Hepatoprotective study of Indonesian water kefir against CCl 4 -induced liver injury in rats

Context : Water kefir is a fermented beverage that is typically made in the home by inoculating a sugar-rich solution with a microbial community (water kefir grains). Several studies on the metabolite content and hepatoprotective effects of water kefir have been published, but carbon tetrachloride (CCl 4 )-induced acute liver injury has not been studied. Aims : To evaluate the efficacy of water kefir in vivo against hepatoprotective CCl 4 -induced acute liver injury and to in silico investigate metabolites that play an important role in hepatoprotective mechanisms. Methods : The present study aimed to investigate the hepatoprotective activity of water kefir in an animal model caused by CCl 4 . Furthermore, using molecular docking, the metabolites found in water kefir were evaluated for their role in the NF-κB and Nrf2 signaling pathways . Results : Water kefir significantly and dose-dependently alleviated acute liver injury caused by CCl 4 . Water kefir administration at all doses produced results comparable to the positive control ( Curcuma extract). Molecular docking simulations showed that, compared to Nrf2, the 25 metabolites were more likely to interact with the NF- B receptor. Fumaric acid is the strong metabolite that interacts with the NF-κB receptor with a free energy of binding and an inhibition constant of -6.66 kcal/mol and 13.22 µM, respectively. Conclusions : Water kefir administration improved the condition of liver damage, characterized by a decrease in serum levels of AST, ALT, TNF- , TGF- , and an improvement in the liver tissue profile. In silico evaluation showed that the metabolites in water kefir were able to interact with target proteins in the NF- B and Nrf2 pathways. It was concluded that water kefir improves the condition of the liver by reducing the level of necrosis and fibrosis.


INTRODUCTION
In most cases, making water kefir involves combining dried fruit, sugar, and water kefir grains in a container.Water kefir's exact origins are unknown; however, two hypotheses have been proposed regarding its history: the first suggests that water kefir grains were brought to Europe from the Caucasus by soldiers returning from the Crimean War in the late nineteenth century (Ward, 1892); the second theory proposes that water kefir grains originated in Mexico from the Opuntia cactus through natural processes (Moinas et al., 1980).Sugary kefir grains, Balm of Gilead, African bees, Japanese beer seeds, Ale nuts, and California bees are some other names for water kefir.Tibi grains and ginger beer plants are other names for water kefir (Kebler, 1921;Moinas et al., 1980).Water kefir is appealing to both consumers and researchers due to the variety of microbiota it contains, the fact that it is an alternative to dairy products, the versatility with which it can be flavored, the fact that it is low in calories and sugar, the ease with which it can be produced, and the health benefits it offers.
Studies have shown that acute liver injury is frequently accompanied by high levels of oxidative stress and inflammatory responses (Dai et al., 2021).These findings have been found in several studies.
The most important signaling pathways that are involved in the regulation of inflammation and antioxidation are the nuclear factor (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways, respectively.It has been shown that activating Nrf2 and inhibiting NF-κB can reduce the amount of damage done to the liver.For instance, curcumin protects against aflatoxin B1-induced liver injury by increasing the expression of Nrf2 and related downstream antioxidant molecules (such as superoxide dismutase (SOD), catalase (CAT), heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO-1) (Wang et al., 2022b).Also, it has been shown that the molecule methoxy eugenol, which comes from nutmeg and Brazilian red propolis, protects the liver both in vitro and in vivo.This may be attributed to the fact that it targets the NF-κB signaling pathway, which has been shown to have anti-inflammatory effects (De Souza Basso et al., 2021).
Ethanol and lactic acid are the primary metabolites found in fermented water kefir, while lower quantities of glycerol, mannitol, and acetic acid can also be found in the beverage.Additionally, a variety of aromatic and volatile compounds are produced, including ethyl acetate, isoamyl acetate, ethyl octanoate, ethyl hexanoate, and ethyl decanoate, among others (compared to their threshold levels) (Laureys and De Vuyst 2014).The chemical constituents of both phytochemicals and secondary metabolites in natural products, including water kefir, are certainly capable of providing various pharmacological effects for the body (Asnawi et al., 2022a;Nursamsiar et al., 2022).However, an in silico study to evaluate the metabolite content in water kefir has not been reported yet.Because of its capacity to speed up the process of identifying and optimizing lead compounds, the in silico method has become the front-runner in the race to improve the speed and accuracy of the process of discovering new drugs.This is because the in silico method can identify and optimize lead compounds more quickly.Techniques such as molecular docking and molecular dynamics (MD) were able to directly indicate a small number of compounds that have high affinity and selectivity by analyzing how the ligand and target interact with one another (Febrina et al., 2021).
Water kefir has been used for an extensive period of time and has been recognized for its widespread benefits, especially in Indonesia.However, its level of popularity falls short in comparison to that of milk kefir.Furthermore, there is a scarcity of studies specifically focused on the hepatoprotective activity of water kefir made with Indonesian grains.Therefore, the purpose of this study was to evaluate the hepatopro-tective effects of water kefir in CCl4-induced rats while also investigating the stability interactions of its metabolites within the NF-κB and Nrf2 receptors using molecular docking studies.

Experimental sample and reference extract
The water kefir grain was obtained from Yogyakarta, Indonesia.The water kefir solution was produced using a fermentation procedure.The initial stage involved the preparation of 60 g of sugar, 2 g of raisins, 100 g of water kefir grains, and 1 L of mineral distillated water.The sugar and warm distilled water were mixed in a beaker, followed by the addition of water kefir grains and raisins to the resulting sugar solution.The fermentation procedure was conducted over a duration of forty-eight hours at ambient temperature.A small cloth was used to cover the beaker glass.The kefir grain was utilized in future production, while the filtrate was employed for the purpose of evaluation and analysis.(Aligita et al., 2020;2021) The rhizome extract of Curcuma (Curcuma zanthorrhiza Roxb, (family Zingiberaceae) was employed as a reference drug.The utilized product was a standardized herbal medicine with the brand name Tulak, manufactured by the Borobudur Company Herbal Medicine Industry.The primary purpose of Tulak capsules was to support and preserve optimal liver functionality.

Animals and experimental design
Rats (Wistar strain, male, 200-250 g) were maintained on normal pellet food and tap water ad libitum.Four mice in each group were used.All procedures relating to animals and their care conformed to the international guidelines Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985) with the ethical approval number 363/UN6.KEP/EC/2020.In order to develop an animal model with liver injury, the rats received CCl4 (20% in olive oil) at 1.25 mL/kg BW every 2 days via a gavage tube (Yeh et al. 2011).The rats were randomized into five groups after the development of ani-mals with liver injury, which is characterized by a significant increase in serum ALT level, as follows: (1) positive control group, (2) Curcuma extract group 100 mg/kg BW, (3) water kefir 15 mL/kg BW group, (4) water kefir 35 mL/kg BW group, and (5) water kefir 50 mL/kg BW group, with the addition (6) negative control group.Each group received group-specific treatment for two weeks, along with the administration of 1.25 mL/kg BW of CCl4 (20% in olive oil) every three days.
The rats, which had undergone a fasting period of 8-10 hours while being provided with water, had their blood samples collected via the retro-orbital sinus using a hematocrit capillary tube.The blood sample was taken into a microtube and afterwards then to centrifugation.The serum was separated in order to facilitate further measurements (Parasuraman et al., 2010).Serum ALT level, as the main parameter, was measured prior to induction, following induction, and following treatment.Meanwhile, following therapy, serum AST, TNF-α, TGF- levels, and liver histopathological were evaluated.The ALT, AST, TNF-α, TGF- levels measurements are conducted in accordance with the protocols outlined in the reagent kit.
After the euthanasia procedure, the liver specimen was promptly immersed in a 10% formalin solution at room temperature for a duration of 24 hours.Subsequently, the tissues were immersed in paraffin, subjected to sectioning, and affixed onto glass microscope slides.The slices underwent staining with hematoxylin and eosin and were afterwards analyzed using light microscopy (Konstantopoulos et al., 2017).

Molecular docking simulation
Molecular docking experiments were done with the PyRx software (Dallakyan and Olson, 2015) to predict how metabolites, which are small-molecule ligands, bind to biological macromolecules.The NCBI PubChem database (https://pubchem.nlm.nih.gov/,accessed on 3 May 2023) was used to derive the threedimensional structure of water kefir metabolites (Patel et al., 2022).Target proteins like NF-κB (PDB code: 1A3Q) and Keap1 (PDB code: 4L7B) were obtained from the RCSB Protein Data Bank (http://www.rcsb.org/,accessed on 03 May 2023).The PyRx program was used to reduce protein, ligand converted to PDBQT (Asnawi et al., 2023), then maximize GRID parameter (Asnawi et al. 2022b) and perform docking study (Febrina et al., 2022).The BIOVIA Discovery Studio 2017 R2 program was used to view the protein and ligand complex and distance (Ischak et al., 2023).The BIOVIA Discovery Studio 2017 R2 tool was also utilized to find protein active sites.

Statistical analysis
All of the information is displayed in the form of individual data points as well as the mean along with the standard error of the mean (SEM).The statistical analysis was carried out with the help of Minitab software (version 19.0), and to make comparisons between several different groups, a one-way analysis of variance with Dunnett's post hoc test was utilized.All statistical graphs were created with Microsoft Excel 2019 in their respective versions.The level of significance that were considered to have been reached was p<0.05.

In vivo evaluation of hepatoprotective activity
The serum ALT levels, as the main parameter for the liver damage, were measured prior to and following induction, as well as following treatment (Fig. 1).Meanwhile, after treatment AST, TNF-α, and TGF- levels were also evaluated.These findings were analyzed using a one-way ANOVA with a 95% confidence level and a two-sided confidence interval.A significant rise in blood ALT levels demonstrated the establishment of an animal model with liver injury, according to statistical analysis, following the administration of CCl4.When compared to the positive con-trol group, ALT serum levels decreased significantly after two weeks of therapy with curcuma extract or water kefir (p<0.05).The three doses of water kefir groups demonstrated equivalent activity when curcuma extract was used as the standard treatment, and there was no significant difference between the three doses of water kefir (p>0.05).When compared to the positive control group, AST levels were also reduced dramatically following treatment with curcuma extract or water kefir.TNF-α levels in the water kefir group were significantly lower than in the positive control group at dosages of 35 and 50 mL/kg body weight (p<0.05).Even though there was no statistically significant difference in TGF- levels, the group that received the treatment demonstrated a decrease in TGF- levels.
A histological examination of a normal liver group revealed a typical central vein bordered with endothelial cells and normal hepatocytes, resulting in hepatic cords of cells with distinct cell borders and sinusoidal gaps (Fig. 2A-B).The CCl4-induced group developed centrilobular hepatic necrosis level 4, which was characterized by massive vacuolization and necrosis of epithelial cells in the liver's centrilobular region (blue arrow) (Fig. 2C-D).The groups that received either Curcuma extract or water kefir treatment improved in varied necrotic conditions ranging  from level 1 (water kefir 50 mL/kg BW) (Fig. 2K-L) to level 2 [Curcuma extract (Fig. 2E-F), water kefir 15 mL/kg BW (Fig. 2G-H), and water kefir 30 mL/kg BW (Fig. 2I-J)].

Molecular docking
Molecular docking studies are considered a powerful tool for predicting the potential targets of bioac-tive molecules.In order to carry out molecular docking simulations, one of the most critical steps is to identify the target active site.If the target protein is crystallized with a native ligand, in many instances, the location of the active site can be established without any difficulty (Li et al., 2019b).However, the NF-κB (PDB ID 1A3Q) and nrf2 Keap1 (PDB ID 4L7B) proteins do not have a native ligand, so the active site  was determined.Active site prediction in docking is a computational method for predicting the location and orientation of a receptor protein's binding site for a ligand molecule.The active site prediction was based on a protein structural analysis and the identification of amino acid residues that are likely to interact with the ligand.The projected binding site is then utilized as a starting point for molecular docking, a computer method for predicting a ligand molecule's binding affinity and orientation to a receptor protein.The active site prediction for target proteins (Keap1 and NF-κB) gives the grid box coordinates (x y z) of 17.500880 Å, 62.323000 Å, and 0.973748 Å; and -1.751769 Å, -20.743853Å, and -29.010438Å, respectively (Fig. 3).
The docking results indicated that the 25 metabolites could interact with the target proteins (Keap1 and NF-κB) (Table 1).In general, all metabolites could interact with both NF-κB receptors (PDB ID 1A3Q) and nrf2 Keap1 (PDB ID 4L7B) (Table 1).The interaction of metabolites with nuclear factor-kappa B receptors (PDB ID 1A3Q) revealed that 13 metabolites possessed free energy for binding that was greater than that of curcumin.On the other hand, there was not a single metabolite that had a free energy binding that was more powerful than that of nrf2 Keap1 (PDB ID 4L7B) (Table 1).
For volatile compounds, 2-phenyl ethanol and benzaldehyde interacted most strongly with the NF-κB receptor (PDB ID 1A3Q) with almost the same binding energy of -4.19 kcal/mol.As for organic acids, succinic acid, fumaric acid, and citric acid provided nearly the same strong interactions.Bond energy values of fumaric acid, succinic acid, and citric acid were -6.66, -6.24, and -6.25 kcal/mol, respectively.In sugars, glucose provided the strongest interaction with the NF-κB receptor (PDB ID 1A3Q) where the binding energy was -3.71 kcal/mol.As for the nrf2 Keap1 receptor (PDB ID 4L7B), 2-phenylacetaldehyde from volatile compounds was able to interact most strongly with the Keap1 receptor (PDB ID 4L7B) with a binding energy of -3.46 kcal/mol.As for organic acids, succinic acid provided nearly the same strong interaction with a bond energy value of -3.02 kcal/mol, respectively.In sugars, glucose showed the strongest interaction with the Keap1 receptor (PDB ID 4L7B) where the binding energy was -3.09 kcal/mol.
The theoretical binding modes of the top three metabolites with their target proteins (Keap1 and NF-κB) are shown in Figs. 4 and 5, respectively.The molecular docking results suggested that these metabolites interacted with the Keap1 and NF-κB to form a complex through hydrogen bonds with various residues.The interaction of 2-phenyl ethanol with the active site of NF-κB was formed by two hydrogen bonds and one Pi-Alkyl interaction with amino acid residues of PRO223, LYS252, and LYS252, respectively.The interaction of benzaldehyde with the active site of NF-κB was formed by a hydrogen bond with the amino acid residue of LYS252.The interaction of fumaric acid with the active site of NF-κB was formed by six hydrogen bonds with the amino acid residues of LYS221, LYS252, and TYR285.The interaction of glucose with the active site of NF-κB was formed by six hydrogen bonds with the amino acid residues SER220, LYS221, SER226, and LYS252.Although glucose and fumaric acid were able to be formed by six hydrogen bonds, different types of amino acid residues were involved in the interaction, so fumaric acid interacted more strongly with the active site of NF-κB (Fig. 4).The interaction of 2-phenylacetaldehyde with the active site of Keap1 was formed by one hydrogen bond and one Pi-Alkyl interaction with amino acid residues of GLN530 and ALA556, respectively.The interaction of succinic acid with the active site of Keap1 was formed by four hydrogen bonds with the amino acid residues of ARG336, HIS575, and PHE577.The interaction of glucose with the active site of Keap1 was formed by six hydrogen bonds with the amino acid residues LYS551, ARG553, and ASP573.Although glucose can form more hydrogen bonds than 2-phenylacetaldehyde, the different types of amino acid residues involved have not been able to have a significant effect on the binding energy of its interaction with the active site of Keap1 (Fig. 5).
Curcumin (the reference compound) created three hydrogen bonds with the amino acid residues ARG52, https://jppres.comJ Pharm Pharmacogn Res ( 2023) 11( 6   GLU58, and LYS252 to interact with the active site of NF-κB.Meanwhile, curcumin established three hydrogen bonds with the amino acid residues ARG336, TYR572, and GLY600 to interact with the active site of nrf2 Keap1 (Figs. 4 and 5).Despite the fact that curcumin could create three hydrogen bonds at both the active sites of NF-κB and nrf2 Keap1, its interaction with nrf2 Keap1 had a lower free energy of binding than phenylethanol, benzaldehyde, and fumaric acid (Table 1).Overall, the metabolites of water kefir prefer to interact with NF-κB and nrf2 Keap1 receptors.

DISCUSSION
Increased liver enzyme production is one of the abnormalities indicating liver damage.This increase in secretion increases the leakage of enzymes into the circulation.Transaminase is a frequent enzyme used to evaluate liver disorders.The transaminases AST and ALT catalyze the transfer of the -amino group from alanine and aspartic acid to -ketoglutaric acid.AST is found in the liver, heart, and skeletal muscle, as well as the kidney, brain, pancreas, lung, leukocytes, and erythrocytes.While ALT is predominantly found in the liver, it is also found in low concentrations in other tissues (Lee et al., 2012).Consequently, ALT was used as the principal hepatotoxicity criterion in this study.
Carbon tetrachloride (CCl4) is a component of the hepatotoxin, which acts after metabolic activation and is extensively employed as a liver-damaging agent.In this study, the administration of CCl4 to animals caused hepatotoxicity, which was characterized by a significant increase in ALT after several administrations.CCl4 is metabolized by the enzyme cytochrome p450 (CYP2E1) in the endoplasmic reticulum to a highly reactive trichloromethyl radical.The trichloromethyl radical rapidly reacts with oxygen to form the trichloromethyl peroxyl radical, which rapidly reacts with lipids to form lipid peroxidation products.Free radicals will disrupt the endoplasmic reticulum (ER) and lead to lipid accumulation, decreased protein synthesis, and increased oxidase activity.CCl4 hepatotoxicity is characterized by hepatocellular necrosis with fat deposition (Ritesh et al., 2015).At the molecular level, administration of CCl4 can activate tumor necrosis factor (TNF)-α, nitric oxide (NO), and transforming growth factor (TGF)- and - in cells, processes that precipitate cell selfdestruction or fibrosis.TNF-α leads to apoptosis, whereas TGF- leads to fibrosis (Weber et al., 2003).
In terms of its pathophysiological underpinnings, liver illness is linked to a condition known as dysbiosis, which refers to an imbalance in the makeup of the gut microbiota (Laureys and De Vuyst, 2017;Romero-Luna et al., 2020;Zavala et al., 2016).Both qualitative and quantitative changes in the gut microbiome have the potential to affect the composition of products produced by the microbiota, such as short-chain fatty acids and bile acids (Romero-Luna et al., 2020).Qualitative changes include an imbalance between harmful and helpful microbiomes, whereas numeric changes involve changes to the overall microbiota.In addition to these symptoms, intestinal inflammation, disruption of the intestinal barrier, and the transfer of microbial products can all be caused by dysbiosis (Laureys et al., 2018).For this reason, the condition of the gut microbiome is an important factor in the initiation and development of chronic liver disease (Lee et al., 2021).Based on the results of the study, treatment with water kefir for two weeks after the occurrence of liver damage was able to improve the overall condition of the liver, which was marked by a significant decrease in the values of AST, ALT, TNF-α, TGF-β, and significant improvement in liver histology.
Water kefir contains a number of microorganisms that have been linked to health benefits, such as the probiotics L. paracasei and B. cereus (Fijan, 2014).This activity is linked to an increase in antioxidants like glutathione and catalase and a decrease in proinflammatory transcription factors like nuclear Bfactor, lipopolysaccharide, and Toll-like receptor 4 (TLR-4).Improvements in intestinal barrier function and histological integrity were also observed.Increased expression of claudin-1 and occludin-1, two components of tight junctions, occurs simultaneously with the restoration of the p38 MAPK pathway (Fijan, 2014;Yao et al., 2019;Tsai et al., 2020;Ji et al., 2022).Bacillus is a kind of endospore-forming bacterium that can endure extremely cold temperatures and lengthy periods of storage without dying; its spores can even tolerate the acidic environment of the stomach and make it all the way to the small intestine (Elshaghabee et al., 2017).Bacillus cereus has been shown to reduce ALT levels, an indicator of liver healing, in various animal models of liver injury.It protects the liver by reducing inflammation, enhancing the gut flora, and strengthening the tight junctions in the intestines (Kim et al., 2018;Li et al., 2019a;Xue et al., 2020).Also, when Bacillus spores were used first, hepatocyte necrosis and serum levels of ALT, ZO-1, AST, and TAC went down by a lot.This effect is comparable to that of the popular hepatoprotective compound silymarin (Neag et al., 2020).
Beer, vinegar, cider, and cocoa bean masses are just some of the places where Gram-negative acetic acid bacteria have been found.One of the features that sets acetic acid bacteria apart from others is their alkaline-stable lipid membrane (Lynch et al., 2021).Their "oxidative" fermentation metabolism is responsible for the principal metabolic process in these bacteria, the oxidation of ethanol to acetic acid.Important in the food and beverage sector and beyond, fermentation helps mediate the transition of diverse substrates into products.Although lactic acid bacteria have been studied more extensively than acetic acid bacteria (Hong et al., 2021;Semjonovs et al., 2014), various studies have shown promising results concerning the pharmacological effects of acetic acid bacteria, especially as a hepatoprotective agent.Acetic acid, which is the main metabolite of acetic acid bacteria, can lower inflammation and the severity of liver injury in rats with septic shock by increasing the expression of TRIM40.TRIM40 has been shown to minimize liver damage, decrease the synthesis and release of cytokines such as IL-6 and TNF-α, raise the expression of IL-10, improve survival in septic mice, and block the activity of the TLR4 signaling pathway.Acetic acid injection decreased inflammation as well as the production of inflammatory cytokines (Yang et al., 2019).Acetic acid also stimulates the AMPK signaling pathway, which leads to enhanced lipid oxidation and reduced hepatic lipid and body fat deposition (Kondo et al., 2009;Li et al., 2018).
Apart from microorganisms that directly provide hepatoprotective effects, the metabolites produced from these microorganisms also have the potential to be hepatoprotective.Molecular docking is a technique that is utilized in the context of NF-κB and Nrf2 to make predictions regarding the affinity and orientation of small-molecule inhibitors to their active sites.The transcription factor known as NF-κB is an essential component in the management of both the immune system and the inflammatory response (Dai et al., 2021).The expression of important inflammatory genes can be inhibited by small-molecule inhibitors that impair the interaction between NF-κB and DNA.These inhibitors have the potential to be used in therapeutic applications.The Nrf2 binding site is Kelchlike ECH-associated protein 1 in the context of nuclear factor erythroid 2-related factor 2, also known as Nrf2 (Keap1) (Jiang et al., 2019;Zhao et al., 2017).Small-molecule inhibitors that disrupt the link between Keap1 and Nrf2 can activate the Nrf2-ARE signaling pathway, which has been demonstrated to have cytoprotective effects.Keap1 is a negative regulator of Nrf2, and this association can be disrupted by small-molecule inhibitors (Zhao et al., 2017).
Binding energy and Ki are important parameters used in molecular docking to evaluate the strength of the interaction between a ligand and a receptor pro-tein.Binding energy is the energy released when a ligand binds to a receptor protein, and it is calculated as the difference between the energy of the bound complex and the energy of the unbound ligand and protein (Meng et al., 2011).Ki, on the other hand, is the dissociation constant of the ligand-receptor complex.It shows how much ligand is needed to bind 50% of the receptor binding sites.Both binding energy and Ki are used to predict the binding affinity and selectivity of a ligand to a receptor protein (Du et al., 2016).Ki, on the other hand, is the dissociation constant of the ligand-receptor complex.It shows how much ligand is needed to bind 50% of the receptor binding sites (Du et al., 2016).
Based on the results of an in silico study of water kefir metabolite compounds, it was known that fumaric acid and 2-phenylacetaldehyde have the strongest interactions with NF-κB (PDB ID 1A3Q) and nrf2 Keap1 (PDB ID 4L7B) receptors, respectively.Fumaric acid has been studied for its potential as a hepatoprotective agent.Fumaric acid protects rat livers against cadmium toxicity by decreasing oxidative stress and improving hepatic serum.Fumaric acid prevented cadmium-accrued hepatocellular damage and showed the potential to avert hepatic injury against cadmium in rats ( Kaur et al., 2020).Fumaric acid esters were found to ameliorate inflammation and oxidative stress in rats, which was associated with less adiposity and ectopic fat accumulation (Šilhavý et al., 2014).NF-B and Nrf2 are two transcription factors that play important roles in regulating inflammation and cell survival.While NF-B is involved in the inflammatory response, and Nrf2 is involved in the antioxidant response (Ganesh Yerra et al., 2013;Wang et al., 2022a).Both transcription factors have been investigated as potential targets for the development of hepatoprotective agents (Gao et al., 2022;Li et al., 2023;Rahman et al., 2021;Wang et al., 2022a).There is evidence of crosstalk between the Nrf2 and NF-kB pathways (Ganesh Yerra et al., 2013;Gao et al., 2022).The Nrf2 pathway inhibits the activation of the NF-B pathway by increasing antioxidant defenses and HO-1 expression, which efficiently neutralizes ROS and detoxifies (Ganesh Yerra et al., 2013).The crosstalk between Nrf2 and NF-B could be a new therapeutic target against hepatotoxicity (Gao et al., 2022).Researchers have tried to identify molecule activators of Nrf2 as chemoprevention for ROS-dependent carcinogenesis, while others have focused on identifying Nrf2 inhibitors to increase the sensitivity of cancer cells to chemotherapy (Sharifi-Rad et al., 2023).While NF-B and Nrf2 are involved in different cellular processes, they have both been investigated as potential targets for the development of hepatoprotective agents.Molecular docking studies have been used to investigate the interaction of potential hepatoprotective agents with these transcription factors.There is also evidence of crosstalk between the Nrf2 and NF-B pathways, which could be a new therapeutic target against hepatotoxicity.
In the pathogenesis of hepatocellular damage, liver fibrosis, and HCC, NF-κB plays a crucial role in regulating inflammation and cell death (Luedde and Schwabe, 2011).In response to many stimuli that may pose a threat to the host, NF-κB is activated, setting in motion processes such as inflammation, immunity, wound healing, and pathogen clearance (Luedde and Schwabe, 2011).Pathogen-derived chemicals that activate Toll-Like Receptors (TLRs) include lipopolysaccharide (LPS), viral and bacterial DNA and RNA, and inflammatory cytokines, including tumor necrosis factor (TNF) and interleukins (IL)-1 (Luedde and Schwabe, 2011).When NF-B is activated, a lot of genes with B-binding sites are transcribed.These genes play important roles in controlling inflammation, the immune response, and cell survival.In an NF-κB-dependent manner, lipopolysaccharide (LPS) activates TLR4 on dormant HSCs by reducing BAMBI expression (an inhibitory TGF-β pseudoreceptor) and increasing Kupffer cell chemotaxis.Due to low levels of BAMBI, recruited Kupffer cells secrete TGF-β, which stimulates unrestrictively.When HSCs have been activated, NF-κB serves a second crucial function by increasing their chances of survival.LPS, Kupffer cell-derived mediators (such as IL-1 and TNF), and angiotensin II, which is produced and acts on HSCs in an autocrine way, all play roles in activating NF-κB in activated hepatic stellate cells.More activated HSCs and extracellular matrix are deposited in the liver as a result of greater HSC activation and survival (Luedde and Schwabe, 2011).
TNF-α and TGF-β can stimulate one another's production, and TNF-α has been shown to influence TGF-β expression in a variety of cells and tissues (Liu et al., 2022).TNF-α is an inflammatory cytokine that contributes to liver inflammation, and chronic liver inflammation results in liver fibrosis.TNF-α exerts its effects on liver fibrosis via multiple mechanisms, including TGF-β signaling activation (Yang and Seki, 2015).Targeting TNF-α and TGF-β signaling pathways may, therefore, have therapeutic potential for treating liver diseases.In regard to hepatoprotective effects, the relationship between TGF-β and TNF-α is complex and not completely understood.

CONCLUSION
This study evaluated the hepatoprotective qualities of Indonesian water kefir in rats with CCl4induced liver damage.Water kefir administration improved the condition of liver damage, characterized by a decrease in serum levels of AST, ALT, TNF, TGF, and an improvement of liver tissue profile.In silico evaluation showed that the metabolites in water kefir were able to interact with target proteins in the NF-B and Nrf2 pathways.It was concluded that water kefir improves the condition of the liver by reducing the level of necrosis and fibrosis.
Fig. 1. (A) Before induction, after induction, and after treatment serum ALT level; (B) After treatment serum AST level; (C) After treatment serum TNF- level; (D) After treatment serum TGF- level.Data represent mean ± SEM (n = 4).*p<0.05significantly different result when compared to the positive control group.

Fig. 3 .
Fig. 3. Binding pocket (colored in red) generated using the BIOVIA Discovery Studio 2017 tool, coupled with the sequence containing the highlighted residues that constitute the binding pocket.

Fig. 4 .
Fig. 4. 2D and 3D representation of the kind of interaction on the formation of the NF-B active site (PDB ID 1A3Q).

(Fig. 5 .
Fig. 5. 2D and 3D illustration of the type of interaction on the formation of Keap1's binding pocket (PDB ID 4L7B).

Table 1 .
The free energy of binding and inhibition constant of water kefir metabolite active site interaction on NF-B (PDB ID 1A3Q) and Keap1 (PDB ID 4L7B).