The modulatory effect of bee honey against diethyl nitrosamine and carbon tetrachloride instigated hepatocellular carcinoma in Wistar rats

Abstract

Hepatocellular carcinoma (HCC) is a serious threat to human health that has attracted substantial interest. The purpose of this study was to investigate the modulatory effect of bee honey against induced HCC by diethylnitrosamine/carbon tetrachloride (DEN/CCl4) in rats. HCC was induced by a single intraperitoneal dose of DEN (200 mg/kg B.W). Two weeks later, CCl4 (1 ml/kg) was intraperitoneally injected (three times a week). Bee honey was administered orally at 2 g/rat before and after the induction of HCC. The results showed that bee honey administration significantly increased body weight, decreased liver weight, and relative liver weight compared to those in the HCC-induced group. Moreover, a significant decrease in serum alpha-fetoprotein (AFP) as well as AST, ALT, GGT, ALP activities were observed in bee honey administration rats compared with those in HCC-induced group. Also, the hepatic MDA was significantly decreased; in addition, SOD, CAT, and GPx activities were significantly increased in groups treated with bee honey compared with those in the HCC group. The hepatic histopathology alterations caused by DEN/CCl4 injection were ameliorated by bee honey treatment. Likewise, the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), transforming growth factor (TGF-β1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), glypican (GP-3), thioredoxin (TRX), and glutaredoxin (GRX) were downregulated, and caspase-3 was upregulated by bee honey treatment compared with untreated HCC-induced group. In conclusion, bee honey has remarkable beneficial effects against HCC induced in rats through its antioxidant, anti-inflammatory, antifibrotic, and antimetastatic effects.

Practical applications: The current study confirmed that honey has the potential to act as an antimetastatic factor. Bee honey supplementation either before or after combined injection of DEN/CCl₄ exhibited inhibitory and ameliorative effects against DEN/CCl₄-induced HCC through its antioxidant, antiproliferative, anti-metastatic, antifibrotic, and apoptosis properties. To our knowledge, this is the first study to describe the molecular mechanisms underlying honey's effects against DEN/CCl₄-induced HCC in rats.

Keywords: HCC; anti-metastatic; antifibrotic; antioxidant; bee honey.

Graphical abstract

Figure 1

Ameliorative effects of bee honey on the serum biochemical parameters in different study groups (A) alanine transaminase (ALT), (B) aspartate aminotransferase (AST), (C) gamma glutamyl transferase (γGT), and (D) alkaline phosphatase (ALP). Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.

Figure 2

Ameliorative effects of bee honey on the serum α-fetoprotein (AFP) level in different studied groups. Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.

Figure 3

Ameliorative effects of bee honey on the hepatic (A) malondialdehyde (MDA), (B) catalase, (C) superoxide dismutase (SOD), and (D) glutathione peroxidase (GPX) activities.). Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.

Figure 4

Photomicrograph of liver tissue (A) control group showing, normal hepatic parenchyma with the central vein (V), normal blood sinusoid (arrowheads), and the hepatocyte with large spherical vesicular nuclei (arrows). (B) Control+bee honey group showing, the same architecture as a control group; (C) HCC group showing the focal area of lymphoid stroma (thick arrow), congested central vein (arrow head), vacuolar degeneration of hepatocyte with necrosis in some area (thin arrow), (D) HCC group showing sever necrosis (thick arrow), vacuolar degeneration (thin arrows), and dilated blood sinusoids (arrow heads). (E and F) Bee honey before HCC induction group showing, mild congestion of central vein (V), dilated blood sinusoids (arrow heads) with normal hepatic cords (thin arrow), small focal area of lymphoid stroma (thick arrow). (G and H) bee honey after HCC induction group showing, sever congestion of blood vessels (V), pseudo glandular form (thick arrow), hepatocytes with large vacuoles (thin arrows), area of degeneration and necrosis (asterisk) and dilated blood sinusoids (arrow heads).H&E stain.

Figure 5

The degenerative hepatocyte percentage in different study groups. The score of the degenerated hepatocyte, Control(0 scale), control+bee honey (0 scale), HCC (+2 scale), bee honey +HCC (0 scale), and HCC + bee honey (+1 scale). Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.

Figure 6

Ameliorative effects of bee honey on (A) TNF-α, (B) ICAM-1, (C) VCAm-1, and (D)TGF-β1 gene expressions in liver tissues. The mRNA expression levels were measured by qRT-PCR and normalized to β-actin. The primers were listed in (Table 1). Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.

Figure 7

Ameliorative effects of bee honey on (A) Caspase 3, (B) GP3, (C) TRX, and (D) GRX gene expression in liver tissues. The mRNA expression levels were measured by qRT-PCR and normalized to β-actin. The primers were listed in (Table 1). Data were presented as mean ± SEM ^*significantly different at P < .05, ^** significantly different at P < .001, ^*** significantly different at P < .0001, and ≠ nonsignificant using ANOVA followed by Bonferroni’s as a post hoc test.