Polyphenol-Rich Extract from 'Limoncella' Apple Variety Ameliorates Dinitrobenzene Sulfonic Acid-Induced Colitis and Linked Liver Damage

Abstract

Inflammatory bowel conditions can involve nearly all organ systems and induce pathological processes through increased oxidative stress, lipid peroxidation and disruption of the immune response. Patients with inflammatory bowel disease (IBD) are at high risk of having extra-intestinal manifestations, for example, in the hepatobiliary system. In 30% of patients with IBD, the blood values of liver enzymes, such as AST and ALT, are increased. Moreover, treatments for inflammatory bowel diseases may cause liver toxicity. Apple polyphenol extracts are widely acknowledged for their potential antioxidant effects, which help prevent damage from oxidative stress, reduce inflammation, provide protection to the liver, and enhance lipid metabolism. The aim of this study was to investigate whether the polyphenol apple extract from Malus domestica cv. 'Limoncella' (LAPE) may be an effective intervention for the treatment of IBD-induced hepatotoxicity. The LAPE was administrated in vivo by oral gavage (3-300 mg/kg) once a day for 3 consecutive days, starting 24 h after the induction of dinitro-benzenesulfonic acid (DNBS) colitis in mice. The results showed that LAPE significantly attenuated histological bowel injury, myeloperoxidase activity, tumor necrosis factor and interleukin (IL-1β) expressions. Furthermore, LAPE significantly improved the serum lipid peroxidation and liver injury in DNBS-induced colitis, as well as reduced the nuclear transcription factor-kappaB activation. In conclusion, these results suggest that LAPE, through its antioxidant and anti-inflammatory properties, could prevent liver damage induced by inflammatory bowel disease.

Keywords: antioxidant; apple polyphenols; inflammatory bowel disease; liver inflammation; nutraceuticals.

Figure 1

Effect of LAPE on colonic damage in DNBS-treated mice. Effect of LAPE (30–300 mg/kg, given by oral gavage) on colon weight/colon length ratio (A) and, at the dose of 300 mg/kg, on colonic damage (B) in DNBS-induced colitis. All acquisitions were performed 3 days after DNBS injection. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. (A) Data are expressed as the mean ± SEM of 10–12 mice for each experimental group and were statistically analyzed using a one-way ANOVA followed by Dunnett’s test. **** p < 0.0001 versus DNBS alone. (B) Representative H&E-stained colon cross-sections of mice treated with vehicle, DNBS and DNBS+LAPE (300 mg/kg by oral gavage). Original magnification 10×. Scale bars represented 100 μm.

Figure 2

Effect of LAPE on inflammatory parameters in DNBS-treated mice. Effect of LAPE (300 mg/kg, given by oral gavage) on myeloperoxidase (MPO) activity (A) and on colonic IL-1β (B) and IL-6 (C) levels in DNBS-induced colitis. Measurements were performed 3 days after DNBS administration. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. MPO, IL-1β and IL-6 levels were measured in vehicle-, DNBS- or DNBS+ LAPE-treated mice. Data are expressed as the mean ± SEM of 5 (A) or 4 (B,C) mice for each experimental group and were statistically analyzed using a one-way ANOVA followed by Dunnett’s test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. DNBS alone.

Figure 3

Effect of LAPE on intestinal permeability in DNBS-treated mice. Effect of LAPE (300 mg/kg, given by oral gavage) on serum FITC–dextran concentration in DNBS-induced colitis. Measurements were performed 3 days after DNBS injection. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. Data are expressed as the mean ± SEM of 4 mice for each experimental group and were statistically analyzed using a one-way ANOVA followed by Dunnett’s test. * p < 0.05 and ** p < 0.01 vs. DNBS alone.

Figure 4

Effect of LAPE on β-catenin localization in DNBS-treated mice. Representative image of colon cross-sections showing the expression of β-catenin (green) and nucleus (blue) in mice treated with vehicle (CTR), DNBS and DNBS+LAPE. Measurements were performed 3 days after DNBS injection. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. Original magnification 20×. Representative H&E-stained colon cross-sections of mice treated with vehicle, DNBS and DNBS+LAPE (300 mg/kg by oral gavage). The scale bars represented 50 μm.

Figure 5

Effect of LAPE on fatty liver disease in DNBS-treated mice. Effect of LAPE (300 mg/kg, given by oral gavage) on lipid droplets (A) and triglycerides (TG) levels (B) in liver of DNBS-treated mice. Liver sections of mice treated with vehicle (CTR), DNBS or DNBS+LAPE (300 mg/kg, by oral gavage) were stained with Oil Red O. Intense red indicates lipid droplets. Measurements were performed 3 days after DNBS injection. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. Original magnification 20×. ** p < 0.01 vs. DNBS alone. Scale bars represented 100 μm.

Figure 6

Effect of LAPE on liver damage in DNBS-treated mice. Representative H&E-stained liver cross-sections of mice treated with vehicle (CTR), DNBS and DNBS+LAPE (300 mg/kg by oral gavage). Measurements were performed 3 days after DNBS injection. LAPE was given once a day starting from 24 h after DNBS and continued for the following days until the sacrifice. Original magnification 20×. Evaluation of liver injury following arrows; thin arrows = blood sinusoids with lymphocytes infiltration; thick arrows = centrilobular hepatocytes displayed deeply stained acidophilic cytoplasm and darkly stained nuclei. BD = bile duct; CV = central vein; PV = portal vein; HA = hepatic artery. Five random fields per colon of at least four mice per group were chosen to analyze histological alterations [17].

Figure 7

LAPE counteract NK-kB activation, in DNBS-induced colitis in mice. Immunofluorescence analysis showing the expression of NF-kB (red) of liver mice treated with vehicle (control), DNBS and DNBS plus LAPE (300 mg/kg) by oral gavage. Livers were collected three days after the induction of colitis by DNBS. Original magnification 20×. The DNBS-induced colitis in mice was treated for three consecutive days after the inflammatory insults with LAPE (300 mg/kg, by oral gavage). Scale bars represented 10 μm.