The Effect of Retinol Acetate on Liver Fibrosis Depends on the Temporal Features of the Development of Pathology

Abstract

Background

The effect of vitamin A on the manifestations of liver fibrosis is controversial and establishing the causes of its multidirectional influence is an urgent problem. In the work, the functional characteristics of the liver with Cu-induced fibrosis were determined after the restoration of vitamin A to the control level at the F0/F1 stage.

Methods

In animals with liver fibrosis, classical indicators of physiology, functional activity of the liver, histological, and hematological characteristics were determined; the content of calcium and ROS was determined in bone marrow cells.

Results

It was shown that in the liver with Cu-induced fibrosis, the restoration of vitamin A content to control values after per os injections of a retinol acetate solution at a dose of 0.10 mg (300 IU)/100 g of body weight in the early stages of this pathology development (Fо/F1) was accompanied by: a decrease in the number of immunocompetent cells in the bloodstream to control values; normalization of the amount of calcium ions and ROS in bone marrow cells; restoration to the control level of activity of alkaline phosphatase; an increase in the number of binuclear hepatocytes; and restoration of the dynamics of body weight growth in experimental animals, even against the background of the ongoing action of the hepatotoxic factor.

Conclusion

We came to the conclusion that the multidirectional action of vitamin A, which occurs in liver fibrosis, depends not only on the concentration of vitamin A in the liver but also on temporal characteristics of cellular and metabolic links involved in the adaptive response formation. It was suggested that knowledge of the initial temporal metabolic characteristics and the amount of vitamin A in the liver, taking into account the stages of fibrosis development, can be an effective way to restore the altered homeostatic parameters of the body.

As is known, the process of liver pathologies formation goes through a number of stages: hepatitis, fibrosis, and cirrhosis, which is the terminal stage—a dangerous and irreversible condition.^{1, 2, 3} Liver cirrhosis is reportedly the 4th leading cause of death among people aged over 40 years in the United States.⁴ Despite the relevance, intensive study of the mechanisms of liver fibrosis formation, and the availability of a large arsenal drugs, it has not yet been possible to develop a single effective protocol for the treatment of this pathology.⁵

The problem is that pathological changes in the liver occur much more often than they are registered and are often detected at the stage of chronicity of the process. This is explained by several fundamental properties of the liver as a central link in the body's metabolic regulation. First, functional changes in the liver occur under the influence of an extremely wide range of physical, chemical, and biological factors.^{6, 7, 8} In this context, in the liver, as in a “mirror” not only exogenous but also endogenous factors are reflected and transformed, which manifests itself in the form of inflammatory reactions and the adaptive functional response formation.^9,10 Second, since the liver is a “coordinating” and “executive” element of the body metabolic system, that is, it provides vital functions, an effective system of regeneration and repair of liver cellular elements was formed in the evolution. The processes of maintaining and restoring the functional activity of the liver are carried out by polyploidization and hypertrophy of hepatocytes; and regeneration of the liver cellular elements, that is, the liver has a wide arsenal of adaptive mechanisms.^{11, 12, 13, 14} These features provide a pronounced variability in the functional activity of the liver and this “masks” the manifestation of pathological processes. In connection with these features, comprehensive studies of the mechanisms of adaptive reactions formation of the liver to the actions of various toxic environmental factors and the development of methods for their regulation are necessary.

Currently, the methods of treating liver fibrosis are being actively developed, since this stage of fibrosis development can be reversible, according to available data.^{15, 16, 17} For this, a wide arsenal of therapeutic measures is used, which include the elimination of causative factors; the use of medicinal preparations with anticytotoxic activity, hepatoprotective action, antifibrotic activity, antioxidant properties; the use of inhibitors of the renin-angiotensive system, and so on.^{18, 19, 20, 21} However, effective treatments for fibrosis have not yet been developed.⁵

We believe that in the treatment of liver pathology and, above all, fibrosis, we should consider such fundamental concepts as: (1) temporal features of the inflammatory process development, that is, the functional state of the liver at the time of drug exposure and (2) the natural ability of the liver to regenerate, and for this, it is advisable to use not xenobiotics, but natural biologically active substances, which are natural and “fine” regulators of metabolic processes in the liver. Vitamins and, above all, vitamin A are promising in this regard.

Vitamin A is a group of retinoids with similar biological properties, which are primarily characterized by the membranotropy and a fairly wide range of biological activity. Depending on the dose, they can exhibit antioxidant properties; change the structure of lysosomal membranes and thus activate proteolytic enzymes; change the rate of proliferation and even the direction of cell differentiation.²² There are indications that vitamin A is involved in the formation of sulfur-containing molecules, the synthesis of glycoproteins and the metabolism of membrane phospholipids.^23,24 Retinoic acid has recently been shown to have antiplatelet and fibrolytic activity.²⁵ Along with these properties, the effect of vitamin A on the functional activity of the liver (primarily in fibrosis) is of particular interest since 50–80 % of this vitamin is localized in the liver, and 90–95 % of this amount is retained in hepatic stellate cells (HSCs), which play an important and possibly a leading role in the development of fibrosis.^26,27 There is evidence that with fibrosis, the content of vitamin A in the liver decreases, and at the same time, it contributes to the production of triglycerides in hepatocytes.^28,29

Retinoids have been shown to inhibit the formation of fibrotic liver, and maintenance of hepatic vitamin A stores is an important means of minimizing the effects of fibrogenic agents.³⁰ In an experiment on the Cu-induced liver fibrosis model, it was shown that the administration of vitamin A at a dose of 300 IU (0.09 mg)/100 g of body weight to animals with this pathology for four consecutive days was accompanied by a “restoration” of the amount of the vitamin to control values and a significant decrease in the mortality of animals with fibrosis.^31,32 Along with these data, there are works that point out that a significant increase in the content of hepatic vitamin A, on the contrary, accelerates the transition of fibrosis to cirrhosis.^33,34 In this regard, the solution of the problem of inconsistency of experimental data is not only of great practical but also of theoretical importance.

Such inconsistency and ambiguity of the vitamin A action in patients with liver fibrosis may be due to the fact that the direction of its action depends not only on the dose of the vitamin A, but also on the temporal characteristics of the pathological process in the liver. By temporality, in this case, we mean two aspects: the stage of fibrosis development and the temporal characteristic (increase, stationary level, or decrease) of immunological and metabolic patterns that are involved in the adaptive response formation and can form a pathogenetic phenotype. We believe that the direction of action of vitamin A will be determined by the dynamic microenvironment (pattern) that is formed at a given time, which can be explained by the temporal optimality principle,³² and these patterns can have a pronounced individual character.

The work tested the hypothesis that the effect of vitamin A on the liver with fibrosis depends on the fibrosis development stage (that is, the “distance” of functional indicators from the initial homeostatic indicators that take part in the formation of the pathogenetic phenotype), as well as on temporal characteristics: the state of increase, the stationary level, or a state of decrease in indicators involved in the formation of an adaptive response at the time of the action of vitamin A on them. In this case, the following assumptions were made: (1) the metabolic system of the body always “aims” to restore its previous (conditionally stationary) homeostatic state, since it is able to “remember” the previous states, which underlies the mechanism of self-regulation^35,36; and (2) the effect of any regulatory factor will depend on the temporal characteristics (context), that is, the increase, stationary level or decrease of metabolic and immunological parameters involved in the formation of a particular phenotypic manifestation.

MATERIAL AND METHODS

The Design of Experiments

As is known, one of the basic properties of biological systems is their ability to dynamically rebuild their structural and functional characteristics in accordance with constantly changing environmental conditions, that is, to adapt.^{37, 38, 39, 40} However, in the process of adaptation, the body metabolic system “solves” at least two seemingly incompatible tasks: to maintain the homeostatic level that exists at a given point in time (the principle of homeostasis) and to adapt in accordance with new environmental conditions, that is, “to change while maintaining constancy”.

We believe that biological systems are capable of solving such a complex problem based on the unity of several principles of their structural and functional organization: temporal optimality; multifunctionality of molecules (or the ability of one type of molecules to perform different functions depending on the physicochemical conditions of a particular compartment); organization hierarchy (multi-level regulation); and biological memory (the ability of metabolic processes to self-maintenance and reproduction of interrelated cyclic processes—the formation of a systemic response of the body to the actions of various factors) (Figure 1).

Figure 1.

Scheme demonstrates the participation of the four basic principles of the structural and functional organization of biosystems in the formation of a single systemic response at the level of homeostasis in response to the action of various environmental factors; the whole variety of exogenous factors, according to their ability to induce various responses in the body, can be represented as four conditional categories: adaptively irrelevant (a), adaptively significant (b), toxicogenic (c), and lethal (d) (dotted links in the diagram between these categories of factors reflect a wide range of possible variations between them). The signals of exogenous factors are transformed (shown with green dotted arrows) at the level of the structural and functional organization of the biosystem by four basic principles: temporal optimality (1), multifunctionality of biomolecules (2), hierarchical organization (3), and metabolic memory (4), which function as a single system (shown by angular arrows) and provide the formation of adaptive responses at the level of the homeostatic system of the body (marked ∗) in the form of four main metabolic manifestations: fluctuations (short-term deviations from the homeostatic level), quasi-stationary metabolic states (the formation of new homeostatic levels slightly different from the initial indicators), chronic pathologies (lead either to recovery—the formation of new homeostatic characteristics, or to their complete degradation—death).

The dynamics of the adaptive response formation to the action of vitamin A or other factors of influence can be explained on the basis of the proposed concept (the unity of 4 basic principles). Thus, in response to significant changes in the environment, including an increase in the content of biologically active compounds in the body, first of all, new metabolic patterns are formed from the already existing elements (biomolecules) of the system at a given point in time, based on the proposed principle of temporal optimality.³¹ This is possible because almost all macromolecules in biological systems can perform different functions, the switching of which depends on the “context”—the dynamics of the physicochemical characteristics of the corresponding cellular compartments based on the principle of multifunctionality of biomolecules.⁴¹ In case of such a switching of functions does not provide a “solution” to the emerging adaptive problem, the system uses the next level of adaptive response hierarchy—the induction of new elements, including cells, that is, the principle of hierarchy (multilevel) regulation functions: molecular, cellular, tissue, organ, physiological.^42,43 Taking into account the fact that biological systems are characterized by cooperativity in the formation of responses,^{44, 45, 46} and metabolism is formed from cycles and hypercycles,⁴⁷ the formed new variants of homeostatic patterns will be remembered and self-maintained at the metabolic level.^{48, 49, 50} This principle explains the constant tendency of the biological system maintaining or restoring the previous homeostatic state. The retention time (short-term or long-term) of new adaptive metabolic patterns will depend on the characteristics of the acting factor and on the number of combined metabolic cycles into a single hypercycle and will be determined by the characteristics of the formed metabolic memory. We believe that based on the concept of the four basic principles unity of structural and functional organization, the body is able to “constantly change while maintaining its constancy.” Such a sequence of forming a response even to toxicogenic factors that lead to pronounced pathologies is also the only true one at a particular point in time and should be interpreted as adaptive.

Based on these hypothetical ideas, it can be assumed that in the early stages of the liver fibrosis formation, until metabolic memory is formed, the system can ensure the restoration of a homeostatic state. This can be realized by the administration of additional biologically active compounds involved in the “maintenance” of homeostatic balance at the initial stages of pathology development. In this case, the realization is the administration of vitamin A at a dose that corresponds to the restoration of the concentration to the control (initial) values at the initial stages of the Cu-induced liver fibrosis development. It can be assumed that the direction and effect of vitamin A action will depend on the temporal characteristics of the metabolic, cellular, and physiological parameters that are involved in the formation of the body's adaptive response (fibrosis), that is, on the “context” (physicochemical characteristics of the corresponding cellular compartment) at the time of exposure. It can be assumed that such a multifactorial dependence of the body's response to vitamin A, which is also a multifunctional molecule, underlies the inconsistency of the results obtained in previously published experiments.

In order to test this hypothesis about the restoration of the homeostatic state at the early stages of the liver fibrosis formation by the administration of vitamin A, this disease was induced in experimental animals by two consecutive series of injections of copper sulfate (Figure 2A). One series consists of three administrations of copper sulfate every 48 h at a dose of 1 mg/100 g of body weight as described in previously published work.⁵¹ The first series of injections allows to simulate the initial stages of fibrosis development,⁵¹ and a repeated series of intoxication simulated a constantly acting toxigenic factor, which can lead to the formation of a chronic pathological condition (Figure 1). Based on the data obtained earlier, it can be assumed that the administration of vitamin A in the form of retinol acetate three times at a dose of 0.10 mg (300 IU)/100 g of body weight between two series of intoxication can restore the content of vitamin A in the liver to control values, and will also provide an opportunity to evaluate its action against the background of additional hepatotoxic action of copper ions. The dose itself of 0.10 mg/100 g was established by us in a model of liver fibrosis, it provided a therapeutic effect and did not lead to toxic manifestations in preliminary studies, which was also described in previous studies with a study of the action of vitamin A.^31,32 The evaluation of biochemical, cellular, tissue and organismal parameters in these animals made it possible to trace the cooperativity and hierarchy of the adaptive response to the long-term hepatotoxic effect of copper ions (Figure 2B).

Figure 2.

Scheme showing the sequence of procedures with four experimental groups of animals (A) and a list of hematological, cellular, biochemical, and physiological parameters (B) that were determined in these groups of animals: I—intact control animals that did not receive experimental effects; II—experimental animals that received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals that received 1 cycle of administration of copper sulfate as in group II, then three days of physiological saline per os, followed by a 2nd cycle of administration of copper sulfate; IV—experimental animals that received 1 cycle of copper sulfate injections as in group II, then received an oil solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and after that, the 2nd cycle of copper sulfate administration.

Experimental Facilities

The experiments were carried out on young mature (3-month-old) male Wistar rats weighing 80–130 g. Throughout the experiment, the animals were kept under standard vivarium conditions and had free access to food and water. All manipulations were carried out in agreement with the bioethical committee of V. N. Karazin Kharkiv National University (Protocol No. 1 dated March 3, 2022), which is guided by the provisions of the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” (Strasbourg, March 18, 1986). The solution of administered substances was recalculated on body weight.

To test the working hypothesis, 4 groups of animals were formed (Figure 2). Throughout the experiment, the body weight of the animals was recorded. Six days after the last administration of copper sulfate (formation of the body's response), all animals were decapitated for an expert assessment of the connective tissue formations of the intraperitoneal cavity and the isolation of the test material for further analysis: whole blood (hematological parameters), blood serum (biochemical parameters) and liver (histological examination) (Figure 2).

Animal Decapitation

The animals were decapitated under ether anesthesia. The first drops of blood were collected in tubes with EDTA (3-substituted potassium salt of ethylenediaminetetraacetic acid) for the study of hematological parameters (the number of leukocytes, lymphocytes, monocytes, granulocytes, platelets and erythrocytes). The rest of the blood was collected in sterile tubes and incubated at 37 °C for 20 min for further centrifugation at 1500g for 20 min. The supernatant was transferred to clean tubes and used for the determination of serum enzymes.

The number of cells in the blood of animals was determined on a Mindray BC-2800 Vet (USA) hematology analyzer using the impedance method.⁵² The method is based on two independent measurement principles: the Coulter method and colorimetric methods. During each measurement cycle, samples are automatically aspirated, diluted, and the cell content is quantified. The device allows you to determine 18 parameters with differentiation of leukocytes into different populations. Reagents (Mindray Medical International Limited): M-30D Diluent reagent; Reagent M-30R Rinse; Lising reagent for hematology analyzer Mindray bc-2800 Vet.

After opening the abdominal cavity, the degree of connective tissue adhesions formation around the liver lobes was assessed: a low level of adhesive disease was valued as 1–3 points and a high level was valued as 4–5 points in case of complete union of the lobes by connective tissue.

Collection and Use of Liver Samples

For histological examination, liver fragments were taken and fixed in 10 % formalin solution. Samples were processed as described earlier.⁵³ After one or two days of fixation of the liver pieces in 10 % formalin, the samples were dehydrated with ethanol. To do this, the samples were transferred into ethanol with increasing concentration (from 70 % to 96 %) and kept for 12 h at each concentration. After that, the samples were transferred for 1–3 h into xylene, then for 2–3 h into liquid paraffin up to 57°С. Semithin sections were prepared on a microtome and the samples were stained with hematoxylin–eosin. Microscopy: microscope Carl Zeiss, camera SIGETA M3CVOS 14000, magnification 200.

Investigation of the Effect of Retinol Acetate Different Doses on the Resistance of Animals to the Subsequent Action of Large (Lethal) Doses of Copper Sulfate and a Method for Determining Vitamin A in the Liver

In order to determine the possible effect of vitamin A to provide a “protective” effect of the body on the toxic effect of copper sulfate, 4 additional groups of animals were formed, each included 13 rats weighing 80–130 g. The first group received daily vegetable oil instead of vitamin A, the second group—vitamin A (as retinol acetate) at a dose of 0.03 mg (100 IU)/100 g of mass, the third, respectively, vitamin A at a dose of 0.10 mg (300 IU), and the fourth—0.31 mg (900 IU). 24 h after the last administration of vitamin A to all groups of animals, each group was divided into two subgroups; in one of them (3 rats per subgroup), the content of vitamin A in the liver was determined,³² and the animals of the second subgroup (each with 10 rats) received intraperitoneal injections of copper sulfate at a dose of 3.25 mg/100 g of weight (acute lethality) and the time of death was recorded.

The determination of vitamin A content in the liver as described in previously published work.³² The method is based on the vitamin complex formation with boron trifluoride etherate and determination of the decomposition rate of this complex by spectrophotometry.

Isolation of Bone Marrow Cells

Bone marrow cells were isolated from two femurs as described in the works.^54,55

Diaphyses were washed with chilled sodium phosphate buffer containing 137 mmol sodium chloride, 2.7 mmol potassium chloride, 10 mmol sodium hydrogen phosphate, and 1.46 mmol potassium monophosphate (pH = 7.2–7.4) under pressure using a syringe with a large-diameter needle to a total volume of 10 ml from two diaphyses.

The resulting bone marrow suspension of cells with tissue fragments was mechanically disaggregated by resuspension. After that, the cells were passed through a nylon filter with a pore diameter of 100 μm (cell dissociation sieve TGK, Sigma). Then the cell suspension was washed with sodium phosphate buffer by centrifugation for 10 min at 1000 g.

Erythrocytes from the cell suspension were removed by a single treatment with a solution containing 154 mmol ammonium chloride, 10 mmol sodium bicarbonate, 0.082 mmol EDTA (KhimLaborReaktiv, Ukraine) (the prescription is given below) for 10 min at room temperature.

To remove erythrocytes from the suspension, a lysing solution containing: 4.15 g of NH₄Cl, 0.5 g of KHCO₃, and 15 mg of EDTA was prepared. Suspended salts were mixed in a dry test tube, then dissolved in 20–30 ml of autoclaved distilled water, brought up to pH = 7.2–7.4 and sterilized by filtration. The volume of the solution was brought up to 500 ml.

The bone marrow cell suspension was washed twice with 5 ml of chilled sodium phosphate buffer. After that, 2 ml of chilled sodium phosphate buffer was added to the obtained cell suspensions and aliquots of 50 μl were taken into Eppendorf tubes to count the total number of cells in the Goryaev chamber.

To count cells, 50 μl of a solution of 0.4 % trypan blue (Sigma, USA) prepared on the basis of sodium phosphate buffer (pH = 7.1–7.4) and 100 μl of sodium phosphate buffer were added to aliquots and cells were counted. Next, the concentration of cells per ml of uterine suspension was calculated according to the formula for the Goryaev chamber, taking into account the dilution, and the volume of the aliquot was determined so that in each Petri dish the concentration of bone marrow cells was the same and amounted to 2 × 10⁶ cells/ml. The content of ROS and Ca²⁺ was determined in bone marrow cells.

ANALYTICAL METHODS

Determination of ROS and Calcium Ions in Bone Marrow Cells

The ROS content in bone marrow cells was measured by adding Kit–Red according to the manufacturer's recommendations (Cellular ROS Assay Kit - Red ab186027). To do this, 5 μL of the working solution was added to the cell culture (6 × 106/ml). The content of calcium ions in bone marrow cells was measured by adding 5 μL of 25 μmol Fluo-3 solution to the cell suspension (6 × 106/ml). Data on the content of ROS and calcium ions in cells were presented in relative units according to the manufacturer's recommendations.^56,86 In both cases, the components were mixed with a pipette and incubated for 15 min in a dark place at room temperature. Cells were visualized by confocal microscopy using an Olympus FV10i-LIV laser scanning confocal microscope (Olympus, Japan) with Olympus FV4.1 software and imaging protocol. The relative intensity of fluorescent emission in each cell was measured using Olympus FV4.1 software, taking into account the background glow.

Aspartate Aminotransferase Activity in Serum

The method is based on the fact that aspartate aminotransferase (AST) catalyzes the transfer of the amino group from l-aspartate to α-Ketoglutarate to yield oxalacetate and l-glutamate (Karmen et al.).⁵⁷ The oxalacetate undergoes reduction with simultaneous oxidation of NADH to NAD in the malate dehydrogenase catalyzed indicator reaction. The resulting rate of decrease in absorbance at 340 nm is directly proportional to the AST activity. Lactate dehydrogenase is added to prevent interference from endogenous pyruvate which is normally present in serum.

Alanine Aminotransferase Activity in Serum

The activity of alanine aminotransferase (ALT) (EC 2.6.1.2) in blood serum was determined as described previously.⁵⁸ ALT catalyzes the transition of the amino group from l-alanine to α-ketoglutarate, which leads to the formation of pyruvate and l-glutamate. The resulting rate in absorption decrease is proportional to ALT activity. The measurements were carried out on a STAT-FAX 1908 spectrophotometer (USA).

Alkaline Phosphatase Activity in Serum

The activity of alkaline phosphatase (EC 3.1.3.1) in blood serum were determined as described previously.⁵⁹ In the reaction for the determination of this enzyme, p-nitrophenyl phosphate is hydrolyzed to p-nitrophenol and inorganic phosphate. The level of hydrolysis of p-NPP is directly proportional to the activity of alkaline phosphatase. Absorption was determined at a wavelength of 405 nm, at a temperature of 37 °C, incubating for 60 s and a determination time of 60 s on a STAT-FAX 1908 analyzer (USA). Activity was expressed in arbitrary units.

γ-glutamylaminotransferase Activity in Serum

The method for determining γ-glutamylaminotransferase (GGT) activity⁶⁰ is based on the fact that γ-glutamylaminotransferase (EC 2.3.2.2) catalyzes the transition of the glutamyl group l-γ-glutamylcarboxy-4-nitroanilide to glycylglycine with the formation of 5-amino-2-nitrobenzoate, the amount of which is directly proportional to GGT activity and is measured kinetically at a wavelength 405 nm (STAT-FAX 1908, USA).

Statistical Analyses

Data are presented as group means and standard error (x ± SE). Data analysis and visualization were performed using Excel 2013 programs (Microsoft Corporation., USA). Significant differences between groups were determined using ANOVA (Kruskal–Wallis H test) with post hoc comparisons of Mann–Whitney U test with Bonferroni correction. Differences between the control and experimental groups were considered significant at P ≤ 0.05 (the Bonferroni correction was 0.008). Lethality of animals was presented as Kaplan–Meier curves (comparison using the logarithmic rank test).

RESULTS

Retinol Acetate Modifies the Cellular Immune Response to Copper Sulfate Induced Hepatotoxicity

Quantitative Characteristics of Immunocompetent Cells

The total number of the main types of immunocompetent cells (leukocytes, lymphocytes, monocytes, and granulocytes) in the bloodstream increased after the first cycle of intoxication by 40 % and after the second cycle of intoxication by 120 % compared with the control (intact animals) (Figure 3A-I–III). The administration of vitamin A (as retinol acetate) to animals between two cycles of copper sulfate intoxication prevented an increase in the number of immunocompetent cells in the blood compared to animals that did not receive vitamin A and their number remained at the level specific to animals with one cycle of intoxication (Fig. 3A-IV). Therefore, the administration of retinol to animals at the initial stages of the liver fibrosis formation (F0/F1) prevented its further development at the level of immunocompetent blood cells, even against the background of the ongoing action of hepatotoxic factors (copper sulfate).

Figure 3.

Changes in the total number of immunocompetent cells in the bloodstream, where intact animals are taken as the initial (zero) level (A), the number of calcium ions (B) and reactive oxygen species—ROS (C) in bone marrow cells, in the studied groups of animals: I — intact control animals (n = 3) that did not receive experimental treatments; II—experimental animals (n = 3) received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 3) received 1 cycle of copper sulfate injections as in group II, then three days of saline per os, followed by a 2nd cycle of copper sulfate administration; IV—experimental animals (n = 3) received 1 cycle of copper sulfate injections as in group II, then received an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and after that, the 2nd cycle of copper sulfate administration; X ± SE; ∗—significant values (P < 0.05) compared with the intact level (Mann–Whitney U test with Bonferroni correction) are noted; data on the content of ROS and calcium ions in cells were presented in relative units according to the manufacturer's recommendations; Tables of averages are presented in Appendix 1.

It was found that the number of bone marrow cells in all studied groups of animals remained at the control level. It can be assumed that the development of Cu-induced liver fibrosis was accompanied by activation of the immune response. In this case, changes in the characteristics of immunocompetent cells may also occur. The content of calcium ions and ROS in cells can serve as indicators of such qualitative characteristics of cells.

Bone marrow cells obtained from animals after the first cycle of intoxication (the initial stages of fibrosis development) turned out to contain significantly less calcium ions compared to those in control animals (Fig. 3B-II). However, after a repeated cycle of intoxication, a slight increase in the number of cells in the bone marrow was observed compared with the control (Fig. 3B-III). In the event that after the first cycle of intoxication vitamin A was administered to the animals, the amount of calcium ions in them did not differ from the control bone marrow cells despite the subsequent intoxication of these animals (Fig. 3B-IV).

Determination of the ROS amount in bone marrow cells showed that their number was reduced after the first cycle of intoxication compared to the cells of the control groups of animals (Fig. 3C-II), and after the second cycle of intoxication, on the contrary, increased (Fig. 3C-III). The amount of ROS in bone marrow cells in animals treated with vitamin A between two cycles of intoxication did not differ from that in bone marrow cells of the control group (Fig. 3C-IV).

It can be assumed that the cells in the bone marrow of animals after the first and second series of intoxication differed in functional characteristics, and these cell characteristics are affected by the amount of vitamin A in the body, if it was administered between two cycles of intoxication.

Temporal Characteristics of the Immunocompetent Blood Cells Number After Injections of Copper Sulfate

In the next series of experiments, the number of leukocytes, lymphocytes, granulocytes, and monocytes in the blood of all experimental groups of animals was determined. We found that two consecutive series of administrations of copper sulfate, with a three-day interval between series, were accompanied by an increase in the number of leukocytes, lymphocytes, monocytes and granulocytes (Figure 4A–D-III). However, the dynamics of their increase for different types of immunocompetent cells was different. So, if the number of leukocytes and lymphocytes increased after the first cycle of intoxication by 47 % and 22 %, respectively (Figure 4A,B-II), then after the second cycle of intoxication with copper sulfate, the number of these cells increased by 156 % and 116 % (Figure 4A,B-III). At the same time, the number of monocytes and granulocytes increased sharply after the first cycle of intoxication, by 100 % and 82 %, respectively (Figure 4C,D-II), while after the second cycle of intoxication, the number of these cells increased slightly (granulocytes) or remained unchanged (monocytes) compared to the first cycle (Figure 4C,D-III).

Figure 4.

The number of leukocytes (A), lymphocytes (B), granulocytes (C), and monocytes (D) in the studied groups of animals: I—intact control animals (n = 7) that did not receive experimental treatments; II—experimental animals (n = 5) that received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 4) that received 1 cycle of copper sulfate injections as in group II, then three days of physiological saline per os, followed by a 2nd cycle of copper sulfate administration; IV—experimental animals (n = 3), which received 1 cycle of copper sulfate injections as in group II, then received an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and after that the 2nd cycle of administration of copper sulfate; X ± SE; ∗—significant values (P < 0.05) compared with the intact level, ∗∗—significant values (P < 0.05) compared with 1 cycle of copper sulfate injections (group II) (ANOVA Kruskal–Wallis H test) are noted; Tables of averages are presented in Appendix 1.

It should be noted that the number of leukocytes after two cycles of intoxication increased by 2.6 times compared with the initial level, the number of lymphocytes—by 2.2, monocytes—by 2.2, and granulocytes—by 2.4 times; that is, as a result, all types of cells studied increased in comparison with the control (Fig. 4A–D-III).

Therefore, during the development of liver fibrosis (successive effect of the copper ions hepatotoxic influence), the temporal nature of the response at the level of immunocompetent cells was different for different cell types.

Temporal Characteristics of the Immunocompetent Blood Cells Number After Injections of Copper Sulfate and Vitamin A

In the event that, after the first cycle of intoxication with copper sulfate, the animals received vitamin A (as retinol acetate) at a dose of 0.10 mg/100 g of body weight daily for 3 days, the number of leukocytes after the second cycle of intoxication increased only by 27 %, and not by 74 % as in variant of two consecutive cycles of intoxication without receiving vitamin A (Fig. 4A-IV). The administration of vitamin A between two cycles of intoxication was accompanied by the same decrease in the number of lymphocytes compared with the group of animals without receiving vitamin A (Fig. 4B-IV). At the same time, the administration of vitamin A between two cycles of intoxication had no effect on the number of monocytes and granulocytes; their number remained the same as in animals that did not receive vitamin A (Figure 4C, D-IV).

Consequently, the effect of retinol acetate on immunocompetent cells depended on the temporal characteristics of one or another type of immunocompetent cells. In the case when the increase in the number of monocytes and granulocytes was completed in the first cycle of intoxication, vitamin A did not affect their number. In the case when retinol acetate is administered to animals in which an increase in the number of leukocytes and lymphocytes is at the growth stage, it partially stops the further growth of their number, induced by the additional administration of copper sulfate. So, the effect of vitamin A on the change in the number of immunocompetent cells depends on the nature of the cellular system dynamics at the time of exposure, that is, their temporal features at the time of exposure.

Retinol Acetate Enhances Anemic Changes in Animals With Cu-Induced Liver Fibrosis

Characteristics of Erythron

The first cycle of intoxication did not show significant changes in the parameters of erythrocyte characteristics (Fig. 5A-II). After the second cycle of intoxication, there was a slight decrease in the number of erythrocytes, the amount of hemoglobin and hematocrit compared with the control (Fig. 5A-III). These results indicate the initial stages of the development of anemia. If the experimental animals received retinol acetate between two cycles of intoxication (Fig. 5A-IV), then the number of erythrocytes was reduced by 20 % compared to the control, the content of total hemoglobin was 19 % less than the control, and anisocytosis was slightly increased. Consequently, the three-fold administration of vitamin A to animals at the initial stage of fibrosis development somewhat increased the manifestation of anemia after further intoxication. At the same time, these changes in the erythron system did not subsequently lead to inhibition of body weight growth in such animals (Figure 9).

Figure 5.

Characteristics of erythrocytes (A) and platelets (B) in the studied groups of animals: I—intact control animals (n = 7) that did not receive experimental treatments; II—experimental animals (n = 5) that received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 4) that received 1 cycle of copper sulfate injections as in group II, then three days of physiological saline per os, followed by a 2nd cycle of copper sulfate administration; IV—experimental animals (n = 3), which received 1 cycle of copper sulfate injections as in group II, then received an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and after that the 2nd cycle of administration of copper sulfate; X ± SE; ∗—significant values (P < 0.05) compared with the intact level (ANOVA Kruskal–Wallis H test) are noted; data are presented as percentages, Tables of averages are presented in Appendix 1.

Figure 9.

Dynamics of body weight in the studied groups of animals: I—intact control animals (n = 6) that did not receive experimental treatments; II—experimental animals (n = 5) that received 1 cycle of copper sulfate (Cu²⁺) injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 5) that received 1 cycle of copper sulfate injections as in group II, then three days of physiological saline per os, and after that the 2nd cycle of copper sulfate administration; IV—experimental animals (n = 5) that received 1 cycle of copper sulfate injections as in group II, then three days of an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os, and after that the 2nd cycle of copper sulfate administration; X ± SE; ∗—significant values (P < 0.05) compared with the intact level, ∗∗—significant values (P < 0.05) of the retinol acetate group (group IV) compared with 2 cycles of copper sulfate injections (group III) (repeated measurements ANOVA test) are noted.

Characteristics of Platelets

The number of platelets in the bloodstream of the animals after the first cycle of intoxication was increased compared to the control group (Fig. 5B-II). It should be noted that when determining the number of platelets, especially after the first cycle of intoxication, there was a pronounced individual difference in this indicator in animals in the group. After the second cycle of intoxication, the platelet count did not differ from the control level (Fig. 5B-III). This indicates that after the first cycle, this indicator stabilized, and a further increase in copper ions did not affect the number of platelets. Retinol acetate administration between two cycles of intoxication did not significantly affect platelet count, trombocrit, and platelet size distribution (Fig. 5B-IV).

Therefore, the administration of vitamin A to animals at the initial stages of fibrosis somewhat increased the manifestation of anemia in such animals and did not affect the characteristics of platelets, which did not differ from those of control animals.

The Action of Retinol Acetate Contributed to the Normalization of the Characteristics of the Functional Activity of the Liver, If It Was Administered at the Early Stages of Fibrosis Development

After the first cycle of intoxication, the activity of AST in the blood serum remained unchanged compared to the control and decreased after the second cycle of intoxication (Figure 6A-I–III), that is, there was a slight decrease in the activity of this enzyme during two successive cycles of intoxication. If the animals after the first cycle of intoxication received retinol acetate, then the activity of AST corresponded to the activity of that in the control group of animals, that is, the repeated administration of copper sulfate to animals against the background of the action of vitamin A did not inhibit its activity (Fig. 6A-IV).

Figure 6.

The activity of enzymes in the blood serum: aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B), alkaline phosphatase (ALP) (C), and γ-glutamyl aminotransferase (GGT) (D), in the studied groups of animals: I—intact control animals (n = 9) that did not receive experimental treatments; II—experimental animals (n = 4) that received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 4) that received 1 cycle of copper sulfate injections as in group II, then three days of physiological saline per os, followed by a 2nd cycle of copper sulfate administration; IV—experimental animals (n = 4), which received 1 cycle of copper sulfate injections as in group II, then received an oil solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and after that the 2nd cycle of administration of copper sulfate; X ± SE; ∗—significant values (P < 0.05) compared with the intact level, ∗∗—significant values (P < 0.05) compared with 1 cycle of copper sulfate injections (group II) (ANOVA Kruskal–Wallis H test) are noted; Tables of averages are presented in Appendix 1.

The activity of ALT in blood serum after the first cycle of intoxication was not significantly reduced compared to the control and remained at the same level after the second cycle of intoxication (Fig. 6B-I–III). In the event, after the first cycle of intoxication the animals received 3 successive injections of retinol acetate, the activity of ALT in the blood serum increased even after repeated administration of copper sulfate and slightly exceeded the control level (Fig. 6B-IV).

ALP activity decreased after the first cycle of copper sulfate administration, and after the second cycle, it remained at the same level (Fig. 6C-II, III). In the event that the animals received retinol acetate after the first cycle of intoxication, the ALP activity was restored to the control level, and repeated administrations of copper sulfate did not affect the activity of this enzyme in the blood serum (Fig. 6C-IV).

Serum GGT activity remained at the control level in all studied groups of animals (Figure 6D). It should be noted that retinol acetate did not have any effect on the indicators, which remained within the homeostatic values.

Therefore, the administration of retinol acetate at the initial stages of the Cu-induced liver fibrosis development ensured the restoration of the functional activity level of this organ, at least according to the studied indicators of enzymatic activity. At the same time, the effect of retinol acetate on the functional parameters of the liver depended on the “degree” of deviation of one or another indicator from the homeostatic norm and the temporal characteristics of enzyme activity during the action of vitamin A.

Effect of Retinol Acetate on Anatomical and Morphological Changes in the Liver With Cu-Induced Fibrosis

Formation of Adhesions Around the Liver

The morphology of the liver in the intact group of animals corresponded to the accepted norm in terms of the shape of the lobes and their color for all experimental animals of this group (Figure 7A,D-I). In the event that the experimental animals underwent one cycle of intoxication, then in 60 % of the animals proliferation of connective tissue around the liver lobes and their partial “refusing” due to the connective tissue were observed, which was estimated at 1–3 points (Figure 7B,D-II); and in 40 % of this group animals, the proliferation of the connective tissue and the refusing of the lobes was more pronounced, and such changes were estimated at 4–5 points (Figure 7C,D-II). In the event that the animals underwent two cycles of intoxication, then the number of animals with a pronounced proliferation of connective tissue around the liver lobes increased by 10 % compared with one cycle of intoxication; and the distribution on scores 1–3 and 4–5, respectively, was 50 % each (Fig. 7D-III).

Figure 7.

The appearance of the liver of the control groups of animals (A), the appearance of the liver of animals in which there are adhesions, which were estimated at 1–3 points (B) and the appearance of the liver in which the presence of adhesions was estimated at 4–5 points (C); the number of animals in percent (D) in which no adhesions were detected in the liver, adhesions of 1–3 points were present and adhesions of 4–5 points were present, as well as the relative weight of the liver (X ± SE) (E) in the studied groups of animals: I—intact control animals (n = 9) that were not exposed to experimental effects; II—experimental animals (n = 5) that were exposed to one cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 4), which were subjected to two consecutive cycles of administration of copper sulfate, as well as group II; IV—experimental animals (n = 4), which received 1 cycle of copper sulfate injections, as in group II, received an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os for three days, and then the 2nd cycle of copper sulfate administration; Tables of averages are presented in Appendix 1.

If between two cycles of intoxication the animals were injected three times with retinol acetate, then in 25 % of the animals there were no connective tissue formations between the lobes of the liver, that is, they did not differ in anatomical features from intact animals, while in 75 % of the animals, on the contrary, the growth of connective tissue around the liver lobes was strongly marked and scored 4–5 points (Figure 7C,D-IV); the liver lobes in these animals formed a single “conglomerate”, and there were no variants with a slight formation of connective tissue formations (1–3 points) (Fig. 7D-IV).

Consequently, retinol acetate had two opposite effects on the connective tissue adhesions formation around the liver lobes, if it was administered at the initial stages of fibrosis development: both their degradation and the induction of connective tissue proliferation; which may be associated with the individual characteristics of the structural and functional organization of the liver and the temporal characteristics of the indicators involved in the formation of adhesions at the time of the sequential action of copper sulfate and retinol acetate.

Relative Mass of the Liver

The relative mass of the liver in the studied groups of animals did not significantly differ from each other (Figure 7E); however, it should be noted that in the case when between two cycles of intoxication the animals were injected with retinol acetate three times (0.10 mg/100 g of body weight), the liver weight increased slightly, however, compared with the intact group (Fig. 7E-IV). Such an increase in the relative mass of the liver may be associated with some increase in regenerative processes and/or hypertrophy of liver cells.

Some Histological Features of the Liver

After the first cycle of intoxication of the animals, morphological changes in the Glisson's capsule were revealed (1), it was thickened and didn't have ruptures compared to the control sample, the number of binuclear hepatocytes was less common compared to the intact control (Figure 8F). There were no differences between intact animals and animals after intoxication: in blood filling (2)—vascular hyperemia; in number of HSC (3) (Figure 8A, B).

Figure 8.

Histological sections of the liver (A–D) (scale bar = 20 μm) and the number of binuclear hepatocytes (E) (X ± SE) in the studied groups of animals: I—intact control animals (n = 3) that did not receive experimental treatments; II—experimental animals (n = 3) that received 1 cycle of copper sulfate injections (three intraperitoneal injections every 48 h at a dose of 1 mg/100 g of body weight); III—experimental animals (n = 3) that received 1 cycle of copper sulfate injections as in group II, then three days of physiological saline per os, and after that the 2nd cycle of copper sulfate administration; IV—experimental animals (n = 3) that received 1 cycle of copper sulfate injections as in group II, received an oily solution of vitamin A (as retinol acetate, 0.10 mg/100 g of body weight) per os, and after that the 2nd cycle of copper sulfate administration; the following structures were noted: 1—liver capsule, 2—blood vessel, 3—hepatic stellate cells (HSC); hematoxylin-eosin stain, microscope Carl Zeiss, camera SIGETA M3CVOS 14000, magnification 200; ∗—significant values (P < 0.05) compared with the intact level, ∗∗—significant values (P < 0.05) compared with 1 cycle of copper sulfate injections (group II) (ANOVA Kruskal–Wallis H test) are noted; Tables of averages are presented in Appendix 1.

After two cycles of intoxication, these indicators remained without pronounced changes: Glisson's capsule (1) was not changed, with multiple ruptures, there was no blood supply (2), except for the central vessels; HSC (3) are rare, the number of binuclear hepatocytes is less than that in the control variants (Figure 8C,E-III).

In the event that retinol acetate was administered to animals between two cycles of intoxication, then Glisson's capsule (1) had no ruptures. Blood supply (2): in the central vessels—hyperemia compared with the second cycle of intoxication; HSC (3) are as few as after two cycles of intoxication. It is important that the number of binuclear hepatocytes was increased by 2 times compared with intoxication and to a lesser extent compared with the control (Figure 8D,E-IV).

Consequently, the most pronounced histological changes in the liver were manifested at the level of structural changes in the Glisson's capsule, blood filling, and the number of binuclear hepatocytes, which reflects the stimulation of regenerative processes in the organ after the administration of retinol acetate and the normalization of its functional activity.

The Effect of Retinol Acetate on the Dynamics of Body Weight in Animals With Cu-Induced Liver Fibrosis

The intensity of body weight growth in animals is an integral indicator of the functional state of the organism. Body weight in the intact group of animals during the experiment (20 days) increased by 20–25 % compared with the initial (Figure 9, curve I), which corresponds to the accepted standard for rats. If the animals received 3 injections of copper sulfate (the first cycle of intoxication), then their body weight growth stopped (Figure 9, curve II). If the animals after 3 days underwent the second cycle of intoxication, they lost the ability to grow and their body weight loss continued even after they were transferred to the standard living conditions (Figure 9, curve III).

If the animals received retinol acetate after the first cycle of intoxication, then the second cycle of intoxication did not inhibit the growth rate of such animals, they were superior in growth rate to animals with liver fibrosis and did not significantly differ from control animals in growth rate (Figure 9, curve IV).

Consequently, the administration of retinol acetate to animals during the development of liver fibrosis, that is, between two cycles of intoxication with copper sulfate, normalized the growth rate of such animals, and they did not differ from intact animals and significantly exceeded the growth rate of animals with Cu-induced liver fibrosis.

Effect of Different Doses of Retinol Acetate on Resistance to Subsequent Action of Large (Lethal) Doses of Copper Sulfate

The results of the work showed that the most pronounced effects of the action of retinol acetate were manifested at the integrative, physiological level. Previously, it was shown that the preliminary administration of biologically active compounds obtained from different sources can ensure the survival of such animals from the subsequent action of lethal (toxic) doses of copper sulfate.^{61, 62, 63} It was of interest to determine the possible protective effect of different concentrations of vitamin A in the liver on the subsequent effect of toxic doses of copper sulfate in experimental rats.

It was found that the liver of young adult rats (3–4 months old) on a standard diet contained about 4.5 μg/1 g of tissue of vitamin A (Figure 10A-I). If such (intact) animals were injected per os with retinol acetate at a dose of 0.03 mg/100 g of body weight daily for 3 days, then its content in the liver was more than 10 μg/1 g of tissue (Figure 10A-II), however, these values did not differ significantly from the control. An increase in the dose of retinol acetate to 0.10 mg (/100 g of body weight) was accompanied by an increase in the content of this vitamin in the liver by 3 times compared with a dose of 0.03 mg/100 g of body weight (Figure 10A-III). In the event that animals received retinol acetate at a dose of 0.31 mg/100 g of body weight also daily for 3 days, then its amount in the liver increased by 4 times compared to a dose of 0.10 mg (/100 g of body weight); that is, not proportional to the administered dose (Figure 10A-IV). In this case, the content of vitamin A in the liver was increased by 27 times compared with the initial (control) level (Figure 10A-IV).

Figure 10.

The content of vitamin A in the liver of rats (A) and survival curves according to Kaplan–Meier (B&C) in the studied groups of animals: I—control animals (n = 13), which were injected with saline three times, and on the 4th day they were decapitated (n = 3) to determine the content of vitamin A in the liver or a lethal dose (n = 10) of copper sulfate (3.25 mg/100 g of body weight) was administered to study acute toxicity; II, III, IV—experimental animals (n = 13 for each group), which were injected three times with retinol acetate at a dose of 0.03, 0.10 and 0.31 mg/100 g of body weight, respectively; and on day 4 they were decapitated (n = 3 for each group) to determine the content of vitamin A in the liver or injected with a lethal dose (n = 10 for each group) of copper sulfate (3.25 mg/100 g body weight) to study acute toxicity with different doses of vitamin A; X ± SE; ∗—significant values (P < 0.05) compared with the control level (ANOVA Kruskal–Wallis H test) are noted; log-rank test for survival; Tables of averages are presented in Appendix 1.

In the next series of experiments, the effect of an increased content of vitamin A in the liver on the survival of animals to the toxic effect of a lethal dose of copper sulfate for animals with a normal content of vitamin A was determined.

It was found that if the control group of animals was injected with copper sulfate at a dose of 3.25 mg/100 g of body weight, then after an hour they began to die and after 3 h 20 % remained alive, after 24 h only 10 % remained (there were 10 animals in the group) (Figure 10B-I).

In the event, that the same dose of copper sulfate was received by animals that were previously injected with vitamin A at a dose of 0.03 mg/100 g of body weight, then 30 % remained alive throughout the entire experiment (Figure 10B-II). Therefore, an increase in the amount of vitamin A in the liver up to 10 μg/1 g of tissue was accompanied by an increase in the number of surviving animals after exposure to high doses of copper sulfate, although insignificant. A further increase in the dose of vitamin A to 0.10 mg/100 g of body weight did not affect the survival of experimental animals that received high doses of copper sulfate compared to the intact variant, i.e. 20 % of all animals survived in the group, there were also 10 animals (Figure 10 C, III).

However, if the content of vitamin A in the liver was increased to 123 μg/1 g of tissue by administering vitamin A at a dose of 0.31 mg/100 g of body weight, then all 10 experimental animals died within 60–80 min after the administration of a high dose (3.25 mg/100 g of body weight) of copper sulfate (Figure 10C, IV).

Therefore, for vitamin A, there is a U-shaped dose dependence in the manifestation of resistance to a lethal dose of copper sulfate; that is, a relatively small increase in the amount of vitamin A in the liver was accompanied by some increase in resistance to the toxic effects of copper; moderate increases in the content of the vitamin had no effect; and a significant increase in the content of vitamin A (up to 123 μg/1 g) in the liver reduced resistance to the subsequent action of toxic copper ions, however, it is currently difficult to speak of significant differences and additional studies are needed. These data may demonstrate the presence of a dose dependence of the vitamin A action on the processes of adaptation of the body to the action of toxic compounds; however, further studies are needed to find doses with a more pronounced effect.

DISCUSSION

The results of the work are consistent with the proposed concept of the unity of the four basic principles for the formation of an adaptive response of the body to the action of vitamin A, in the case of Cu-induced liver fibrosis model.

Thus, two consecutive cycles of copper sulfate administration to rats (each cycle consisted of 3 injections with an interval of 3 days between cycles, experimental group III) induced an inflammatory reaction, which manifested itself in the form of liver fibrosis, at the stage (F1/F2).

In such animals, there was a two-fold increase in immunocompetent cells in the bloodstream, while the total number of cells in the bone marrow remained the same as in the control, however, in the bone marrow cells, the content of calcium ions and ROS was increased compared to the control; that is, there was a change in the structural and functional characteristics of immunocompetent cells of the bone marrow. After two cycles of intoxication, a decrease in the number of binuclear hepatocytes, a slight inhibition of the functional activity of the liver, the emergence of pronounced connective tissue formations around the liver lobes (adhesive disease) and inhibition of the growth rate of such animals were observed. Such changes occurred against the background of a decrease in the content of vitamin A in the liver.^29,31,32 At the same time, there was a phenomenon of staging in the development of liver fibrosis against the background of two consecutive cycles of intoxication with copper sulfate; that is, after the first cycle of intoxication, these changes were less pronounced compared with two consecutive cycles of intoxication.

Consequently, the chronic-toxicogenic effect of copper ions on the body at the level of homeostasis was accompanied by the formation of a chronic state (Figure 1). It should be noted that the formation of chronic pathology was not manifested in all experimental animals, although in most of them. However, in the experimental group, there were animals in which fibrotic formations were weakly expressed or almost not detected, i.e. there was a pronounced “group” variability in the action of the same toxicogenic factor (Figure 7), which was shown earlier in this experimental model.^31,32

We believe that such differences in the response of an organism of the same sex and age to the same exogenous factor can be explained by the fact that at the time of exposure, these animals differed at least in the temporal characteristics of metabolic parameters and in the features of metabolic memory, which was “formed” as a result of individual differences in the previous “adaptive experience”, since the characteristics of metabolic memory are influenced by the genetic, epigenetic and metabolic characteristics of the organism.

It is clear that the greater the initial structural and functional differences in metabolic patterns between individuals are, the more diverse the responses to the same acting factor will be, since the integral response of the organism is formed as a combination of various constituent elements.

In the event that the experimental animals after the first cycle of intoxication, i.e. at the initial stages of the development of liver fibrosis (F1/F2), were administered vitamin A, then the content of vitamin A in the liver was restored to the control level,³² and this was accompanied by a recovery in the growth of the body weight of such animals, even if they underwent additional intoxication (Figure 9). In such animals, the number of immunocompetent cells decreased compared to animals with Cu-induced fibrosis and it corresponded to the control level, or was close to normal, the amount of calcium ions and ROS in bone marrow cells also corresponded to the norm, which may indicate the restoration of their functional characteristics. The functional parameters of the liver, which are most often assessed by the activity of specific liver enzymes, did not differ from those of the control group of animals (Figure 6). Previously, it was shown that the formation of Cu-induced liver fibrosis was accompanied by a change in the pattern of immunocompetent cells and a number of their functional characteristics, including their lifespan in culture.⁶⁴

It should be noted, in the present study, it was found that during the development of liver fibrosis, there was not an increase in the activity of ALT, AST in the blood serum, but, on the contrary, a decrease in these indicators (Figure 6). As is known, for more than 50 years, the level of activity of ALT and other transferases in the blood serum has been used as an indicator of hepatocellular damage. However, at present, a fairly large amount of experimental data has been accumulated, indicating the absence of a correlation between the degree of damage to hepatocytes and the rate of ALT release and other hepatic enzymes into the bloodstream. This can be explained by various reasons: inhibition of ALT synthesis in the liver (the half-life of this enzyme is 59 ± 9 h),⁶⁵ a decrease in the activity of the enzyme in the liver with age and other functional changes, the absence of cytolysis with suppression of liver function, and so on.

In a series of studies on the effect of relatively high concentrations of copper ions on the development of Cu-induced liver fibrosis, it was shown that serum ALT activity of young animals is reduced compared to control,⁶⁶ and this was also confirmed in the present study. This fact may explain the ability of copper ions to inhibit ALT activity in the liver and, possibly, in the blood. This is supported by the fact that in Cu-induced liver fibrosis, there was a slight violation of the plasma membranes of hepatocytes (Figure 8). An increase in the content of vitamin A in the liver against the background of a high content of copper ions was accompanied by the restoration of ALT activity to the level of control values,³² which may indicate the restoration of the functional activity of the liver with fibrosis.

Differences in the change in ALT activity aroused interest in the study of the functional features of this long-known enzyme. It has been shown that ALT is present in the cells of bacteria, fungi, plants, and animals, along with its participation in the regulation of amino acid and carbohydrate metabolism (glucose–alanine cycle).⁶⁷ This enzyme can exhibit antibacterial activity by binding to LPS and destroying gram-negative bacteria, that is, it can also participate in the development of acute phase inflammation reactions.⁶⁸ It can be argued that ALT, like most other enzymes and vitamins, is capable of performing various functions in the body and the direction of their action will be determined not only by concentration but also the characteristics of their microenvironment, i.e. functional state of the body at a given time.

Consequently, the normalization of the content of vitamin A in the liver at the initial stages of the development of fibrosis “cancelled” the further negative effect of copper sulfate on the body. The normalization of liver function probably also took place due to an increase in the processes of liver regeneration, which may be evidenced by an increase in binuclear hepatocytes compared to the norm, and this is especially pronounced compared to fibrosis (Figure 8). At the same time, in 75 % of the animals, there was an increase in connective tissue formations, and in 25 %, they were not detected and/or were probably destroyed even after the second cycle of intoxication with copper sulfate (Figure 7).

Data on the important role of “maintaining the concentration of vitamin A in the liver” on the development of fibrosis are confirmed by a number of works that indicate the ability of vitamin A to prevent the development of fibrosis.^30,69,70 This effect of vitamin A is explained by the fact that hepatics stellate cells (HSCs) of the liver,^26,27 which are the main depot for vitamin A, play a central role in the formation of liver fibrosis. The localization of vitamin A in HSC is provided by specific receptors that bind complexes of “retinol–retinol binding proteins”, and in HSC, vitamin A is metabolized into esters.^71,72 It has been shown that against the background of the inflammatory process, the formation of ROS in the liver increases, the content of vitamin A decreases, and HSC are transformed into myofibroblasts, which actively produce the connective tissue matrix.^26,27

It remained unclear why, against the background of the inflammatory process, the content of vitamin A in HSC decreased. Recently, it was shown that a decrease in the amount of vitamin A in HSC of the liver may be due to the activation of lipogenesis and the accumulation of triglycerides in hepatocytes.²⁹

It can be assumed that the restoration of vitamin A in liver cells in the presence of additional factors contributing to the activation of HSC (the presence of oxidative stress, an increased content of cytokines, etc.) can maintain a balance between the production of connective tissue elements and the subsequent activation of metalloproteinases, which are involved in the degradation of excess connective tissue, i.e. regulation of biogenesis. In other words, against the background of a low content of vitamin A in HSC and in the presence of other activation factors of HSC, there is no activation of metalloproteinases,⁷³ which is accompanied by the transition of fibrosis to a chronic state or by disturbing the balance between the synthesis and degradation of connective tissue.

This assumption is indirectly supported by the fact that in some animals (about 25 %) that received retinol acetate between two cycles of intoxication, connective tissue formations around the liver were not detected (Fig. 7D-IV); while in animals that underwent two complete cycles of intoxication without administration of retinol acetate, connective tissue formations around the liver were present in all ones (Fig. 7D-III).

These results suggest that vitamin A can both enhance the fibrogenic functions of HSC, along with the action of oxidative stress products, and possibly activate the synthesis of metalloproteinases; and the balance between these two oppositely directed the processes of connective tissue biogenesis will determine the increase in the development of fibrosis or, on the contrary, the hydrolysis of connective tissue formations and the transition of chronic pathology to a new homeostatic state; which is observed in some animals and can be interpreted within the framework of the concept of 4 basic principles of the organization of biosystems (Figure 1).

We suppose that an important effect in the action of vitamin A on the Cu-induced liver fibrosis model is the excessive accumulation of connective tissue formations around the liver at the initial stages of fibrosis development (Fig. 7C-IV).³¹

As is known, connective tissue induction is a primary response to cellular tissue damage, and this plays a key role in the formation of the cytoskeleton and induces morphogenesis in the damaged tissue, that is, such growth of connective tissue at the initial stages of the inflammatory process has a pronounced adaptive character.⁷⁴ The adaptive role of connective tissue formations in the restoration of liver function is indicated by the fact that not only HSC but also hepatocytes, fibroblasts, macrophages, and other cell types participate in their formation,⁷⁵ that is, there is a multi-level system of adaptive manifestations. It was shown and confirmed in the present study that the administration of retinol acetate to animals with experimental liver fibrosis was accompanied not only by the restoration of liver function but also by the induction of connective tissue, and this correlated with the restoration of liver function and the dynamics of body weight growth in such animals.³²

We believe that the problem of the liver fibrosis formation is associated with a “violation” of the staging of fibrogenesis. At the initial stages of the inflammatory process development, the launch of fibrogenesis as the primary reaction of the adaptive response of the body contributes to the process of regeneration of the liver tissue, after which the process of activation of metalloproteinases and stabilization of connective tissue homeostasis close to the initial level should begin. However, an imbalance between the induction and hydrolysis of connective tissue, which may also be regulated by vitamin A, leads to two different consequences: the transition to cirrhosis or the restoration of liver function (Figure 1).

Therefore, when studying the effect of vitamin A on fibrotic liver, we are faced with the problem of ambiguity or non-reproducibility of the results of the vitamin A action on fibrotic tissue. Such inconsistency of the results reflects the real-life multidirectional response of the body to the same active agent, in this case, vitamin A.

Long-term studies of the mechanism of vitamin A action have shown that it is able to exhibit various functions, that is, it is a multifunctional molecule and its action is manifested at various hierarchical levels of biological organization (4 principles) (Figure 1); that is, there is a manifestation of systemic changes in the body, some of which are shown in Figure 11.

Figure 11.

Scheme that demonstrates that vitamin A is a multifunctional molecule^{29,31,32,41, 42, 43, 44, 45, 46, 47, 48, 49, 50,76, 77, 78, 79, 80, 81, 82, 83, 84}. It is involved in the regulation of gene expression and in the activity of enzymes—this is manifested at all levels of organization: molecular, cellular, tissue, and organism (I–IV); The “direction” of the action of vitamin A in fibrosis will depend on the formation of a single integral response, which is implemented on the basis of the cooperation of four basic principles of the functioning of biological systems (Fig. 1): 1—temporal optimality (TOP), 2—multifunctional molecules, 3—the principle of hierarchy, 4—biological memory.

The complexity of the problem experimental solution of the vitamin A multidirectional action also lies in the fact that biological systems are highly dynamic, compartmentalized; and these compartments are represented not only by “chambers” edged by membranes but also by microcompartments, which represent a high localization of certain types of molecules, as a rule, forming certain metabolic cycles.⁸⁵ The most difficult thing about this is to understand how different compartments in different cell types respond to “disturbance” factors, simultaneously as a single cooperative system.

Since the “molecular context” or microcompartment is variable, that is, it changes over time, the rate and direction of temporal changes in the microcompartment are also different and can be conditionally represented as (1) states of increasing the concentration of elements or reaction rates and (2) decrease or stationarity of these parameters in certain compartments per unit time.

So, if the number of leukocytes and lymphocytes increased or decreased during the development of fibrosis (there was a temporal change in these indicators), then retinol acetate prevented the further development of these indicators, that is, restored the altered indicators of cellular homeostasis to the initial homeostatic level (Figure 4). Similar actions of retinol acetate also took place in the case of a pronounced temporal change in enzyme activity during the development of fibrosis (Figure 6). It is well known that in cases where homeostatic indicators correspond to the normal homeostatic level, then biologically active compounds or xenobiotics do not have any noticeable effect on them; and vice versa, if the indicator differs from the norm, then, as a rule, changes in this indicator are recorded in response to the actions of certain drugs. Of course, the available data cannot serve as a rigorous proof of these assumptions, but they allow us to outline ways to find answers to these complex problems of modern biomedicine.

The direction of the action of vitamin A on certain metabolic systems depends not only on the temporal characteristics of the metabolic processes involved in the formation of an adaptive response, but also on its concentration in biological tissues. This paper shows that high concentrations of vitamin A in the liver increased the mortality of animals after administration of high doses of copper sulfate, while small doses of vitamin A may provide, although not significant, protection against this toxicant (Figure 10).

Further research into the role of vitamin A at different stages of fibrosis development, including its later stages, is promising in resolving the issue of the reversibility of this pathological process.

• 1.The second cycle of administration of copper sulfate (3 injections every 48 h at a dose of 1 mg/100 g of body weight) to animals at the initial stages of fibrosis development, which is induced by the first cycle, simulates the transition of fibrosis from the F0/F1 stage to the F1/F2 stage.
• 2.The administration of vitamin A to animals at the initial stages of fibrosis development (Fо/F1) in the form of retinol acetate at a dose of 0.10 mg/100 g of body weight (this ensured the restoration of its concentration in the liver to control values) prevented the transition of fibrosis to the F1/F2 stage, even against the background of continued exposure to copper ions; this was manifested by a decrease in the number of immunocompetent cells to the control level; normalization of the amount of calcium ions and ROS in bone marrow cells; restoration of the activity of liver enzymes (ALP) to the level of control; an increase in the number of binuclear hepatocytes; and restoration of the growth dynamics of the body weight of such animals.
• 3.The “direction” of the action of vitamin A depends on the stages of fibrosis development and temporal characteristics (increase, decrease or standard level) of cellular and metabolic parameters involved in the formation of the body's adaptive response to the action of vitamin A against the background of fibrotic changes.

Credit authorship contribution statement

Anatoly I. Bozhkov: experiment idea, writing a manuscript;

Rustam A. Akzhyhitov: work with animals, participation in the design and editing of the manuscript;

Svitlana G. Bilovetska: conducting histological and morphological studies;

Evgeny G. Ivanov: carrying out biochemical analyzes;

Nataliia I. Dobrianska: conducting hematological studies;

Anastasiia Yu. Bondar: bone marrow research.

Conflicts of interest

All authors have none to declare.

Funding

This work was carried out with the financial support of V.N. Karazin Kharkiv National University. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript.

Appendix 1.

Figure 3 B, C.

Groups	Ca2+, conv. units		ROS, conv. units
	X	SE	X	SE
I	17,938	987	13,196	456
II	7229	309	10,071	140
III	22,810	298	20,148	286
IV	18,010	1553	13,397	819

ROS, reactive oxygen species; SE, standard error.

Figure 4 А, B, C, D.

Groups	Leukocyte content, × 109/L		Lymphocyte content, × 109/L		Granulocyte content, × 109/L		Monocyte content, × 109/L
	X	SE	X	SE	X	SE	X	SE
I	7.27	0.85	4.27	0.58	2.79	0.51	0.18	0.03
II	10.70	0.57	5.24	0.60	5.10	0.68	0.36	0.05
III	18.67	0.64	9.23	2.35	6.63	1.18	0.40	0.04
IV	13.63	1.86	6.70	1.48	6.50	0.61	0.43	0.09

SE, standard error.

Figure 5 А, B.

Groups	Red blood cells, × 1012/L			Hemoglobin g/L			Hematocrit, %
	X	SE	%	X	SE	%	X	SE	%
I	9.43	0.38	100.00	154.86	7.84	100.00	47.14	2.2	100.00
II	8.20	0.10	86.96	134.4	1.44	86.79	42.6	0.32	90.37
III	8.90	0.68	94.38	134.67	5.93	86.96	44.2	3.94	93.76
IV	7.64	0.39	81.02	125.67	8.37	81.15	40.7	1.25	86.34
Groups	Mean corpuscular volume, fL			Mean corpuscular hemoglobin concentration, g/L			Anisocytosis, %
	X	SE	%	X	SE	%	X	SE	%
I	49.94	0.36	100.00	327.71	4.71	100.00	13.16	0.61	100.00
II	52.06	0.50	104.25	315.20	5.29	96.18	14.20	0.42	107.90
III	49.60	0.58	99.32	335.00	4.08	102.22	12.60	0.58	95.74
IV	53.53	1.52	107.19	307.67	11.70	93.88	15.47	0.58	117.55
Groups	Platelets, × 109/L			Mean platelet volume, fL			Hematocrit, %
	X	SE	%	X	SE	%	X	SE	%
I	294.80	80.10	100.00	5.30	0.09	100.00	0.19	0.05	100.00
II	467.00	139.39	158.41	5.30	0.11	100.00	0.25	0.08	131.58
III	259.25	43.25	87.94	5.40	0.20	101.89	0.14	0.02	73.68
IV	296.67	42.15	100.63	5.20	0.13	98.11	0.15	0.02	78.95

SE, standard error.

Figure 6 A, B, C, D.

Groups	AST, U/L		ALT, U/L		ALP, U/L		GGT, U/L
	X	SE	X	SE	X	SE	X	SE
I	163.65	12.03	61.29	5.93	316.41	45.00	2.17	0.29
II	151.50	12.93	47.55	3.97	101.70	16.03	2.55	0.32
III	137.50	6.15	46.28	7.09	137.90	26.38	2.85	0.66
IV	154.28	5.91	78.50	11.17	291.43	64.19	2.60	0.10

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamylaminotransferase; SE, standard error.

Figure 7 E; Figure 8 E.

Groups	Relative liver weight to body weight, %		Number of binuclear hepatocytes, n/100 cells
	X	SE	X	SE
I	4.41	0.30	16.00	0.58
II	4.60	0.32	10.00	0.00
III	4.96	0.36	10.33	2.60
IV	5.10	0.19	23.33	1.67

SE, standard error.

Figure 10 A.

Groups	The dose of retinol acetate that the animals received	Vitamin A content, μg/g
		X	SE
I	0.00 mg	4.50	0.10
II	0.03 mg	10.58	3.81
III	0.10 mg	30.00	3.31
IV	0.31 mg	122.85	8.35

SE, standard error.