Astaxanthin Is Able to Prevent Alcohol-Induced Dysfunction of Liver Mitochondria

Abstract

The search for new targets for the pathological action of ethanol remains an urgent task of biomedicine. Since degenerative changes in the liver are associated with the development of oxidative stress, antioxidants are promising agents for the treatment of alcohol-related diseases. In this work, we studied the ability of the carotenoid antioxidant, astaxanthin (AX), to prevent ethanol-induced changes in the liver of rats. It was shown that AX is able to protect the structure of mitochondria from degenerative changes caused by ethanol to improve mitochondrial functions. AX positively influences the activity and expression of proteins of the mitochondrial respiratory chain complexes and ATPase. In addition, a protective effect of AX on the rate and activity of mitochondrial respiration was demonstrated in the work. Thus, studies have shown that AX is involved in protective mechanisms in response to ethanol-induced mitochondrial dysfunction.

Keywords: astaxanthin (AX); chronic ethanol intoxication; rat liver mitochondria (RLM); the activity of respiratory chain complexes.

Figure 1

Histological samples of liver tissue in rats of the control and experimental groups. (a)—Fragments of histotopograms of periportal areas of rat liver tissue. Light microscopy; main images—Masson’s trichrome stain (collagen/fibrosis—blue, non-collagen components—pink, cell nuclei—dark crimson), ruler 100 µm; Inserts—Malory trichrome stain (collagen/fibrosis—blue, non-collagen components—orange/red, cell nuclei—red), ruler 50 µm; (b)—Photomicrographs of perivascular rat liver hepatocytes. Light microscopy; H&E (cell nuclei in purple, erythrocytes in red, cell cytoplasm in pink), ruler 20 µm; (c) is a diagram showing the growth of deposited collagen in the periportal region of the liver of each group of rats. The data are presented as the means ± SD of three independent experiments. * p < 0.05 significant difference in the protein levels in comparison with the control (group 1). # p < 0.05 compared to RLM isolated from ethanol-administrated rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using the Student–Newman–Keul test.

Figure 2

The effect of AX and Ethanol on the content of ALT, AST, LDG, CNPase, and TSPO in rat liver tissue. (a)—upper part: immunostaining with ALT, AST, and LDG antibodies; low past: quantification of immunostaining by computer-assisted densitometry presents as a ratio of proteins to GAPDH. GAPDH was used for normalization of proteins. (b)—upper part: immunostaining with CNPase and TSPO antibodies; low part: quantification of immunostaining by computer-assisted densitometry presented as a ratio of proteins to GAPDH. The data are presented as the means ± SD of three independent experiments. * p < 0.05 significant difference in the protein levels in comparison with the control (group 1). # p < 0.05 compared to RLM isolated from ethanol-administrated rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using the Student–Newman–Keul test, while ethanol treatment (group 3, red columns on all diagrams) significantly reduced the protein content. Thus, we showed a decrease in ALT levels by 60%, AST by 70%, and LDG by 40% compared with the control group (red columns 3 vs. black columns 1). At the same time, in the liver of rats treated with both ethanol and AX (group 4, scarlet columns in all diagrams), the indicators increased approximately to the control values (scarlet columns 4 compared to black 1). Thus, AX is able to abolish ethanol-induced changes in the liver tissue lysates.

Figure 3

The effects of AX and Ethanol on the change of respiratory activities in RLM. (a)—The curves of respiratory activities in state 2 (V_st.2), 3 (V_st.3), 4 (V_st.4), and uncoupled respiration rate (V_u). Arrows showed the additions to RLM. The concentration of ADP was 200 µM. DNF was 30 µM. (b)—quantitative analysis of RLM respiration rate in states 2 (V_st.2); (c)—quantitative analysis of RLM respiration rate in states 3 (V_st.3); (d)—quantitative analysis of RLM respiration rate in states 4 (V_st.4); (e)—quantitative analysis of the uncoupled respiration rate (V_u); (f)—respiratory control index (RCI) values and phosphate/oxygen (P/O) ratio. Oxygen consumption rates (V_st.2, V_st.3, V_st.4, and (V_u)) were estimated as ng-atom O min⁻¹ mg⁻¹ protein. The data are presented as the means ± SDs of four independent experiments. * p < 0.05 significant values compared with control (group 1); # p < 0.05 significant values compared with RLM isolated from ethanol-administrated rats (group 3).

Figure 4

The influence of AX and Ethanol on the change in the levels of the main subunits of the complexes of respiratory chain in RLM. Tom20 was used as a protein load control. (a)—immunostaining with an antibody cocktail of OXPHOS; (b–f)—quantification of immunostaining by computerized densitometry presented as a ratio of subunits to Tom20. The data are presented as mean ± SD of three independent experiments. * p < 0.05 indicates a significant difference in the protein level compared to the control (group 1). # p < 0.05 compared with the corresponding value in the RLM isolated from ethanol-administrated rats (Group 3). Statistical significance was assessed using the ANOVA type 2 test (Student–Newman–Keuls).

Figure 5

The effect of AX and Ethanol on the activity of respiratory chain complexes and F_oF₁-ATP synthase. (a)—The native intact RLM isolated from each group was solubilized in the buffer for BNE samples (Materials and Methods Section). Mitochondrial samples were separated by the first dimension BNE, and the gel was stained for the detection of the activity of respiratory chain complexes and F_oF₁-ATP synthase (Materials and Methods Section); (b)—the change of activity I complex; (c)—the change of activity V complex; (d)—the change of activity III complex; (e)—the change of activity IV complex. The data are presented as mean ± SD of three independent experiments. * p < 0.05 indicates a significant difference in the complex activity compared to the control (Group 1). # p < 0.05 compared with the corresponding value in the complex activity in RLM from ethanol-administrated rats (Group 3). Statistical significance was assessed using the ANOVA type 2 test (Student–Newman–Keuls).

Figure 6

The effect of AX and Ethanol on the change of proteins associated with respiratory chain complexes and F_oF₁-ATP synthase. The native RLM were separated to the first dimension by BNE. Bands containing the corresponding complexes were cut out from the gel and applied to the lanes of 12.5% PAAG and separated in the second dimension. The proteins were transferred from the gel to the nitrocellulose membrane. The membrane was stained with the corresponding antibodies. (a)—the separation of mitochondrial samples to the first dimension by BNE; (b)—the proteins associated with complex I. Upper part: the gel stained with Coomassie, the Western blot stained with CNPase and NDUFB8; low part: the changes of proteins content (absolute units). (c)—the proteins associated with complex V. Upper part: the gel stained with Coomassie, the Western blot stained with ATP5F1, ATP5G, and ATP5A; low part: the changes of proteins content (absolute units). (d)—the proteins associated with complex III. Upper part: the gel stained with Coomassie, the Western blot stained with CNPase and UQCRC2; low part: the changes of protein content (absolute units). (e)—the proteins associated with complex IV. Upper part: the gel stained with Coomassie, the Western blot stained with CNPase, COXIV, and MTCO1. The data are presented as mean ± SD of three independent experiments. * p < 0.05 indicates a significant difference in the protein levels compared to the control (Group 1). # p < 0.05 compared with the corresponding value in the protein levels in RLM from ethanol-administrated rats (Group 3). Statistical significance was assessed using the ANOVA type 2 test (Student–Newman–Keuls).