Protective effects of honey and bee venom against lipopolysaccharide and carbon tetrachloride-induced hepatoxicity and lipid peroxidation in rats

Abstract

In the present study, the protective effects of honey and bee venom (BV) either independently or in combination against lipopolysaccharide (LPS) and carbon tetrachloride (CCl₄)-induced hepatoxicity, lipid peroxidation, and hematological alterations in male albino rats were investigated. In addition, histopathological alterations of hepatic tissues induced by LPS/CCL₄ were recorded. Sixty-four of male albino rats of average weight 120–150 g were included in this study. Rats were divided into eight equal groups of eight. The obtained results demonstrated that treatment with LPS/CCl₄ caused an increase in the levels of alpha-fetoprotein, which was accompanied by changes in the hepatic function biomarkers that characterized by the increased levels of transaminases (AST, ALT). The results showed oxidative stress as assigned by the increase in lipid peroxide. Meantime detraction in the antioxidants, including glutathione peroxidase was observed. Interruptions in biochemical parameters accompanied by disturbances in hematological parameters and liver histopathology were resulted due to exposure to LPS/CCl₄. This study showed the use of honey and BV provided a protective effect on hepatotoxicity induced by LPS/CCl₄. This might have been occurred through the reduction of hepatic transaminases and the “Alpha-fetoprotein” in serum and the equilibration of the antioxidation system, thereby, inhibiting the reactive oxygen species accumulation. Honey and BV administration reestablish disturbed hematological parameters and liver histopathology persuaded by LPS/CCl₄. More interesting, we demonstrated that using a combination of the honey and BV showed promising enhancement in their protective effects over the use of just one of the two reagents.

Graphical abstract

Introduction

Honey is a sweet, semifluid, viscous substance made from nectar. Honey contains mainly water and carbohydrates. Moreover, it comprises trace amounts of several vitamins and minerals. It contains calcium, copper, riboflavin, iron, magnesium, potassium, and zinc [1]. Honey is a good resource of antioxidants due to its abundant polyphenolic content, such as flavonoids and phenolic acids. Previous studies reported that honey has many beneficial effects that acting against several chronic diseases caused by oxidative injury. Recently, it has been confirmed the potential protective effect of honey against liver injury induced by oxidative stress occurring by chemical compounds [2]. Besides, the acidity of honey and its content of sugars and other nutrients are vital for the elevation of the healing process. It also increases the release of oxygen from hemoglobin (Hb) in the blood, oxygenation of the tissues being essential for the growth of new tissue [3]. In most cases, honey is used when conventional antibacterial treatment with antibiotics and antiseptics are unsuccessful [4].

Studies have shown that treatment with antioxidants reduces some of the complications arising from diabetes [5]. Several studies have also shown, diabetes (type 1) leads to reactive oxygen species (ROS) generation, which increases oxidative stress in the hippocampus of rats resulting in neuronal loss [6]. Anarkooli et al. [ 7] has concluded that treating rats with insulin, honey, alone, or in combination can inhibit the progression of neuronal damage in the diabetic hippocampus of rats as honey can fight cell membrane damage by neutralizing free radicals.

Erejuwa et al. [8] examined the impact of honey as an adjunct to glibenclamide or metformin (hypoglycemic agents) on glycemic control in streptozotocin-induced diabetic rats. They concluded that the combination of oral hypoglycemic agents with honey could be a useful adjuvant therapy to achieve and/or maintain glycemic control and probably reduce or delay the onset of diabetics. As daily supplementation of diabetic rats with honey, sufficient concentrations of certain minerals, such as zinc, selenium, copper, calcium, potassium, chromium, and manganese, may be achieved to stimulate pharmacological reactions and improve the secretion of insulin from the pancreas. Also, they exhibited that there were no significant alterations in the serum glucose concentrations in non-diabetic rats treated only with honey (1.0 g/kg b. wt. for 4 weeks) compared with non-diabetic control rats treated with distilled water.

The honey bee develops bee venom (BV) that represents a big part of the bee colony’s protection because it is a mixture of natural toxins. It can also protect bees from a wide range of pests, as it has an efficient and numerous mixes of constituents considered for that role. BV consists of different peptides including mast cell degranulating peptide, adolapine, melittin, and apamine. This also includes enzymes consisting of phospholipase A2 (PLA2), hyaluronidase, lysophospholipase, and α-D-glucosidase as well as non-peptides such as histamine, dopamine, norepinephrine, and protease inhibitors [9].

Previous studies have shown BV’s efficacy in treating other pathological disorders, such as cancerous tumors and arthritis and pain. Melittin consists of 26 residues of amino acids, representing almost half of BV’s dry mass. It was also shown that melittin has numerous properties in various cell types, particularly antibacterial, antiviral, and anti-inflammatory. In addition, melittin can cause the arrest of the cell cycle, inhibition of cell growth and apoptosis in various tumor cells [10]. Melittin has a significant inhibitory effect on the spirochete of Lyme disease at very low dosages [11]. Hyun Park et al. [12] indicated melittin may be used as a therapeutic agent for hepatic fibrosis treatment. While adolapin and protease inhibitors have anti-inflammatory activity, these substances are present in the whole BV in very small amounts. In addition, dopamine and noradrenaline that constitute around 12% of the BV increase the rate of rhythm [13].

In various studies on different pathological conditions, Ram et al. [14] and Hwang et al. [15] confirmed BV’s antioxidant potential. At the same time, Hanafi et al. [16] concluded that BV decreases the inflammation and the oxidative stress and induce insulin sensitivity which in turn ameliorates liver function parameters; ALT, AST, GGT, and bilirubin levels. Furthermore, the previous conclusion indicated that non-alcoholic fatty liver rats treated with BV showed significantly lower dose-dependent hepatic levels of malondialdehyde (MDA) compared to untreated rats. BV also seems to play a role in maintaining the lipid profile values, this effect was more prominent with cholesterol, LDL C, and HDL C, which were completely normalized even with the lowest dose [17].

Insulin use in poorly controlled type 1 diabetes may result in a medical condition known as glycogenosis or glycogenic hepatopathy, characterized by varying degrees of hepatomegaly, abdominal pain, and serum aminotransferase elevations [18]. A further medical problem increased insulin resistance is often associated with chronic liver failure and is a pathophysiological feature of hepatogenic diabetes [19]. Interestingly, Hassan et al. [20] showed that BV might have therapeutic and protective effects on the management of glucose level in diabetic rats enhance insulin secretion, and improved several biochemical and histological disorders resulted due to diabetes metabolic disorder. This could be either through varying ways including suppression of the inflammation of pancreatic β-cell, antioxidant activity, or insulin secretion promotion.

Carbon tetrachloride (CCl₄) is a colorless liquid produced by the reaction of chloroform with chlorine, also it is mainly produced from methane [21]. Previously, carbon tetrachloride was widely used in fire extinguishers, as a precursor to refrigerants and as a cleaning agent, but because of toxicity and safety worries, it has been gradually stopped. Exposures of humans to higher levels of inhalation or oral exposure to carbon tetrachloride induces several damages, especially in the liver and kidney. As carbon tetrachloride induces free radicals and triggers a peroxide chain reaction, it is usually used to induce liver injury models in animals [22].

Lipopolysaccharide (LPS) are glycolipids found on the outer membrane of all gram-negative bacteria and have the capability to incite a strong inflammatory response [23]. It is reported that the bacterial LPS has been commonly used in experimental models of endotoxic shock [24]. Throughout endotoxic shock, the oxygen-derived radicals are produced and induce tissue damage. In addition, nanograms of LPS injected into the blood stream of human can result in all the physiological indicators of septic shock [25]. Several studies demonstrated that during endotoxic shock, the liver is the main organ of bacterial endotoxin LPS detoxification [26]. Moreover, this process is accompanied by hepatocytes damage being started by the LPS induced stimulation of phagocytic cells, such as polymorphonuclear neutrophils and Kupffer cells [27].

To the best of our knowledge, no study has been conducted on using both LPS and CCl₄ to induce hepatotoxicity and lipid peroxidation in rats and the use of honey and/or BV as protective agents. Therefore, our current study focuses on exploring the predictable therapeutic benefits of the co-administration of honey and BV on hepatotoxicity and lipid peroxidation, as well as hematological and liver histopathology induced by LPS and CCl₄. In addition, the efficiency of using only one of the protective reagents in improving the injury induced by LPS and CCl₄ is investigated.

Materials and Methods

Experimental animals

Ethics of animal study

All experimentation, transportation, and care of the animals of this study were performed in compliance with the formal approval of the Faculty of Science, Minia University’s policy on animal use and ethics. All required laboratory health and safety measures have been complied with while conducting the experimental work described in this study.

Sixty-four adult male albino rats (Dawely), with an average weight 120–150 g were used at the current work. The rats were obtained from the Animal House of the Faculty of Agriculture, Minia University, Minia, Egypt. Rats were provided a commercial rodent diet containing all the necessary healthy elements. Food and water were available throughout the experiment. The rats were housed in a well-ventilated room at 25 ± 3°C in clean plastic cages for 3 weeks before the commencement of the experiment as an acclimatization period.

Chemicals

LPS were extracted from (Escherichia coli serotype O127:B8), purchased as a lyophilized powder from Sigma-Aldrich chemical, and purified by phenol extraction. Carbon tetrachloride (CCl₄, > 99.5%) was purchased from Sigma Aldrich. Honey (moisture 60%), lyophilized whole BV, and olive oil were purchased from GHADA Company, Borgalarb, Alexandria, Egypt. All other chemicals and reagents used were of analytical grade.

Treatment

LPS was prepared immediately by dissolving in 0.9% saline and was injected intraperitoneally at a single dose (1 mg/kg b. wt.) [27]. The LPS solution was prepared by dissolving 3 mg of LPS in 15 mL saline and then 0.5 ml of this solution was injected per 100 g of rats. Moreover, CCl₄ (0.5 mg/kg b. wt.) was injected intraperitoneally two times per week for 4 weeks [28–31]. A solution of CCl₄ in olive oil in a ratio of [1, 1] was prepared by dissolving 100 mg of CCl₄ in 100 ml of olive oil then 0.5 ml of the obtained solution was injected intraperitoneally per 1Kg b. wt. of rats. We have chosen these doses of LPS and CCl₄ based on previous studies on rats showing that intraperitoneal injection of these toxic agents produced a pronounced liver injury [27–31].

Honey (25 mg/kg b. wt.) diluted in sterile distilled water and administered orally to rats every day for 2 months [32]. The honey solution was prepared by dissolving 40 mg of honey in 16 ml of sterile distilled water then 1 ml of the resulted solution was given orally per 100 g of rats. In addition, BV was dissolved in sterile distilled water and then injected intraperitoneally at a dose of (1 mg/kg b. wt.) every day for 2 months [20, 32]. The BV solution was obtained by dissolving 1.6 mg of BV in 4 ml sterile distilled water than 0.25 ml of the prepared solution was injected per 100 g ofrats.

Experimental design

The rats were divided into eight groups of eight animals each as follows:

Group 1 (control group): rats were fed on a balanced diet during the experimental period without any treatment.

Group 2 (honey treated group): rats were administered orally with honey (25 mg/kg b. wt.) every day for 2 months.

Group 3 (BV treated group): rats were injected intraperitoneally with BV (1 mg/kg b. wt.) every day for 2 months.

Group 4 (honey + BV treated group): rats were treated with orally administration of honey (25 mg/kg b. wt.) along with intraperitoneally injection of BV (1 mg/kg b. wt.) every day for 2 months.

Group 5 (LPS + CCl₄ group): initially, rats were injected intraperitoneally with a single dose of LPS (1 mg/kg b.wt.) followed by an intraperitoneal injection of CCl₄ (0.5 mg/kg b.wt.) two times a week for 4 weeks (to assure liver injury models in animals).

Group 6 (LPS + CCl₄ + honey treated group): rats received the same treatment as described above in Group 5 and honey was co-administered orally (25 mg/kg b. wt. daily) for 2 months.

Group 7 (LPS + CCl₄ + BV treated group): rats were administered with LPS + CCl₄ as previously cited and BV was co-administered intraperitoneally injection (1 mg/kg b. wt. daily) for 2 months.

Group 8 (LPS + CCl₄ + honey + BV treated group): rats received the same treatment as described above in Group 5 then honey + BV were co-administered for 2 months under the same procedure and dosage, as described above.

Measurement of body weights

All animals were weighed by automatic balance (ANDGX-600, Japan) at the beginning of the experiment and before dissection.

Collection of blood samples

By the end of the experimental period, all animals were scarified under diethyl ether anesthesia at fasting state, after which blood samples are taken. Whole blood samples were immediately collected with anticoagulant EDTA for hematological analysis. Plasma samples were separated through centrifugation of the blood for 10 min at 4000 rpm using EDTA as an anticoagulant. Serum samples were separated via centrifugation of the blood samples at 4000 rpm for 10 min. Plasma and serum samples were kept frozen at −80°C for consequent biochemical analysis.

Biochemical estimation

Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were determined calorimetrically by spectrophotometer according to Gella et al. [33] using reagent kits purchased from Bio-Systems chemical, Egypt. Plasma lipid peroxide (LPx) levels and glutathione peroxidase (GPx) activities were determined calorimetrically according to Ohkawa [34], Paglia, and Valentine [35], respectively, using Bio-Diagnostic kits. Alpha-fetoprotein (AFP) quantitative measurement was performed in serum by using ELISA kit according to the method described by Sato et al. [36] using an enzyme immunoassay kit purchased from CALBIOTECH, a life science company.

Evaluation of hematological parameters

Hematological parameters [total number of red blood cells (RBCs), Hb content, mean values of packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), platelets (PLTs), the mean corpuscular hemoglobin concentration (MCHC) and the total number of white blood cells (WBCs)] were analyzed using an automatic hematology analyzer (Celltac, NEK-6510 k). Each sample has been run in duplicate.

Histopathology

For histopathological examination and optical microscopic examination, the rats were dissected and the liver samples were fixed in 30% formal saline for 24 h, processed using a graded ethanol series, and embedded in paraffin, sectioned (5 μm), and finally stained with hematoxylin and eosin. The sections were viewed and photographed using an Olympus optical microscope (Olympus CH20BIMF200, Olympus Optical Co. Ltd., Japan) with an attached photograph machine (MicroCam PHD-5MP).

Statistical analysis

The results of the present study were analyzed using SPSS version 22 for Windows. The significance was designed via a one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison procedure. The results were expressed as the mean ± SE, and P < 0.05 was measured as the level of significance.

Results

Morbidity and mortality

During the experiment, nearly all treated rats from Group 5 (LPS + CCl₄) showed some toxicity signs such as bleeding of eyes and nose with closed eyes and general body weakness. In addition, the bodyweight increase in examined rats with LPS + CCl₄ was less as compared to the rest of the experimental rats. Moreover, no death was observed in any of the experimental groups.

Evaluation of biochemical parameters

Changes in ALT, and AST activities

As shown in Figures 1 and 2, the results of the present study showed that there were no significant alterations in ALT, AST levels in groups treated with honey (Group 2), BV (Group 3), or their combination (Group 4) compared to the normal control group (Group 1). Whereas ALT, AST levels in the LPS + CCl₄ group (Group 5) were demonstrated a significant increase (P ˂ 0.05) compared to the control group. The percentages of increase were 660.34% and 545.27% respectively. However, in LPS + CCl₄ + honey group (Group 6) and in the LPS + CCl₄ + BV group (Group 7), ALT and AST levels were shown to be significantly decreased compared to the LPS + CCl₄ group (Group 5). The percentages of decrease were 54% and 62%, respectively. In addition, the treatment of the LPS + CCl₄ group with honey plus BV (Group 8) showed a significant decrease in ALT and AST levels, and the percentage decreased was 77% as compared to the LPS + CCl₄ group (Group 5). This decrease was demonstrated by marked improvements that were almost attainable to normal levels.

Figure 1.

Serum ALT enzyme activities of control and experimental rats, data are presented as (mean ± S.E, n = 8), significance at P ˂ 0.05, (a) significantly different from control group, (b) significantly different from LPS + CCl₄ group.

Figure 2.

Serum AST enzyme activities of control and experimental rats, data are presented as (mean ± S.E, n = 8), significance at P ˂ 0.05, (a) significantly different from control group, (b) significantly different from LPS + CCl₄ group.

Changes in AFP activity

The findings of this study (Figure 3) demonstrated non-significant changes in the level of AFP in groups treated with honey, BV, or their mixture (Groups 2, 3, and 4) compared with the control group. Whereas a significant increase in AFP level was observed in the LPS + CCl₄ group (Group 5), and the percentages of increase was 421.29% as compared to the control group. Treatment of the LPS + CCl₄ group with honey (Group 6), BV (Group 7), and both (Group 8) helped to bring the AFP values close to the control group value (group 1). The percentages of decrease in the treated Groups 4, 5, and 6 were 71, 76, and 82%, respectively, compared to LPS + CCl₄ treatedrats.

Figure 3.

Serum AFP enzyme activity of control and experimental rats, data are presented as (mean ± S.E, n = 8), significance at P ˂ 0.05, (a) significantly different from control group, (b) significantly different from LPS + CCl₄ group.

Changes in oxidative stress parameters

No statistically significant changes in LPx and GPx levels were found in groups treated with honey and BV alone or together compared to the normal untreated group. Changes in LPx and GPx levels in the treated experimental groups were summarized in Figures 4 and 5, respectively. A significant increase in LPx level was observed in the LPS + CCl₄ group (Group 5), and the percentages of increase were 230.08% as compared to the control group. Whereas a significant decrease in GPx level was observed in the LPS + CCl₄ group (Group 5) with the percentages of decrease 73.69% as compared to the control group. In addition, the administration of honey and BV to the LPS + CCl₄ group as represented in Groups 6 and 7, respectively, showed a significant improvement in the oxidative stress parameters examined compared to the LPS + CCl₄ group (Group 5). In addition, the administration of honey together with BV (Group 8) prevented the increase and decrease in LPx and GPx levels significantly correspondingly after comparison with LPS + CCl₄ group. The LPx levels decreased to 47, 39, and 52% but the GPx levels increased to 101,131, and 327% in the treated groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treatedrats.

Figure 4.

Plasma Lipid peroxide (LPx) activity of control and experimental rats, data are presented as (mean ± S.E, n = 8), significance at P ˂ 0.05, (a) significantly different from control group, (b) significantly different from LPS + CCl₄ group.

Figure 5.

Plasma glutathione peroxidase (GPx) of control and experimental rats, data are presented as (mean ± S.E, n = 8), significance at P ˂ 0.05, (a) significantly different from control group, (b) significantly different from LPS + CCl₄ group.

Our results in Figure 6 summarized the percentage changes in the biochemical parameters examined in the LPS + CCl₄ + honey group, LPS + CCl₄ + BV group, and CCl₄ + LPS + honey + BV group relative to LPS + CCl₄ group.

Figure 6.

The percentage changes in the biochemical parameters examined in the LPS + CCl₄ + honey group, LPS + CCl₄ + BV group and CCl₄ + LPS + honey + BV group relative to LPS + CCl₄ group.

Changes in hematological parameters

The obtained data concerning the changes in hematological parameters are presented in Table 1. A non-significant alteration in RBCs count, Hb concentration, MCV, MCH, MCHC, PCV values, WBCs count, and PLTs count in groups treated with honey (Group2), BV (Group 3), and their combination (Group 4) compared to the normal control group. Also, the results showed a significant decrease in RBCs count, Hb concentration, MCV, MCH, MCHC, and PCV values in LPS + CCL₄ treated group when compared with control group at (P < 0.05), and the percentages of decrease were 31.33, 31.36, 14.64, 16.30, 23.53, and 22.36%, respectively. While this treated group showed a significant increase in WBCs count and PLTs count compared with control group at (P < 0.05) with the percentages of increase 49.92% and 37.83%, respectively.

Table 1.

Hematological parameters of control and different experimental groups

Parameters	Groups
	G1	G2	G3	G4	G5	G6	G7	G8
RBCs (106/μl)	6.681 ± 0.110	6.560 ± 0.202b	6.645 ± 0.200b	6.671 ± 0.174b	4.587 ± 0.196a	5.571 ± 0.213b	6.020 ± 0.145b	6.288 ± 0.128b
Hb (g/dl)	12.18 ± 0.186	11.91 ± 0.156b	12.04 ± 0.152b	12.53 ± 0.159b	8.36 ± 0.298a	10.04 ± 0.178b	10.11 ± 0.117b	11.15 ± 0.121b
MCV (fl)	47.67 ± 0.256	46.34 ± 1.19b	47.64 ± 0.286b	47.66 ± 0.204b	40.69 ± 1.62a	46.40 ± 0.228b	46.01 ± 0.232b	47.34 ± 0.152b
MCH (Pg)	16.69 ± 0.183	16.91 ± 0.151b	16.92 ± 0.124b	17.19 ± 0.112b	13.97 ± 0.220a	16.09 ± 0.145b	16.57 ± 0.153b	17.05 ± 0.191b
MCHC (g/dl)	35.19 ± 0.456	35.25 ± 0.299b	34.93 ± 0.352b	35.98 ± 0.271b	26.91 ± 0.342a	30.09 ± 0.407b	30.66 ± 0.210b	35.40 ± 0.163b
PCV%	41.50 ± 0.885	42.31 ± 0.822b	42.77 ± 0.696b	72.79 ± 0.861b	32.22 ± 0.384a	36.85 ± 0.482b	37.84 ± 0.425b	39.60 ± 0.592b
PLTs (103/μl)	329.80 ± 11.33	321.35 ± 4.92b	335.38 ± 8.08b	324.96 ± 5.54b	454.55 ± 12.97a	343.24 ± 12.25b	385.85 ± 15.67b	326.88 ± 4.85b
WBCs (103/μl)	6.53 ± 0.157	6.57 ± 0.132b	7.34 ± 0.139b	6.96 ± 0.097b	9.79 ± 0.265a	8.43 ± 0.174b	8.49 ± 0.144b	8.19 ± 0.146b

^aSignificantly different from control group.

^bSignificantly different from LPS + CCl4 group.

Data are presented as follows (mean ± SE, n = 8). Significance at P ˂ 0.05. G1: control, G2: honey only, G3: BV only, G4: honey + BV, G5: LPS + CCL4, G6: LPS + CCL4 + honey, G7: LPS + CCL4 + BV, G8: LPS + CCL4 + honey + BV.

The Groups 6, 7, and 8 exhibited a significant increase in RBCs count, Hb concentration, MCV, MCH, MCHC, and PCV values when compared with LPS + CCL₄ treated group. While WBCs count and PLTs count decreased significantly in Groups 6, 7, and 8 when compared with LPS + CCL₄ treated group. No statistically significant changes were observed in all parameters when control group was compared to Groups 6, 7, and 8.

The percentage changes in the hematological parameters examined at the LPS + CCl₄ + honey group, LPS + CCl₄ + BV group, and CCl₄ + LPS + honey + BV group relative to LPS + CCl₄ group described as follow. The percentages of increase in RBCs count were 21.45, 31.24, and 37.08% in the treated groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treated rats. The percentages of increase in Hb concentration were 20.10, 20.93, and 33.37%, also the percentages of MCV increased to 14.03, 13.07, and 16.34% in the treated groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treated rats. MCH percentages increased to 15.18, 18.61, and 22.05%, and MCHC percentages increased to 11.82, 13.94, and 31.55% in the treated groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treated rats. PCV values were changed to 14.37, 17.44, and 22.91% in the treated groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treated rats. However, the WBCs count decreased to 13.89, 13.28, and 16.34% and the PLTs count decreased to 24.49,15.11, and 28.09% in the treated Groups 4, 5, and 6, respectively, compared with the LPS + CCL₄ treatedrats.

Liver histopathology

Liver sections of normal control (Fig. 7A), honey-treated group (Fig. 7B), BV-treated group (Fig. 7C) and honey + BV-treated group (Fig. 7D) showed normal hepatic architecture with normal plates of hepatocytes radiating from the central vein (CV). The hepatocyte plates distinguished by blood sinusoids were lined with endothelial cells and von Kupffer cells. Liver section of LPS + CCl₄ treated rats showed some histopathological changes, such as congested CV with hemolyzed blood, swollen hepatocytes with severe cytoplasmic vacuolation, and increased inflammatory cell infiltration (Fig. 8 A). As shown in Figure 8B, honey administration has reduced some hepatic damage. The liver section showed moderate congestion of the CV with an irregular wall and a slight loss of hepatic architecture in some areas with a very low percentage pattern of hepatocellular vacuolation. The LPS + CCl₄ + BV (Fig. 8C) treated group showed marked improvement in liver damage, such as normally arrangement of hepatocyte strands in many areas with no vacuolations and less congestion in the CV. As shown in Figure 8D, the liver retained its architecture to a large extent. Normal hepatocytes with central rounded nucleus with no cytoplasmic vacuolation observed, indicating an excellent protective effect of the mixture of honey and BV against hepatic lesions compared to LPS + CCl₄ treatedrats.

Figure 7.

Liver sections of (A) control group, (B) honey treated group, (C) BV treated group, and (D) honey + BV treated group showing normal CV, normally arranged hepatocytes cords with intervening non-congested sinusoids (black arrows), with normal hepatocytes with round nucleus (yellow arrows), (H&E, ×400)

Figure 8.

(A) Liver section of LPS + CCl₄ group showing congested central vein with hemolyzed blood with interrupted wall (CV), vacuolation of hepatocytes with clear cytoplasm (yellow arrows), inflammatory cellular infiltrations (red arrows), (H&E, × 200), (B) Liver section of LPS + CCl₄ + Honey treated group showing mild improvement, central vein with an irregular wall (CV), mild vacuolation of hepatocytes (yellow arrows), sinusoidal spaces slightly normal (black arrow), (H&E, × 400), (C) Liver section of LPS + CCl₄ + BV treated group showing less extent of hepatocellular hypertrophy, central vein with an irregular wall with a slight congestion (CV), hepatocytes with central rounded nucleus (yellow arrows), (H&E, × 400) and (D) Liver section of LPS + CCl₄ + Honey + BV treated group showing central vein with few RBCs (CV), normally arranged hepatocytes cords with intervening non-congested sinusoids (black arrow), hepatocytes with central rounded nucleus (yellow arrows), (H&E, × 400).

Discussion

The liver is responsible for many biological roles, including breaking down and digesting food and also detoxifying chemicals. Liver enzymes tests can determine if the liver is working well or not. Both ALT and AST, as indicator enzymes, are also important signs of liver damage. It is well known that ALT activity is the most common biomarker of hepatotoxicity when the level of this enzyme is elevated in serum, so the determination of this enzyme is a more specific test for detecting liver abnormalities, since it mainly distinguishes the necrosis of the hepatocellular [37].

AST is another liver enzyme that also helps in detecting necrosis of hepatocytes, but it is still a less specific indicator enzyme for liver cells damage [38]. Our findings showed a prominent increase in the levels of ALT and AST after the treatment of LPS and CCl₄. This comment came in agreement with several previous reports which had shown that LPS and CCl₄ induced hepatotoxicity with a consequent reduction in enzyme biosynthesis causing permeability variations and leakage of lysosomal enzymes persuading the release of enzymes [39]. In addition, it was reported that the liver is the main goal stimulated by the strong amount of the inflammatory molecules released by LPS leading to liver injury inflammation [40]. So, the elevation of ALT and AST levels in this study suggested a probable liver tissue damage due to LPS and CCl₄ toxicity.

Our results showed that honey can antagonize LPS and CCl₄-induced increase ALT and AST levels to reach normal values. These results are in good agreement with the previously reported studies of Onochie et al. [41] who reported that honey caused a significant decrease in ALT and AST levels due to its organic therapeutic biomolecular agents, such as kaempherol, quercetin, chrysin, luteolin, apigenin, and vanillic acid. Moreover, several research projects have reported that the importance of these organic agents in hepatic and biliary medicine [42]. The abovementioned organic agents probably reduce these liver enzymes by maintaining the biomembrane integrity, reducing free radical species, and regulating the metabolic processes of the liver [43]. In the same line, Wang et al. [2] indicated the hepatoprotective effect of vitex honey against paracetamol-induced liver damage. They suggested that vitex honey may be capable of stimulating the regeneration of hepatic tissue, this, in turn, can impair the cellular structure and the function of the liver cells.

Additionally, our results demonstrated that BV treatment significantly decrease ALT and AST levels to the standard range. These results are in consistent with the previous studies Salman et al. [44] who showed that treatment with BV had significantly decreased the elevation of serum ALT, AST levels in rats exposed to gamma radiation (5 Grays) indicating the hepato-protective effect of BV. This might be described by the reduction of elevated hepatic nuclear factor kappaB (NF-kB) expression in liver [45]. Additional studies demonstrated that BV can inhibit the secretion of pro-inflammatory cytokines and reducing the raised serum aminotransferase enzymes in different models of induced hepatic injury [46]. Moreover, Kim et al. [47] reported that PLA2 (phospholipase A2) as a component of BV may have therapeutic potential in acetaminophen-induced hepatotoxicity through modulation of Tregs (CD4 + CD25 + Foxp3 + T cells) and IL-10 (interleukin-10) in mice. Kim et al. [48] reported that PLA2 induced anti-inflammatory cytokine production in acetaminophen-injected mice, that could afford a protection against hepatic dysfunction. Furthermore, Lee et al. [49] provided evidence that melittin (a major peptide component of BV) may protect against acute hepatic failure, as it decreased the high rate of lethality, alleviated hepatic pathological injury, attenuated hepatic inflammatory responses, and inhibited hepatocyte apoptosis.

Our data obtained in the present study showed that the reduction of investigated liver enzymes levels by the combination of honey and BV was more pronounced and suggested that treatment with honey and BV might be a reasonable therapeutic strategy to deal with LPS/CCl₄-induced liver damage.

AFP is a fetal glycoprotein associated with the tumor. In the present study, serum level of AFP was significantly increased in hepatotoxicity group compared with the normal control group. AFP is considered as a promising marker for early detection of hepatic damage and treatment evaluation [50]. Therefore, the observed increase in AFP is an indicative of the hepatic damage than to the development of hepatocellular carcinoma (HCC) [51]. This is parallel with the previous studies [52, 53] that reported the increase in AFP level in CCl₄ intoxicated rats compared to controlrats.

In the present study, the treatment of intoxicated rats with honey showed a significant decrease in AFP level compared to hepatotoxicity group, which is indicative of improvement of liver function enzymes activity. This may be ascribed to the anticancer effect of honey [54]. The present results are in accordance with Mohamed et al. [50] who displayed that honey either alone or with other anticancer drugs markedly decreased the AFP level compared to rats treated with DEN (diethylnitrosamine)/CCl₄. The reported anticancer effect of honey may result from inhibition of DNA synthesis or down-regulation of matrix metalloproteinase levels (MMP-2 and MMP-9), which are involved in the induction of angiogenesis process, apoptotic, and cytotoxic effects [55]_. Moreover, honey constituents, as polyphenols have shown complementary and overlapping mechanisms of chemoprotective activity in multistage carcinogenesis [56].

From the results of Group 7, it was observed that the treatment with BV reduced the AFP level compared to the hepatoxicity group. Several studies have suggested that BV has potential anticancer effects which can be easily related to the remarkable decrease of AFP as a marker of HCC [57]. Heinen and da Veiga [58] suggested that both BV and its individual constituents especially melittin had potential for cancer therapy, wherever they have shown significant efficacy of inhibition of cancer cell metastasis and invasion. The current study showed that the administration of honey either alone or with BV markedly decreased the AFP level compared to rat treated with each of them separately.

LPS and CCl₄ administration at the current study showed an increase in the level of LPx and a decrease in GPx activity. The level of LPx measured is used to estimate the oxidative damage in patients with liver injury [59], where it is considered as one of the most important indices of oxidative stress. Several previous studies had reported that LPS administration is known to increase the lipid peroxidation in many tissues of rats [60]. Mohamed et al. [50] showed that the increase of LPx together with a significant decrease of antioxidant enzymes including GPx in DEN/CCl₄ group compared to control. Moreover, it is well reported that CCl₄ activated by the liver cytochrome P-450 to generate free radicals such as trichloromethyl free radical (CCl₃^*), and trichloromethyl proxy free radical (CCl₃OO^*) as two metabolites related to ROS generation, lipid peroxidation, and decrease of catalase (CAT), (superoxide dismutase) SOD, glutathione transferases (GST), and GPx enzymatic activities [61].

The current study also showed a significant reduction in LPx level and a significant increase in the antioxidant enzyme GPx after medicative of honey to LPS and CCl₄ (Group 6). This observation may be attributed to the presence of phenolic compounds that hold the key to the antioxidant property of the honey [62]. Oliveira et al. [63] reported that phenolic compounds have hydroxyl groups connected to the aromatic ring that can act as hydrogen donors in the scavenging of free radicals. Wang et al. [2] reported that phenolic compounds show pro-oxidative action by promoting the production of hydrogen peroxide that induces oxidative stress. Moreover, this pro-oxidative action confers cells with resistance to successive oxidative damage and the organization of cell functions. So, the hepatoprotective effect of vitex honey maybe, at least in part, due to its antioxidative property and/or perhaps pro-oxidative action.

Additionally, honey has been found to contain other antioxidants including glucose oxidase, catalase, ascorbic acid, carotenoid derivatives, organic acids, amino acids, and proteins [64, 65]. So, honey may minor the hazards and effects of acute and chronic free radical-induced pathologies in vivo via the cooperative action of its antioxidants [66]. Therefore, according to the current study along with other previous researches, honey has the capability to decrease significantly the levels of LPx [67] and restore activities of GPx [68].

In the present study, the treatment of hepatotoxic treated rats with BV caused a significant decrease in the elevated LPx level associated with an elevation in the declined GPx activities, indicating that BV has potential antioxidant and anti-inflammatory properties. According to recently published data, BV has strong antioxidant activities such as free radical scavenging activity and inhibition of lipid peroxidation [69].

Salman et al. [44] also stated that BV therapy is a potent antioxidant led to a decline in the levels of ROS, which may be related to the observations of BV affecting glutathione, superoxide dismutase, and catalase. The antioxidant potential of BV had been confirmed in different studies carried on various pathological conditions [16, 70]. Furthermore, constituents of BV such as melittin, mast-cell degranulating peptide, phospholipase A2 (PLA2)-related peptide and apamin have been reported to possess anti-inflammatory responses, as they have the ability to decrease pro-inflammatory cytokines such as tumor necrosis factor and interleukins, along with other inflammatory mediators including prostaglandin and nitric oxide, which are synthesized by cyclooxygenase and inducible nitric oxide synthase, correspondingly. Sobral et al. [69] reported that tissue inflammation in many diseases correlated with the production of such mediators.

According to the results of Group 6 and through the antioxidative stress of honey, it was found that treatment with honey alone increased the reduced value of GPx as a major antioxidant enzyme, which contributes to ROS scavenging, accordingly providing protection against oxidative stress injury. Additionally, the prior induced effects were more effective in the case of treatment with honey and BV (Group 8).

Our results showed that the treatment of LPS + CCL₄ in rats resulted in a significant decrease in RBCs count along with Hb, MCV, MCH, MCHC, and PCV parameters and a significant increase in WBCs count and platelet count compared to the control group. Chew and Park [71] reported that because of the plasma membranes of the cellular elements of the blood contain a high percentage of polyunsaturated fatty acids, they are sensitive to oxidative stress. Therefore, the decrease in RBCs count along with other measured hematological parameters might be attributed to the hepatic damage induced by free radicals such as trichloromethyl free radical (CCl3^*), and trichloromethyl proxy free radical (CCl3OO^*) [61]. Also, Hussein et al. [72] showed that decreased Hb content confirmed the damage caused by free radicals to the erythrocyte membrane, which may also contribute to the eventual release of Hb from the cells. Following treatment with LPS + CCL₄, decreased MCV, MCH, and MCHC recommended that microcytic hypochromic anemia may have been involved [73]. The increase in WBCs count in LPS + CCL₄ treated rats observed in this study may be considered to be a defensive mechanism of the immune system verifying Hoeney’s report [74]. In the same line, reports of Elshater et al. [75] stated that the ability of such free radicals to affect the defense mechanism of treated rats has led to disruptions in the WBCs count.

The results of the present study suggested that the treatment of honey, BV, and the mixture of both (Group 6, 7, and 8, respectively) improved the hematological blood parameters as compared to Group 5. This observation was reflected by a significant increase (P ≤ 0.05) in RBCs count, Hb, MCV, MCH, MCHC, and PCV levels and a decrease in WBCs count and PLTs count.

Honey is a natural antioxidant, which may contain flavonoids, ascorbic acid, tocopherols, catalase, and phenolic compounds all of which work together to provide a synergistic antioxidant effect, scavenging, and eliminating free radicals [76, 77].

Our results are consistent with previous reports [78, 79] that have shown that honey attenuates gentamicin-induced hematological disturbances, as well as it significantly recovered Hb, RBC, PCV, platelet, and WBC levels close to control in Wister albino rats fed hydrocarbon-contaminated diets [80]. Also, honey and royal jelly have a defensive function against several drugs, wherever honey serves to protect organs by improving some hematological parameters (RBCs, WBCs, and Platelets) due to decreased LPS in rats [81]. More interesting, Wayar et al. [82] showed that the administration of honey and royal jelly prevented injury to the blood cells by maintaining the integrity of the cells.

The tested dose of BV improved the impairment of hematological parameters alone or mixed with honey. Zainab and Ahmed [83] have been reported that BV attenuates the development of arthritis by improving some blood and biochemical parameters. The results showed a significant increase (P ≤ 0.05) in RBCs, Hb, and non-significant increase in hematocrit value in the arthritis group treated with BV at a dose of 1 mg/kg B.W compared to the arthritis group. It has previously concluded that BV has the ability to increase coronary and peripheral circulation and improve blood circulation in micro blood vessels, as well as a significant role in stimulating erythrocyte building was detected [84]. The previous conclusion is supported by the results of Salman et al. [44], who showed that BV plays a preventive role in the protection of the body from radiation and contributes to the improvement of RBCs in rats that are exposed to gamma radiation. BV decreased the total number of WBCs in the arthritis group treated with BV compared to the arthritis group, which may be attributed to the immunosuppressive effect of BV [85].

BV has antioxidant effect, where antioxidants act to mitigate the damage caused by oxidative stress [86], oxidative stress happens as a result of inflammatory cytokines, in particular IL-6, which cause an increase in the number of PLTs as it works on the maturation of generated cells of PLTs, More interesting, BV tends to inhibit the activity of IL-6 and therefore less PLTs are found in the BV-treated group [86].

Histopathological observations of the liver were further supported by biochemical studies. In this study, the control, honey, BV-treated groups showed normal liver tissue, while the LPS and CCL₄-treated groups (Group 5) caused impressive liver damage, marked by congested and CV with hemolyzed blood, swollen hepatocytes with severe cytoplasmic vacuolation, increased inflammatory cell infiltration and loss of architecture. However, treatment with honey, BV and their combination reduced histopathological changes with further improvement.

Previous study of Iida et al. [87] showed that LPS intoxication provides symptoms of hepatotoxicity and liver failure, like disturbance of the structural stability of the hepatic cell membrane and release of several enzymes including AST, ALT, lactate dehydrogenase, and alkaline phosphatase into the bloodstream of the impacted necrotic and dead hepatocytes. In our present study, the observation of elevated serum AST and ALT levels in Group 5 is in agreement with this study.

As previously reported by Uskokovic-Markovic et al. [88], CCl₄ induced severe injury to the liver due to oxygen-free radicals, leading to necrosis and cirrhosis, which may explain inflammation in the liver. The ultrastructural conclusions of Shah et al. [89] also showed that the CCl₄-treated group exhibited several pathological alterations in the organelle structure and edema cytoplasmic matrix likely due to changes in the cellular membrane caused by lipid peroxidation. Our results are consistent with these conclusions, where the LPx level of Group 5 was higher than the normal control group, which consequently supports these histological results. From our results, the administration of LPS along with CCL₄ was effective in the preparation of experimental animals (Group 5–8) with acute liver injury for our study purposes.

Our results showed that the administration of honey (25 mg/kg b. wt.) and BV (1 mg/kg b. wt.) daily for 2 months reduced hepatic damage caused by LPS + CCL₄. However, the greatest improvement was in Group 8, where the mixture of honey and BV improved histological disorders to become more similar to normal control rats. In Group 8, there was no obvious vacuolation of hepatocytes and inflammatory cell infiltration, but the CV with an irregularwall.

As previously reported the administration of Sumbawa forest honey (75 mg/kg b.wt.) can decrease hepatic cells damage induced by Lead acetate [90]. In the same line, Mohamed et al. [50] reported that the treatment of DEN/CCL₄-intoxicated rats with honey caused less CV congestion and a clear enhancement in the cell structure. The main components of the honey are polyphenol compounds, such as flavonoids. They are capable of inhibiting oxidation reactions through a radical scavenging mechanism by donating one electron to unpaired electrons in free radicals to reduce the number of free radicals [91]. Flavonoids are also assumed to have an impact on inhibiting liver failure by binding free radicals to reduce their impact on the liver. Therefore, in our study, the degeneration of the hepatocytes was decreased as the trichloromethyl free radical (CCl3^*), and trichloromethyl proxy free radical (CCl3OO^*) could not react with polyunsaturated fatty acid liver cell membrane to initiate lipid peroxidation.

Lee et al. [49] reported that BV inhibits CCL₄-induced hepatic fibrosis by suppression of fibrogenic cytokines in models of induced liver fibrosis. PLA2 is an important component of BV, triggered anti-inflammatory cytokine manufacturing, and therefore protected against liver failure in acetaminophen-treated mice [47]. Also, Park et al. [92] has shown that melittin (the major component of BV), prevented D-galactosamine/LPS-induced liver failure through suppressing apoptosis and inflammatory response in the liver of the animal. The results of the present study showed the hepatoprotective effect of both honey and BV against LPS and CCL₄ liver damage. The protective effect may be due to their antioxidant properties and inhibition of the secretion of pro-inflammatory cytokines, such as TNF-α and IL-1β.

Conclusions

From this current research, the administration of honey and/or BV showed a significant hepatoprotective potential effect against LPS and CCl₄ induced hepatoxicity in rats. The statistical analysis obtained in the current study showed positive percentages of improvement in liver function enzymes to some extent. Our findings also showed that the administration of honey and BV to the LPS/CCL₄ group resulted in the enhancement in GPx and the oxidative stress biomarker, LPx, suggesting their antioxidant role. It was also revealed that the administration of honey and BV individually or together showed the highest percentages of improvement in the hematological and histopathological changes in the liver as a result of LPS/CCL₄ exposure.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This research received no external funding.