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Anthocyanin-Rich Extract of Red Cabbage Attenuates Advanced Alcohol Hepatotoxicity in Rats in Association with Mitochondrial Activity Modulation

V. Buko
V. Buko
, 
E. Belonovskaya
E. Belonovskaya
, 
T. Kavalenia
T. Kavalenia
, 
T. Ilyich
T. Ilyich
, 
S. Kirko
S. Kirko
, 
I. Kuzmitskaya
I. Kuzmitskaya
, 
V. Moroz
V. Moroz
, 
E. Lapshina
E. Lapshina
, 
A. Romanchuk
A. Romanchuk
 und 
I. Zavodnik
I. Zavodnik
   | 23. Aug. 2022
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Article Category: Original Paper
Online veröffentlicht: 23. Aug. 2022
Seitenbereich: 5 - 16
Eingereicht: 03. Nov. 2021
Akzeptiert: 24. Feb. 2022
DOI: https://doi.org/10.2478/afpuc-2022-0014
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© 2022 V. Buko et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Histological evaluation of the liver of rats with alcoholic steatohepatitis without or after the red cabbage extract (RCE) treatment. (a) Control; (b) Alcohol-treated group (ASH); (c) ASH + RCE (11 mg/kg), (d) ASH + RCE (22 mg/kg). Representative microphotographs of liver sections. The control group displayed normal liver architecture. The group administered with ethanol alone showed macro- and microvesicular liver steatosis, ballooning vacuolisation, scattered lymphocytic infiltration of portal tracts, and inflammatory foci. Macrovesicular fatty dystrophy and inflammatory foci were shown in the ASH group (black arrows). The signs of liver damage, degeneration, and inflammation were substantially reduced with the RCE treatment. The group treated with the larger dose of RCE was characterised mainly by microvesicular steatosis and scattered lymphoid infiltration. Rat liver sections were stained with Haematoxylin and Eosin (magnification: ocular 10×, objective 40×).
Histological evaluation of the liver of rats with alcoholic steatohepatitis without or after the red cabbage extract (RCE) treatment. (a) Control; (b) Alcohol-treated group (ASH); (c) ASH + RCE (11 mg/kg), (d) ASH + RCE (22 mg/kg). Representative microphotographs of liver sections. The control group displayed normal liver architecture. The group administered with ethanol alone showed macro- and microvesicular liver steatosis, ballooning vacuolisation, scattered lymphocytic infiltration of portal tracts, and inflammatory foci. Macrovesicular fatty dystrophy and inflammatory foci were shown in the ASH group (black arrows). The signs of liver damage, degeneration, and inflammation were substantially reduced with the RCE treatment. The group treated with the larger dose of RCE was characterised mainly by microvesicular steatosis and scattered lymphoid infiltration. Rat liver sections were stained with Haematoxylin and Eosin (magnification: ocular 10×, objective 40×).

Figure 2

Effect of red cabbage extract (RCE) on the respiration parameters of isolated rat liver mitochondria. (a) The respiration rates V2 (1, 2) and V3 (3, 4); and (b) the coefficient of phosphorylation (ADP/O ratio) (1, 2) and the respiratory control ratio (RCR) (3, 4) (b) in the absence (1, 3) or in the presence of Ca2+ ions (60 μM) (2, 4). Mitochondria (0.5 mg protein/ml) were incubated in the medium containing 0.125 M KCl, 0.05 M sucrose, 0.01 M Tris-HCl, 0.0025 M KH2PO4, 0.005 M MgSO4, pH 7.4, at 25 °C, in the absence or in the presence of the anthocyanins (1.66–13.6 μg/ml). 5 mM succinate as substrate and 200 μM ADP were added. Significant difference (p < 0.05): * vs. that in the absence of RCE.
Effect of red cabbage extract (RCE) on the respiration parameters of isolated rat liver mitochondria. (a) The respiration rates V2 (1, 2) and V3 (3, 4); and (b) the coefficient of phosphorylation (ADP/O ratio) (1, 2) and the respiratory control ratio (RCR) (3, 4) (b) in the absence (1, 3) or in the presence of Ca2+ ions (60 μM) (2, 4). Mitochondria (0.5 mg protein/ml) were incubated in the medium containing 0.125 M KCl, 0.05 M sucrose, 0.01 M Tris-HCl, 0.0025 M KH2PO4, 0.005 M MgSO4, pH 7.4, at 25 °C, in the absence or in the presence of the anthocyanins (1.66–13.6 μg/ml). 5 mM succinate as substrate and 200 μM ADP were added. Significant difference (p < 0.05): * vs. that in the absence of RCE.

Figure 3

Representative traces of time-dependences of mitochondrial membrane potential dissipation. Control (1); 1.66 μg/ml anthocyanins (2); 3.34 μg/ml anthocyanins (3); 6.67 μg/ml anthocyanins (4); 13.6 μg/ml anthocyanins (5). The mitochondrial membrane potential was detected using the fluorescent dye safranin O (8 μM) at λex/λem 495/586 nm at 25 °C and 5 mM succinate as energising substrate. Mitochondria (0.3 mg of protein/ml) were added at constant gentle stirring to ethylene glycol tetraacetic acid (EGTA)-free medium: 0.05 M sucrose, 0.01 M Tris-HCl, 0.125 M КCl, 2.5 мM KH2PO4, 0.5 мM MgSO4, pH 7.4, 25° C, with or without red cabbage extract (RCE).
Representative traces of time-dependences of mitochondrial membrane potential dissipation. Control (1); 1.66 μg/ml anthocyanins (2); 3.34 μg/ml anthocyanins (3); 6.67 μg/ml anthocyanins (4); 13.6 μg/ml anthocyanins (5). The mitochondrial membrane potential was detected using the fluorescent dye safranin O (8 μM) at λex/λem 495/586 nm at 25 °C and 5 mM succinate as energising substrate. Mitochondria (0.3 mg of protein/ml) were added at constant gentle stirring to ethylene glycol tetraacetic acid (EGTA)-free medium: 0.05 M sucrose, 0.01 M Tris-HCl, 0.125 M КCl, 2.5 мM KH2PO4, 0.5 мM MgSO4, pH 7.4, 25° C, with or without red cabbage extract (RCE).

Figure 4

Effect of red cabbage extract (RCE; 1.66–13.6 μg/ml) on the rate of Ca2+-induced mitochondrial permeability transition (MPT). Dependences of mitochondrial swelling rate v (ΔD520/min) on RCE concentration. Energised by 5 mM succinate isolated rat liver mitochondria (0.5 mg protein/ml) were incubated in the medium containing 0.125 M sucrose, 0.06 M KCl, 0.02 M Tris-HCl and 0.001 M KH2PO4, pH 7.2 (ethylene glycol tetraacetic acid [EGTA]-free medium), with or without RCE at 25 °C. The reaction was started by addition of 60 μM of Ca2+ ions. The rate of the MPT pore formation was determined from the changes in the absorbance of mitochondrial suspension at 520 nm. Significant difference (p < 0.05): * vs. that of the control, # vs. that in the presence of 60 μM Ca2+.
Effect of red cabbage extract (RCE; 1.66–13.6 μg/ml) on the rate of Ca2+-induced mitochondrial permeability transition (MPT). Dependences of mitochondrial swelling rate v (ΔD520/min) on RCE concentration. Energised by 5 mM succinate isolated rat liver mitochondria (0.5 mg protein/ml) were incubated in the medium containing 0.125 M sucrose, 0.06 M KCl, 0.02 M Tris-HCl and 0.001 M KH2PO4, pH 7.2 (ethylene glycol tetraacetic acid [EGTA]-free medium), with or without RCE at 25 °C. The reaction was started by addition of 60 μM of Ca2+ ions. The rate of the MPT pore formation was determined from the changes in the absorbance of mitochondrial suspension at 520 nm. Significant difference (p < 0.05): * vs. that of the control, # vs. that in the presence of 60 μM Ca2+.

Blood immunological parameters in alcohol-administered rats treated with red cabbage extract (RCE) anthocyanins.

Parameters Control ASH ASH + 11 mg/kg RCE ASH + 22 mg/kg RCE
Nitro blue tetrazolium test, % 21.4 ± 1.2 7.7 ± 2.4a 38.3 ± 6.9b 38.3 ± 6.5b
Phagocytic index, % 55.4 ± 3.5 21.1 ± 1.5a 39.9 ± 3.5b 37.1 ± 4.6b

Morphometric evaluation of hepatic damage and blood serum triglycerides, bilirubin levels, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase activities and liver triglycerides content during long-term alcohol administration and red cabbage extract (RCE) anthocyanins treatment.

Parameters Control ASH ASH + 11 mg/kg RCE ASH + 22 mg/kg RCE
Square of sudanophilic area, % of the slide square 2.2 ± 0.2 16.8 ± 1.8a 13.8 ± 1.5a 12.6 ± 1.4ab
AST, U/l 67.0 ± 3.5 170.1 ± 2.6a 165.4 ± 3.5a 165.1 ± 3.1a
ALT, U/l 53.0 ± 2.2 78.8 ± 2.1a 63.2 ± 3.4ab 56.4 ± 2.4b
Alkaline phosphatase, U/l 185.9 ± 13.7 406.9 ± 41.4a 300.4 ± 19.2ab 271.0 ± 14.6ab
Bilirubin, total, μmol/l 4.0 ± 0.2 6.0 ± 0.3a 4.6 ± 0.4b 4.6 ± 0.3b
Bilirubin, bound, μmol/l 1.9 ± 0.2 2.7 ± 0.3a 1.7 ± 0.2b 1.6 ± 0.1b
Serum triglycerides, mmol/l 1.8 ± 0.2 2.8 ± 0.2a 2.0 ± 0.1b 1.9 ± 0.2b
Liver triglycerides, mg/g tissue 16.8 ± 0.9 39.1 ± 2.7a 35.5 ± 1.9ab 30.3 ± 1.9ab

Rat blood cytokines tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ), interleukin-6 (IL-6), and leptin levels during alcohol administration and red cabbage extract (RCE) anthocyanins treatment.

Parameters Control ASH ASH + 11 mg/kg RCE ASH + 22 mg/kg RCE
TNFα, pg/ml 8.4 ± 0.8 49.9 ± 3.8a 50.8 ± 4.7a 35.6 ± 3.2ab
TGFβ, ng/ml 57.3 ± 7.6 141.9 ± 21.0a 152.9 ± 36.2a 125.0 ± 25.3a
IL-6, pg/ml 50.4 ± 4.9 147.0 ± 13.2a 107.3 ± 12.5ab 80.9 ± 6.2ab
Leptin, ng/ml 1.1 ± 0.1 1.9 ± 0.1a 1.9 ± 0.1a 1.3 ± 0.2b

Respiratory parameters of rat liver mitochondria during alcohol administration and red cabbage extract (RCE) anthocyanins treatment.

Parameters Control ASH ASH + 11 mg/kg RCE ASH + 22 mg/kg RCE
V3, ngatom O/min/mg protein 94.8 ± 8.9 59.7 ± 6.2a 104.7 ± 9.2b 110.8 ± 7.2b
V4, ngatom O/min/mg protein 41.4 ± 4.7 29.7 ± 4.8a 45.2 ± 4.1b 39.5 ± 4.5b
Respiratory control ratio (RCR) 2.3 ± 0.3 2.0 ± 0.2 2.1 ± 0.3 2.2 ± 0.2
ADP/O 1.6 ± 0.2 1.1 ± 0.2a 1.7 ± 0.3b 1.8 ± 0.2b
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