Role of sulfide anion in the development of chronic alcoholic hepatitis under the conditions of modulation of adenosine monophosphate kinase – a correlational study

Introduction and aim. Hydrogen sulfide (H 2 S) has attracted the attention of researchers as a novel signaling molecule that affects vascular metabolism, immune function, stress and inflammation. It plays an important role in pathophysiological disorders under the conditions of the development of obesity, diabetes, non-alcoholic fatty liver disease and cardiovascular diseases. The purpose of this work is to establish correlation ratios of H 2 S concentration with markers of oxidative-nitrosative stress and extracellular matrix metabolism of the liver during chronic alcoholic hepatitis modeling and AMPK modulation by phen-formin and doxorubicin. Material and methods. The experiments were performed on 36 white, sexually mature male Wistar rats, weighing 180-220 g. Alcoholic hepatitis was modelled by alcohol administration, on the background of alcoholic hepatitis animals received phen-formin orally at a dose of 10 mg/kg or doxorubicin at a dose of 1.25 mg/kg intraperitoneally. Statistical processing of the results of biochemical studies was carried out using the non-parametric method of Spearman to determine correlations. Results . H 2 S during alcoholic hepatitis inversely proportionally strongly correlates with the concentration of nitrites, oxypro-line and arginase activity. Phenformin administration during alcoholic hepatitis leads to formation of inversely proportionally strongly correlation of H 2 S with the production of superoxide anion radical, the concentration of malondialdehyde, activities of constitutive NO-synthases, nitrite reductases, nitrate reductases, and arginase. Doxorubicin administration during alcoholic hepatitis leads to formation of directly proportional strongly correlation of H 2 S with the activity of constitutive NO-synthases, nitrite reductases, nitrate reductases. Conclusion . Administration of phenformin or doxorubicin expands correlations between H 2 S and indicators of oxidative-ni-trosative stress.


Introduction
In recent years, hydrogen sulfide (H 2 S) has attracted the attention of researchers as a novel signaling molecule that affects vascular metabolism, has influence on immune function, changes stress and inflammation progression.It plays an important role in pathophysiological disorders under the conditions of the development of obesity, diabetes, non-alcoholic fatty liver disease and cardiovascular diseases. 1H 2 S exerts physiological functions by targeting proteins, enzymes, and transcription factors through a post-translational mod-ification known as persulfidation. 2] Enzymatic formation of H 2 S is catalyzed by cystathionine γ-lyase (EC 4.4.1.1,CSE), cystathionine β-synthase (EC 4.2.1.22,CBS) and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2,MST). 5 All these three enzymes are present in the liver and through the synthesis of H 2 S regulate its functions.A small part of endogenous H 2 S is formed by the non-enzymatic reduction of sulfur contained in certain metabolites (persulphides, thiosulphates and polysulphides). 2Hepatic H 2 S metabolism affects glucose metabolism, insulin sensitivity, lipoprotein synthesis, mitochondrial biogenetics, and biogenesis.] Several pathways are involved in the pathogenesis of ethanol-induced liver disease.One of the central pathways involves the induction of cytochrome P450 2E1 by ethanol, which leads to the induction of lipid peroxidation in hepatocytes.The second pathway involves ethanol regulation of transcription factors associated with lipid metabolism.Ethanol also affects the activity of enzymes involved in energy metabolism, including AMP-activated protein kinase (AMPK) and sirtuin-1 (SIRT1) [7].Ethanol-mediated dysregulation of hepatic AMPK, a master regulator of lipid metabolism, is one of the main mechanisms in the pathogenesis of alcoholic fatty liver disease, because impaired AMPK signaling accelerates lipid accumulation and inhibits lipid catabolism, ultimately leading to the development of alcoholic fatty liver disease in animals. 82] Doxorubicin has a powerful inhibitory effect on AMPK activity.The use of doxorubicin at a dose of 2.5 mg/kg has a persistent inhibitory effect on AMPK activity in the heart. 13A single intraperitoneal injection of doxorubicin at a dose of 20 mg/kg also causes a persistent decrease in AMPK expression and leads to development of oxidative stress due to a decrease in the expression of antioxidant enzymes (superoxide dismutase and catalase). 14][17] It has been reported, that diallyl disulfide (DADS) has hepatoprotective effects against alcoholic liver dis-ease (ALD), while the underlying mechanisms of action of H 2 S remain largely unknown.Research by Shi-Xuan Liu et al. (2022) reported that DADS ameliorated ethanol-induced downregulation of peroxisome proliferator-activated receptor α (PPARα), of carnitine palmitoyltransferase 1 (CPT1) and phosphorylated AMP-activated protein kinase in mouse liver and AML12 cells.These results demonstrate that DADS can prevent ethanol-induced hepatic steatosis and early inflammation by regulating the gut-liver axis and supporting fatty acid catabolism. 18he search for ways to reduce oxidative-nitrosative damage to the liver under the conditions of the development of chronic alcoholic hepatitis led us to believe that the modulation of the AMPK cascade plays an important role in the pathogenesis of this disease.Considering the antioxidant and regulatory potential of hydrogen sulfide, which undoubtedly changes its metabolism under conditions of chronic alcoholic hepatitis, the question arises as to what is the role of hydrogen sulfide in changing metabolism of the hepatocyte.Establishing correlations between indicators of oxidative-nitrosative stress and indicators of metabolism of the extracellular matrix of the liver will bring us closer to establishing the role of hydrogen sulfide in the pathogenesis of chronic alcoholic hepatitis.

Aim
We aimed to establish correlation ratios of H 2 S concentration with markers of oxidative-nitrosative stress (total NO-synthase activity, activity of constitutive and inducible isoforms of NO-synthase, concentration of nitrosothiols and nitrites, concentration of peroxynitrites of alkali and alkaline earth metals, the activity of nitrite-and nitrate reductases, arginases, superoxide dismutase and catalase, concentration of malondialdehyde, oxidation-modified proteins and production of superoxide anion) and parameters of extracellular matrix metabolism of the liver (total concentration of glycosaminoglycans, concentrations of heparin-heparan, keratan-dermatan and chondroitin fractions, concentration of free oxyproline and sialic acids) during AMPK modulation by phenformin and doxorubicin under conditions of chronic alcoholic hepatitis modeling.

Sample and experimental groups
The experiments were performed on 36 white, sexually mature male Wistar rats, weighing 180-220 g.The animals were divided into 6 groups: І -control (n=6).The control group included animals that were subjected to similar manipulations throughout the study period, but were injected with a physiological solution.
V -doxorubicin group (n=6) which received doxorubicin hydrochloride according to doxorubicin injection protocol.
VI -alcoholic hepatitis + doxorubicin group (n=6), was subjected to chronic alcoholic hepatitis and doxorubicin injection protocols.
The conditions for keeping animals in the vivarium were standard.Animals were removed from the experiment on the 63 rd day by blood sampling from the right ventricle of the heart under thiopental anesthesia.Devices used for research have passed metrological control.

Phenformin injection protocol
Phenformin hydrochloride (phenformin, Sigma-Aldrich), as activator of AMP-activated protein kinase was introduced orally at a dose 10 mg/kg daily for 63 days. 19

Chronic alcoholic hepatitis modelling protocol
Chronic alcoholic hepatitis in rats was modeled by the method of forced intermittent alcoholization for 5 days, with a repeat after two days by intraperitoneal injection of 16.5% ethanol solution in 5% glucose solution, at the rate of 4 ml/kg of body weight.After that, they were transferred to 10% ethanol as the only source of drinking. 20Modelling lasted for 63 days.

Doxorubicin injection protocol
Doxorubicin hydrochloride (doxorubicin, S.C. Sindan-Pharma S.R.L.), as inhibitor of AMP-activated protein kinase, was injected intraperitoneally at a dose 1,25 mg/kg four times a week for 63 days. 21

Biochemical analysis
For biochemical analysis we used 10% liver tissue homogenate and blood serum.Liver tissue homogenate was obtained after homogenization of 1 g of rat liver with 9 ml of 0.2 M Tris-buffer solution (Trisaminomethane-hydrochloric acid buffer, pH=7.4).Then it was centrifugated at 3000 g for 10 minutes.Upper layer (supernantant) was used for further biochemical analysis.Blood plasma was obtained after addition of 0,109 М sodium citrate at ratio 9:1 and subsequent centrifugation at 3000 g for 10 minutes.
In the blood plasma of rats, the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using diagnostic kits, produced by NPP "Filisit-Diahnostyka".We also calculated the de Ritis coefficient (AST/ALT).
Concentration of sulfide anion (calculated as H 2 S concentration) specifically reacts with N-N-dimethyl-para-phenylenediamine in the presence of Fe 3+ ions and excess of hydrochloric acid to form a red-pink chromogen with a maximum light absorption at a wavelength of 667 nm. 22otal NO-synthase activity (gNOS) was evaluated by the increase of nitrites after incubation of 10% tissue homogenate (0.2 ml) for 30 min in the incubation solution (2.5 ml of 0.1 M trisbuffer, 0.3 ml of 320 mM aqueous solution of L-arginine and 0.1 ml of 1 mM NA-DPH + H + solution).4] The activity of iNOS was calculated by the formula: іNOS= gNOS-сNOS. [28][29][30][31][32][33]

Statistical analysis
Statistical processing of the results of biochemical studies was carried out using the non-parametric method of Spearman to determine correlations (with the exception of groups where the studied parameters corresponded to a normal distribution with very small values of the standard deviation, where the Pearson method was used).All statistical calculations were performed in the Microsoft Office Excel program and its extension Real Statistics 2019.Correlation was considered statistically significant at p<0.05.

Results
The role of the AMPK cascade in the development of chronic hepatitis remains unclear.But it is known that modulation of AMPK activity leads to changes in the pathogenesis of chronic alcoholic hepatitis.Establishing correlational and pathogenetic relationships between the concentration of hydrogen sulfide and biochemical indicators of oxidative-nitrosative stress and markers of the metabolism of the extracellular matrix of the liver under the conditions of chronic alcoholic hepatitis will allow the use of donors and scavengers of hydrogen sulfide in the pathogenetic therapy of alcoholic liver disease.Blood biochemical markers of chronic alcoholic hepatitis (AST, ALT activity and de Ritis coefficient) under the conditions of modulation of AMPK cascade are shown in Fig. 1.Activity of AST, ALT and de Ritis coefficient proved a presence of cytolytic process in rat liver in chronic alcohol hepatitis group.In the control group of animals, no statistically significant correlations were found between the concentration of sulfide anion and other biochemical parameters.In the group of animals injected with phenformin, it was found that the concentration of sulfide anion is inversely proportionally strongly correlated with the activity of superoxide dismutase and inversely proportionally strongly correlated with the concentration of the keratan-dermatan fraction of glycosaminoglycans (Table 1).In a group of rats with chronic alcoholic hepatitis, it was found that sulfide anion is inversely proportionally strongly correlated with the concentration of nitrites, oxyproline and arginase activity.During phenformin correction of chronic alcoholic hepatitis in rats, it was found that sulfide anion is inversely proportionally strongly correlated with the production of superoxide anion radical, the concentration of malondialdehyde and the chondroitin frac-tion of glycosaminoglycans, as well as with activities of constitutive NO-synthases, nitrite reductases, nitrate reductases, and arginase.It was also found that the sulfide anion is directly proportional strongly correlated to the concentration of peroxynitrite and the keratan-dermatan fraction of glycosaminoglycans (Table 1).
Doxorubicin injection to rats on background of chronic alcoholic hepatitis led to following changes: concentration of sulfide anion directly proportional strongly correlated to the concentration of nitrites, the activity of constitutive NO-synthases, nitrite reductases, nitrate reductases and the concentration of the keratan-dermatan fraction of glycosaminoglycans.It was also found that sulfide anion is inversely proportionally strongly correlated with the concentration of nitrosothiols, free oxyproline and the heparin-heparan fraction of glycosaminoglycans (Table 2).

Discussion
The summary of correlation ratios between H 2 S and biochemical parameters of rat liver are presented in Fig. 2. In brief, during excessive alcohol intake sulfide anion receives strong negative correlation bonds with nitrite content, concentration of free L-oxyproline and arginase activity (ARG), which were absent under normal conditions.Stimulation of AMPK cascade by phenformin leaves correlation of sulfide anion with arginase intact, but removes its correlation with nitrites and L-oxyproline, while creating new correlations with nitrite reductases (NiR), nitrate reductases (NaR), MDA, cNOS, peroxynitrite (ONOO -) and superoxide.Despite its inversed correlation with superoxide production, sulfide anion can potentially cause increased production of superoxide from NiR and NaR (as parts of xanthine oxidoreductase complex), thus leading to in-crease of peroxynitrite formation, which may explain appearance of direct correlation between sulfide anion and peroxynitrite during combined influence of phenformin and alcohol.Inhibition of AMPK cascade by doxorubicin leaves correlation of sulfide anion with L-oxyproline intact, but removes its correlation with nitrites and arginase, while creating new correlations with NiR, NaR, S-NO, and cNOS.It is worth mentioning, that correlations between sulfide anion and NiR, NaR, and cNOS in doxorubicin+alcoholic hepatitis group have different vector compared to phenformin+alcoholic hepatitis group.In doxorubicin+ alcoholic hepatitis group sulfide anion can potentially create conditions under which main source of NO production will shift towards predominance of L-arginine-independent pathway.
The absence of statistically significant correlations in the control group of animals may indicate the non-linearity of the relationship between the sulfide anion content and the investigated biochemical parameters.At the same time, modulation of AMPK activity leads to the appearance of statistically significant correlations.The appearance of an inversely proportional strong relationship between SOD activity and H 2 S concentration under the conditions of stimulation of AMPK activation by phenformin may be associated with a decrease in the production of reactive oxygen species (ROS) by mitochondria under the influence of AMPK. 34The appearance of a correlation relationship similar in direction and strength between H 2 S and the concentration of the keratan-dermatan fraction of GAG is related to the ability of AMPK to directly affect the concentration of different fractions of sulfated GAG. 35Reduction of the degree of AMPK activation by doxorubicin leads to the appearance of a direct strong relationship between H 2 S and nitrite concentration, which may indicate the ability of H 2 S to enhance gene expression of constitutive and inducible NOS isoforms. 36nder the conditions of chronic alcoholic hepatitis, the appearance of an inversely proportional strong relationship between H 2 S and nitrite concentration is noted, which may be related to the ability of ethanol to induce the expression of genes of the inducible isoform of NOS, regardless of H 2 S concentration. 37] The inversely proportional strong relationship between the concentrations of H 2 S and L-oxyproline under conditions of chronic alcoholic hepatitis can be explained by the origin of free L-oxyproline, which under conditions of excessive accumulation of alcohol is released from collagen fibers during oxidative stress, while H 2 S is a powerful antioxidant. 40imulation of the activation of the AMPK cascade in the background of simulation of alcohol intoxication significantly enhances the effect of H 2 S on the nitric oxide system.2][43] Considering the fact that inhibition of AMPK cascade activation by doxorubicin completely reverses the relationship between H 2 S and the enzymes described above to a directly proportional strong one, it can be assumed that AMPK can affect the biological function of H 2 S in relation to the xanthine oxidoreductase complex.
H 2 S has the ability to stimulate the conversion of the xanthine oxidoreductase complex into nitrite reductase and promote the formation of nitric oxide from this source. 44The blockade of the transition of the xanthine oxidoreductase complex, due to the activation of AMPK, to nitrite reductase can contribute to the excessive formation of ROS from the oxidase domain, which explains the directly proportional strong relationship between H 2 S and ONOO -in the group of combined exposure to phenformin and chronic alcoholic hepatitis.Conversely, blockade of AMPK activation and H 2 S-dependent stimulation of conversion of the xanthine oxidoreductase complex to nitrite reductase may explain the directly proportional strong relationship between H 2 S and nitrosothiols in a group of animals under combined exposure to doxorubicin and chronic alcoholic hepatitis.
The disappearance of the relationship between H 2 S and arginase activity in a group of animals with combined exposure to doxorubicin and chronic alcoholic hepatitis may be associated with a redistribution of the effect of H 2 S on the nitrate-nitrite reductase pathway of nitric oxide formation towards the predominance of the effect on nitrosothiols, which can modulate the activity of arginase by releasing nitrous oxide. 45odulation of AMPK activity does not change the direction and strength of the relationship between H 2 S and the concentration of free L-oxyproline under conditions of chronic alcoholic hepatitis.Changes in the relationships between H 2 S and the concentration of different GAG fractions depend to a greater extent on the influence of AMPK on the concentration of the latter and require further investigation. 35he limitation of this study is that we did not access the expression of AMPK in studied groups.changes in biochemical parameters with which it has shown strong correlations.On the studied models of modulation of AMPK cascade during alcohol intoxication we can establish dependence of abovementioned biochemical parameters, especially those, that showed significant correlations, from changes in concentration of sulfide anion in liver, which can be achieved by addition of sulfide donors and/or scavengers.Estimation of pathogenetic role of sulfide anion in development of alcoholic hepatitis and its interplay with AMPK cascade may open a path for usage of sulfide anion as a pathogenetically sound treatment of alcoholic hepatitis, free from negative impacts of direct influence on AMPK by specific modulators.

Conclusion
Modeling of chronic alcoholic hepatitis leads to the appearance of correlations between the concentration of endogenous H 2 S and the activity of arginases, the concentration of nitrites and free L-oxyproline in the liver of rats.
Administration of phenformin under the conditions of chronic alcoholic hepatitis modeling expands correlations between endogenous H 2 S and indicators of oxidative-nitrosative stress in the liver of rats, due to new correlations with superoxide anion-radical, peroxynitrite, nitrate-nitrite reductases, constitutive NO-synthases and malondialdehyde.However, the administration of phenformin leads to the loss of the correlation between endogenous H 2 S and the concentrations of free L-oxyproline and nitrite.
Administration of doxorubicin under the conditions of chronic alcoholic hepatitis modeling expands correlations between endogenous H 2 S and indicators of oxidative-nitrosative stress in the liver of rats, due to new correlations with peroxynitrite, nitrate-nitrite reductases, constitutive NO-synthases and nitrosothiols.However, administration of doxorubicin leads to a loss of correlation between endogenous H 2 S and arginase activity and nitrite concentration.

Fig. 1 .
Fig. 1. Biochemical indicators of blood plasma of rats under the conditions of modeling chronic alcoholic hepatitis and modulation of AMPK cascade: (a) activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); (b) the de Ritis coefficient (AST/ALT); * -indicates that difference is statistically significant compared to control group; # -indicates that difference is statistically significant compared to alcoholic hepatitis group

Fig. 2 .
Fig. 2. Correlation of H 2 S concentration with markers of oxidative-nitrosative stress and extracellular matrix metabolism of the liver during chronic alcoholic hepatitis modeling and AMPK modulation: (a) influence of alcohol intoxication and doxorubicin on H 2 S correlation with biochemical parameters, (b) influence of alcohol intoxication on H 2 S correlation with biochemical parameters, (c) influence of alcohol intoxication and phenformin on H 2 S correlation with biochemical parameters

Table 2 .
Correlation analysis of biochemical indicators of the liver of rats under the conditions of modeling chronic alcoholic hepatitis and blockade of AMPK-cascade activation