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

Calcium chloride dehydrate, succinic acid disodium salt hexahydrate, 4-nitro blue tetrazolium chloride, ethylene glycol tetraacetic acid (EGTA), adenosine diphosphate (ADP), safranin O, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), cyclosporine A (CsA), and other chemicals were from Sigma-Aldrich (St. Louis, MO, USA, and Steinheim, Germany). All other reagents and organic solvents were purchased from Reakhim (Moscow, Russia). All the solutions were made with water purified in the Milli-Q system.

Red cabbage antocyanin-rich extract was prepared from fresh red cabbage (Brassica oleracea var. capitate f. rubra), collected from an experimental farm of the Agricultural University of Grodno, Belarus, early in autumn and characterised as described earlier (Buko et al., 2018). Briefly, red cabbage leaves were sliced in an equivalent amount of water containing 0.03% hydrochloric acid with a blender and the mixture was treated by ultrasound. The extract was cooled at −20 °C to precipitate polysaccharides, filtered, centrifuged, and lyophilised under vacuum in cryoconditions. Phenolic compounds from the RCE were analysed using a UHPLC+ Dionex UltiMate 3000 liquid chromatographic system and an ultrahigh-resolution hybrid quadrupole/time-of-flight mass spectrometer (UHR-Q-TOF–MS/MS, Bruker Daltonics GmbH, Bremen, Germany).

The care, use, and procedures performed in this experiment were approved by the Ethics Committee of the Institute of Biochemistry of Biologically Active Compounds, National Academy of Sciences of Belarus (Protocol No 29/16 of 23.05.2016) and complied with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the National Institutes of Health (NIH) guide for the care and use of laboratory animals (NIH Publication No. 80-23; revised 1978).

Male albino Wistar rats (the initial body weight was 200–230 g) were used. The animals were divided into four groups (8–10 rats per group). The rats from all of the groups were fed on a high-fat diet (the content of the fat component was 32%) and tap water ad libitum during 8 weeks and kept under controlled conditions at a temperature of 22 °C ± 2 °C and a relative humidity of 55% ± 5% with a 12-hour light/darkness cycle. Groups 2 through 4 (ASH) rats received ethanol solution (30%, vol/vol) via gastric tube (4 g/kg body weight, between 9:00 and 10:00 a.m.) throughout the experiment (8 weeks). Group 1 (control) rats received the same volume of physiological saline intragastrically. Group 3 (ASH + 11 mg/kg RCE) rats received RCE intragastrically (11 mg phenolic compounds/kg, between 4:00 and 5:00 p.m.) and Group 4 (ASH + 22 mg/kg RCE) rats received RCE intragastrically (22 mg phenolic compounds/kg) throughout 8 weeks of the experiment. These doses of RCE were selected based on our previous studies on the hypolipidemic and antioxidant RCE potential against alcoholic steatohepatitis in rats (unpublished observation). Earlier we showed the efficacy of similar doses of cranberry flavonoids in prevention of chronic liver damage in rats during ASH (Zavodnik et al., 2019). Dry lyophilised RCE was dissolved ex tempore in water.

Severity of alcoholic liver damage in animal modelling depends on the duration and dosage of the consumed ethanol. Because 4-week administration of the generally accepted liquid ethanol-containing Lieber-DeCarli diet (Lieber & DeCarli, 1989) induces only mild liver injury with steatosis and low-grade liver inflammation (Mathews et al., 2014), we applied intragastric administration of ethanol over the course of 8 weeks to induce a more advanced stage of alcoholic liver disease, as we described earlier (Lukivskaya et al., 2012). The rats were decapitated under pentobarbital anaesthesia after 12 hr starving. Blood samples were collected before sacrificing for biochemical and immunological measurements, and serum was obtained by centrifugation at 1,500 g. Part of the liver samples were immediately used for isolation of mitochondria and biochemical investigations. Liver samples for histopathological examination were taken from the left lobe.

Liver samples were randomly selected, fixed in Bouin's solution, and embedded in paraffin wax. Histological sections were prepared and stained with hematoxylin and eosin. Other parts of liver samples were fixed in Becker solution for histochemical lipid determination. Cryostat sections were fixed and stained with Sudan black. Tissue sections were imaged with an Olympus CX-41 light microscope and the digital images were captured with an Olympus C-5060 camera (Olympus, Japan). Computer-assisted morphometry of the sudanophilic area on liver slides was performed with the Image J morphometric analysis software (NIH, USA).

Mitochondria were isolated from the rat liver by the method of differential centrifugation (Johnson & Lardy, 1967). We used the isolation medium containing 0.125 M KCl, 0.05 M sucrose, 0.01 M Tris-HCl, 0.0025 M KH₂PO₄, 0.005 M MgSO₄, and 0.0005 M EDTA, pH 7.4. The mitochondrial pellet was resuspended in the medium to a protein concentration of 35 to 40 mg/ml. Mitochondrial respiration was measured at 25 °C using a laboratory-made oxygen Clark-type electrode with constant gentle stirring as we described earlier (Lukivskaya et al., 2012; Lapshina et al., 2015). The oxygen consumption rates in respiratory state 2 (V2) in the presence of the substrate added, as well as ADP-stimulated (200 μM) state 3 (V3) and state 4 (V4) after ADP consumption were monitored using succinate (5 mM) as substrate + rotenone (5 μM). The respiratory control ratio (RCR) equal to the ratio of respiratory rates (V3/V4) of mitochondria in state 3 and state 4 and the coefficient of phosphorylation (ADP/O ratio) were calculated. Protein concentrations in isolated mitochondria or in liver tissue were determined by the method of Lowry et al. (1951).

Ca²⁺-induced swelling of respiring mitochondria was measured as we described earlier (Golovach et al., 2017). Briefly, the extent of the mitochondrial permeability transition (MPT) pore formation was determined from the changes in the absorbance of mitochondrial suspension at 520 nm and 25 °C using a medium containing 0.125 M sucrose, 0.06 M KCl, 0.02 M Tris-HCl, and 0.001 M KH₂PO₄, pH 7.2 (EGTA-free medium). Isolated mitochondria (0.5 mg of protein/ml) were incubated in the medium containing respiratory substrate (5 mM succinate) and red cabbage anthocyanins (1.66–13.6 μg/ml) (or without RCE) for 2 min prior to the Ca²⁺ ions (60 μM) introduction for starting the MPT, and the rate of the light scattering intensity decrease (ΔD520/min) was used to determine the extent of mitochondrial pore opening. At the end of the measurements, the uncoupler FCCP (0.5 μM) was added to the mitochondria to control the completion of the MPT process. To evaluate the effect of cyclosporine A (CsA), an inhibitor of MPT pore formation, on mitochondrial swelling, the mitochondria were pretreated with 2 μM CsA at 25 °C for 3 min.

Mitochondrial membrane potential was detected using a Perkin-Elmer LS55 spectrofluorimeter (Great Britain) and the fluorescent dye safranin O (8 μM) at λex/λem 495/586 nm (Akerman & Wikström, 1976; Moore & Bonner, 1982) in the EGTA-free medium containing 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. 5 mM succinate was used as the substrate at constant gentle stirring. The positively charged dye accumulates in mitochondria (0.3 mg protein/ml) depending on their potential, with the intramitochondrial dye accumulation resulting in fluorescence quenching. Complete depolarisation of mitochondria to calibrate the dye fluorescence was achieved by addition of FCCP (0.5 μM). The mitochondrial membrane potential values (mV) were determined by applying a calibration plot that represented the dependence of safranin O fluorescence intensity on the mitochondrial membrane potential value. At the end of the measurements, the uncoupler FCCP (0.5 μM) was added.

Kits for determination of the activities of serum marker enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase) and the contents of liver and serum triglycerides were from LaChema (Brno, Czech Republic). Kits for determination of the transforming growth factor β (TGFβ), the tumor necrosis factor α (TNFa), interleukin-6 (IL-6), and leptin by ELISA technique were from R&D Systems GmbH (Wiesbaden, Germany).

The phagocytic index, a measure of phagocytic activity, was determined as the percentage of phagocytes containing absorbed latex particles during a limited period of rat blood incubation with a latex suspension. A standard suspension of latex particles (1.5 μm in diameter; Sigma, St. Louis, MO, USA) was used as an object of phagocytosis. Quantitative registration of the phagocytic reaction was carried out microscopically (Olympus CX-41 light microscope). The metabolic activity of neutrophils was studied using a histochemical test for the reduction of nitro blue tetrazolium into formazan by phagocytes (nitro blue tetrazolium test). We used a 0.2% solution of nitro blue tetrazolium, incubated with blood in equal proportions for 30 min at 37 °C. The percentage of formazan-positive neutrophils was calculated microscopically.

All the data are expressed as means ± standard error (M ± SEM). The data were analysed statistically by one-way analysis of variance (ANOVA; GraphPad software v.5.0; Prism) using the post hoc analysis with Tukey's test. The level of significance was considered at p < 0.05.

Earlier an anthocyanin-rich RCE was assayed for polyphenols, and we identified 21 compounds, predominantly anthocyanins, representing more than half of the total amount of polyphenols (Buko et al., 2018). The total amount of phenolic compounds in RCE was 834.13 ± 2.57 mg per 100 g. In our experiment, we evaluated the hepatoprotective potential of RCE under alcoholic steatohepatitis in rats. According to the histopathological measurements, the liver of the control animals showed normal architecture, the hepatic lobules had a normal structure, and the portal tracts were without signs of cellular infiltration (Fig. 1a).

The long-term (8 weeks) administration of high-dose ethanol accompanied by the high-fat diet caused steatohepatitis that was characterised by macro- and microvesicular steatosis, scattered parenchymal cells with ballooning degeneration, and multifocal lymphocytic infiltration as a sign of inflammation (Fig. 1b). Signs of small- and medium-drop fatty dystrophy of hepatocytes were observed in all parts of the hepatic lobules as well as discirculatory changes in the form of expansion of the central veins and sinusoids with the presence of a large number of erythrocytes in their lumen were noted. The morphometric evaluation of Sudan black-stained liver preparations showed a 7.6-fold increase in the relative square of the sudanophilic area on the alcohol-treated rat liver preparations (Table 1). The treatment of alcohol-administered rats with RCE at the doses of 11 and 22 mg/kg body weight partially alleviated these pathological changes (Fig. 1c and 1d): The sizes of lipid vacuoles in hepatocytes and the inflammatory signs in the liver were decreased. The hepatoprotective effect was dose-dependent. We observed only a small number of hepatocytes with large fat droplets, attenuation of the sudanophilic area, and a decrease in intralobular lymphocytic infiltration (only a small number of intralobular infiltrates were determined) in animals receiving the larger dose of the extract (Fig. 1d, Table 1).

Figure 1

Structural and functional impairments in the liver during ethanol intoxication were shown by determination of blood biochemical parameters (Table 1). The administration of ethanol significantly elevated the activities of the liver damage marker enzymes, AST (by 2.5-fold) and ALT (by 1.5-fold), as well as the activity of alkaline phosphatase (by 2.2-fold) and the levels of total and bound bilirubin (by 1.4-fold and 1.6-fold, respectively) in the rat serum (Table 1). The treatment of animals during intoxication by RCE caused a partial dose-dependent normalisation of serum ALT (not AST) and alkaline phosphatase activities and a complete reversal of the total and bound bilirubin levels. Significant accumulation of neutral lipids in the liver of intoxicated rats resulted in a rise in the levels of serum (1.6-fold) and liver (2.3-fold) triglycerides (Table 1). The administration of anthocyanins substantially decreased these parameters. The serum contents of the proinflammatory cytokines TNFα (by 5.9-fold) and IL-6 (by 2.9-fold), the profibrogenic cytokine TGFβ (by 2.5-fold), and the hormone leptin (by 1.7-fold) were considerably increased in rats with ASH as compared to the control group (Table 2). The long-term administration of RCE during intoxication resulted in dose-dependent and statistically significant attenuation of the levels of the cytokines TNFα and IL-6 as well as that of the adipokine leptin, with the content of the cytokine TGFβ remaining virtually unchanged.

The long-term administration of ethanol influenced rat liver mitochondria respiratory activity: The rates of ADP-stimulated mitochondrial oxygen consumption V3 and the oxygen consumption after ADP phosphorylation V4 decreased and the ADP/O ratio was lowered in the alcohol-fed group, whereas the RCR was not changed (we used succinate, the substrate of Complex II of the electron transport chain; Table 3). The RCE administration during rat intoxication completely recovered the respiration rates and normalised the ADP/O coefficient but did not affect the RCR (Table 3).

For understanding the mechanism(s) of beneficial effects of anthocyanins during ASH in rats, we determined the functioning of cellular factors of the immune system under intoxication. Table 4 presents the blood immunological parameters of rats with ASH treated by RCE. The process of phagocytosis was usually evaluated by measurements of the phagocytic index, a measure of phagocytic activity determined by counting the number of bacteria ingested per phagocyte. In our experiment, the phagocytic index (the number of phagocytes containing absorbed latex particles) and the metabolic activity of neutrophils (spontaneous reduction of nitro blue tetrazolium by phagocytes, the histological nitro blue tetrazolium test) were considerably decreased during ethanol intoxication in comparison with control animals, thus reflecting the immunosuppressive state of the rats and impairments in phagocytosis (Table 4). The administration of anthocyanins showed beneficial effects on the immunological parameters studied: The phagocytic index increased after RCE treatment as compared to the ASH group, and the metabolic activity of neutrophils was elevated, as was shown by the significant increase in the number of formazan-positive neutrophils (Table 4). It should be noted that these RCE effects were not dose-dependent.

We studied the modulatory effects of anthocyanins on the respiratory activity, membrane potential, and proapoptotic calcium-induced process of MPT pore formations using isolated rat liver mitochondria in vitro. RCE dose-dependently effected the oxygen-consumption rates of mitochondria in the absence or in the presence of Ca²⁺ ions in the EGTA-free medium: In the absence of Ca²⁺, the anthocyanins increased the V2 rate at concentrations of 1 to 3 μg/ml and decreased the V3 rate at all the concentrations studied, most pronounced at concentrations of 2 to 4 μg/ml (Fig. 2a). Consequently, RCE diminished the RCR and the ADP/O phosphorylation coefficient, which was most pronounced at lower concentrations (1–4 μg/ml; Fig. 2b). In our experiment, Ca²⁺ (60 μM) alone decreased State 3 and increased State 2 respiration rates—diminishing the coefficients RCR and ADP/O (Fig. 2a and 2b) in the EGTA-free medium (Fig. 2)—and influenced the effects of RCE on mitochondrial respiration. In the presence of exogenous Ca²⁺ (60 μM), RCE decreased the respiration rates V2 and V3, lowered the RCR at concentrations of 2 to 4 μg/ml and increased it at higher concentrations, and elevated the coefficient ADP/O. The RCE dose-dependently dissipated the mitochondrial membrane potential (Fig. 3), enhanced the rate of Ca²⁺-induced MPT at lower concentrations (1–4 μg/ml), and inhibited this process at higher concentrations (Fig. 4) in the EGTA-free medium.

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