in the curves of coconut, BV, BV then cholesterol (prophylactic) groups in comparison with the control group. The P₅₀ (the oxygen tension at which hemoglobin is 50% saturated with O₂) and the blood oxygen affinity did not significantly affect. The OEC of cholesterol and cholesterol before BV treated groups showed a left shift (increase the affinity of O₂ to hemoglobin), this may be due to alkalosis, which was indicated in the cholesterol treated group, an increase in pH shifts the curve to the left so that P₅₀ decreased so occur reducing in the unload of the O₂  to the tissues ,this finding is agreed with Buchwald (2000) who stated that high patient blood cholesterol concentrations were correlated with decreased blood transportation of O₂ and the curve of hemoglobin dissociation shifted to the left.

CONCLUSION

The present study indicates that BV has protective roles against the deleterious effects of cholesterol due to its high contents of antioxidant substances. In recommendation, The BV may be useful when taken as prophylactic to people who may suffer from high cholesterol levels in the blood.

Ethical statement: The study was conducted according to Approval No. 000051 from the Research Ethics Committee of the Faculty of Medicine, Benha University (REC-FOMBU) which operates according to international guidelines, including the declaration of Helsinki, Islamic Organization for Medical Sciences (IOMS), World Health Organization (WHO), and International Council on Harmonization and Good Clinical Practice (ICH-GCP).

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