Sesame oil ameliorates valproic acid-induced hepatotoxicity in mice: integrated in vivo–in silico study

Abstract Sesame oil (SO) has been exhibited to have anti-inflammatory and antioxidant influences. The goal of this experiment was to look into SO’s hepato-protective properties and underlying processes in valproic acid (VPA)-induced hepatotoxicity. Molecular docking was carried out to clarify the functional and structural underlying mechanism of SO ameliorative effect. Mice were given 8 mL/kg/day of SO (orally) and 100 mg/kg/day of VPA (i.p.) for 21 days. The results revealed that VPA caused a considerable increase in hepatic malondialdehyde levels while decreasing the activity of glutathione peroxidase (GPx) enzyme. There was also a significant rise in serum levels of interleukins 1β and 6 (IL-1β and IL-6) and a significant decrease in hepatic (PXR) gene expression level. SO co-administration with VPA significantly normalized the antioxidant and anti-inflammatory status and upregulated the gene expression level of PXR. In silico docking analysis results confirmed these results. This study concluded that supplementation of SO attenuated VPA-induced oxidative stress and inflammation. Hence, it was recommended as a dietary supplement for protection against VPA-induced hepatotoxicity. Communicated by Ramaswamy H. Sarma


Introduction
Valproic acid (VPA) is commonly prescribed for treatment of seizure disorders, bipolar disorder and migraine prophylaxis. Despite its medical efficacy, hepatotoxicity has been reported as a serious complication after VPA administration. Pediatric patients are more susceptible to VPA-induced hepatotoxicity than adults (Nanau & Neuman, 2013).
VPA is broadly metabolized in the liver. Cederbaum (2017) stated that the biotransformation of VPA produced several metabolites such as hydroxylated, unsaturated and conjugated metabolites that may be responsible for its toxicity. Pregnane xenobiotic receptor (PXR) has been well-established as a main regulator of xenobiotic clearance. It is chiefly expressed in liver and small intestine. PXR expression in other tissues such as stomach, lung, breast, uterus, ovary and bone marrow is minor. Activated PXR upregulates genes encoding enzymes that metabolize drugs and drug transporters (Kodama et al., 2017).
Liver injury may be directly affected by the drug or its metabolites, or injury may result from the subsequent inflammatory reaction (David & Hamilton 2010). Many research focused on the pathophysiology of drug-induced toxicity mediated by reactive metabolites. Oxidative stress may perform a role in the etiology of drug-produced hepatotoxicity via oxidative alteration of biological macromolecules such as nucleic acids, lipids and proteins (Abdelkader et al., 2020). Several factors are contributing to hepatotoxicity caused by VPA, including development of oxidative stress and VPA metabolites generate reactive metabolites, which then covalently bond to cellular proteins. During druginduced hepatotoxicity, inflammation of the liver is common (Ramadori & Armbrust, 2001). Inflammatory cytokines were found to be higher in rats with liver damage. The production of inflammatory mediators by activated hepatic macrophages has been shown to aggravate drug-induced hepatic damage (Jin et al., 2014).
For balancing the extreme production of free radicals, mechanisms of enhancing redox homeostasis should be maintained. Many natural compounds have been tested as antioxidants as part of these mechanisms (Abdella et al., 2014). Sesame oil (SO), derived from Sesamum indicum L., is effective against numerous diseases and it possesses antiaging effects. The lignans contained in SO, in addition to sesaminol which is the primary antioxidant component, contribute to its antioxidant and antimutagenic capabilities. SO has a high concentration of phenol, sesamin, sesamol and sesamolin, which contributes to its better oxidative stability (Ahmad et al., 2006). As its antioxidant activity, SO has long been used as a daily dietary supplement to boost cell resistance to lipid peroxidation. Moreover, previous studies suggested that SO enhances the ability of liver to detoxify chemicals and xenobiotics (Hsu et al., 2008). Nevertheless, there is no previous experimental proof reporting the ameliorative influence of SO against valprioc acid-induced hepatotoxicity.
Thus, the purpose of this research is to look at the hepato-protective impact of SO as an antioxidant and antiinflammatory agent on VPA-induced hepatic cell damage in mice. In addition, a docking study was carried out to gain functional and structural insight into the binding mode of the VPA and some SO constituents (sesamin, sesaminol, sesamol and sesamolin), and glutathione peroxidase (GPx), interleukin (IL)-1b, IL-6 and PXR.

Chemicals
El-Borg pharmacy in Beni-Suef, Egypt, sold DepakineV R (sodium valproate, Sanofi Synthelabo, France), each containing 200 mg of sodium valproate. Harraz for food industry and natural products Co., Cairo, Egypt, provided the SO. Malondialdehyde (MDA) and GPx commercial kits were purchased from Biodiagnostic company, Cairo, Egypt. ElabscienceV R in the United States provided ELISA kits for determining IL-1b and IL-6 serum levels.

Treatments for animals
Thirty 10-week-old male mice were obtained from a laboratory animal farm in Egypt's Beni-Suef governorate. Mice were given a balanced commercial meal and free access to water, and were housed at a chamber temperature of 25 � C, 45% moisture. All experimental procedures were in accordance with the guidelines of local Animal Care and Use Committee established at the Beni-Suef University (BSU-IACUC). The study was performed after obtaining an approval number (022-280) to conduct the animal experiments.
After a week of acclimatization, the mice were haphazardly assigned into three groups of ten mice each.
Control group (C group): Mice served as control and were intraperitoneally injected with equal volumes of 0.9% saline.
Valproic acid group (VPA group): VPA at dosage of 100 mg/ kg body weight (dissolved in distilled water) was selected according to Abdella et al. (2014). Mice were intraperitoneally injected with VPA (100 mg/kg/day) for 21 days.
Sesame oil group (SO group): Mice were given 8 mL/kg of SO orally every day (Hsu et al., 2008) concurrent with i.p. injection of VPA all over the period.

Sampling and biochemical analysis
Blood samples were taken through retro-orbital hemorrhage 24 h after the last dosage. Blood samples were left for 30 mins to clot. For serum separation, clotted blood samples were centrifuged at 3000 rpm for 15 min. The sera were maintained at À 20 � C till they were used.

Tissue sample collection
Cervical dislocation was used to sacrifice the mice. The liver tissue was removed and cleaned in physiological saline after that. Two sections of the liver samples were taken. For homogenization, the first part from liver (0.5 g) was put in 5 mL of phosphate buffered saline (Teflon Homogenizer, India). Using a high-speed cooling centrifuge, the tissue homogenate was centrifuged at 3000 rpm for 10 min at 4 � C. The supernatants were kept at -20 � C until the oxidative/antioxidant indices were determined. The other part was preserved at -80 � C to determine the level of PXR gene expression.

Estimation of biochemical, inflammatory and molecular parameters 2.3.2.1. Parameters of oxidative and antioxidant status.
The MDA level and GPx activity in liver tissue homogenate were used to assess the oxidant-antioxidant status of the tissue. The concentration of MDA was determined using the technique of Wills (1987) in which thiobarbituric acid (TBA) reacts with the malondialdehyde in an acidic medium at a temperature of 95 � C for 30 min to form a thiobarbituric acid reactive product. The absorbance of the resultant pink product can be measured at 534 nm. GPx activity measurement was based on the study by Paglia and Valentine (1967). In the glutathione peroxidase assay procedure, glutathione   Values as mean ± SE of mean. The significant difference at (p < 0.05) is represented as different superscript letters between the studied groups. peroxidase oxidizes GSH to produce GSSG (glutathione disulfide) as a part of the reaction in which it reduces cumene hydroperoxide. Glutathione reductase then reduces the GSSG to produce GSH and in the same reaction, consumes NADPH. The decrease of NADPH (measured at OD ¼ 340 nm) is proportional to GPx activity.

Determination of serum levels of IL-1b and IL-6.
IL-1b and IL-6 serum levels were determined using ELISA kits in line with the manufacturer's commands. The micro ELISA plate has been precoated with an antibody specific to mouse IL-1b or IL-6. Standards or samples were added to the micro ELISA plate wells and combined with the specific antibody.
Then a biotinylated detection antibody specific for mouse IL-1b or IL-6 and an Avidin-Horseradish Peroxidase (HRP) conjugate were added successively to each microplate well and incubated. Free components were washed away. The substrate solution was added to each well. Only those wells that contained mouse IL-1b or IL-6 biotinylated detection antibody and Avidin-HRP conjugate appeared blue in color. The enzyme-substrate reaction was terminated by the addition of stop solution and the color turned yellow. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 ± 2 nm. The OD value was proportional to the concentration of the cytokine. The concentration of mouse IL-1b or IL-6 in the samples was calculated by comparing the OD of the samples to the standard curve.

Real time-polymerase chain reaction for detection of PXR gene expression.
Total RNA was extracted from liver according to manufacturer's instructions using Ribozol TM RNA Extraction Reagents with the code N580. A UV spectrophotometer 'Hitachi spectrophotometer, Model U-2000, Tokyo, Japan' was used to quantify the concentration of RNA. cDNA synthesis: Five micrograms of RNA were reverse transcribed with oligonucleotide (dT)18 primer and denatured for 2 min at 70 � C. On ice, denatured RNA was added to a reverse transcription mixture. The reaction tube was kept at 42 � C for 1 h before being heated to 92 � C to end the reaction.
For quantitative real time-polymerase chain reaction (RT-PCR), first-strand cDNA (5 lL) were used in a whole volume of 25 lL, which included 12.5 lL 2� SYBR Green PCR Master Mix and 200 ng of each primer as given in Table 1. On the step one plus RT-PCR system, PCR reactions comprising 95 � C for 10 min (1 cycle), 94 � C for 15 s and 60 � C for 1 min (40 cycles) were performed (Applied Biosystems). The ABI Prism 7500 sequence detection system software was used to assess the data, and PE Biosystems' v1.7 Sequence Detection Software was used to quantify it (Foster City, CA). The comparative threshold cycle approach was used to calculate the relative expression of the genes examined. All of these data were normalized to the beta-actin genes, all of these

In silico molecular docking
Molecular docking was performed for Gpx, IL-1b, IL6 and PXR domains against sodium valproate, sesamin, sesaminol, sesamol and sesamolin as main constituents of SO. Initially, the biological data were collected from both NCBI (ID: EC 1.11.1.9, EC 16176, 16193, 10090) and UniProtKB (ID: P11352, Q3TI17, A2RTD1, O54915) databases for Gpx, IL-1b, IL6 and PXR respectively. Modeling of the 3D structure for each was performed by I-TASSER server. After that, PubChem database was used for obtaining each compound structure. Modification for all compounds structures were done by Swiss PDB Viewer (spdbv) software for energy minimization process. Then, Open Babel (Version 2.3.1) software was used to convert the format from pdb to pdbqt. Before the molecular docking process, each protein and ligand were altered by the addition of hydrogen atoms, and metals were handled with the Discovery Studio software (version 2019). Eventually, Auto Dock Vina (Version 2.0) was used to define the grid box with 1.00 Å spacing and a grid map of 42 54 42 XYZ Å points for Gpx domain, 46 46 48 XYZ Å points for IL-

Statistical analysis
All results were statistically evaluated using one-way analysis of variance (ANOVA) using SPSS software (Chicago, USA) then compared by using the Tukey post hoc test. The data were exhibited as a mean with standard error (SE). At p < 0.05, the differences were judged statistically significant.

Effect of VPA and SO on the antioxidant status
The experimental results revealed that the VPA induced changes in oxidant/antioxidant indices as it significantly increased hepatic MDA level and decreased GPx activity when compared to the control group. Administration of SO modulated these changes as it decreased the hepatic MDA level and increased Gpx activity when compared to the VPA group, as shown in Figure 1.

Effect of VPA and SO on serum proinflammatory mediators
The serum levels of IL-1b and IL-6 were elevated in VPA group as compared to control group. Meanwhile, the co- treatments with SO succeeded to induce a marked improvement in these parameters when compared to VPA group as a result of SO's anti-inflammatory activity (Figure 2).

Docking analysis results
From the docking analysis results, the binding energy ranged from -3.9 to -7.1 among Gpx docked complexes with the five ligands, -4.4 to -7.9 among IL-1b docked complexes, -5.1 to -8.6 among IL6 docked complexes and -5.5 to -8.4 among PXR docked complexes (Table 2). Figures 4-7 illustrate the 2D chemical interactions for each docked protein-ligand complex. Many interactions were identified, such as conventional hydrogen bonds, carbon hydrogen bond, alkyl, Van der Waals, pi-alkyl, pi-sigma, pi-anion and pi-sulfur. The most vigorous interactions appeared through Gpx docked complexes with sodium valproate, sesamol and sesamolin. Also, the best chemical interactions appeared through IL-1b docked complexes with sesamol. On the other hand, through IL-6 docked complexes the best appeared with sodium valproate and sesaminol, sesamol and sesamolin. And finally, through PXR docked complexes the best appeared with sesaminol and sesamolin. Hydrophobicity areas are shown in Figures 8-11 in all docked complexes with the brown color. Gpx-sesaminol, IL1b-sesamin, IL6-sesaminol and PXR-sesamol docked complexes were the most in the hydrophobicity. Also, H-bond areas were shown in Figures 12-15. Gpx-sesamolin, IL1b-sesamol, IL6-sesamin and PXR-sesamin were the most in H-bonds. The solvent accessible surface (SAS) areas are shown in Figures 16-19. Gpx-sesamin, IL1b-sodium valproate, IL6-sesaminol and PXR-sesamin were the most in SAS.

Discussion
VPA is one of the histone deacetylase inhibitors used for management of epilepsy, bipolar disorder as well as migraine. Although the common use of VPA, its clinical use has been linked to hepatotoxicity that has been widely established (Nanau & Neuman, 2013). One of the most essential features of VPA therapy is the development of innovative techniques to avoid or reduce hepatotoxicity. Subsequently in the current study, the preventive influence of SO versus VPA-provoked hepatotoxicity was examined. It was reported that oxidative stress causes excessive formation of free radicals and exhaustion of antioxidants that are linked to liver dysfunction caused by VPA (Kiang et al., 2011). Our results revealed that VPA administration increased considerably hepatic MDA level and decreased Gpx enzyme activity as compared to control group. These results confirm previous work which stated that VPA induced liver lipid peroxidation and decreased hepatic antioxidant enzymes activities (Jin et al., 2014). The critical role of free radicals in cell injury is well understood, and it has been postulated that the covalent attachment of free radicals to macromolecules, in addition to reactive intermediates, may play a role in the severe adverse drug reactions. Several investigations have suggested that hepatotoxic medications produce reactive metabolites and free radicals (Park et al., 2005).
Pre-treatment with SO improved the VPA-induced variations in oxidative stress indices where it significantly reduced hepatic MDA level and increased Gpx activity indicating the hepato-protective effect of SO. These findings were consistent with Hsu et al. (2008) who explained that SO potently inhibited lipid peroxidation by preventing the production of hydroxyl radicals, peroxynitrite and nitric oxide. It is strongly believed that the antioxidant properties of SO is owing to the existence of vitamin E and lignans that are non-fat constituents, such as sesamin, sesamol, sesaminol and sesamolin. Lignans as antioxidant have been observed in numerous studies and vitamin E has also been shown to boost glutathione tissue content (Sharma et al., 2000). In addition, natural products provide protection against free radicals-mediated damage because of their antioxidant properties, which eliminate free radicals from the biological environment (Upadhyay et al., 2010).
The inflammatory reactions associated with VPA administration was clearly obvious during our study via the elevating serum level of IL-1b and IL-6 in mice of VPA group in line with Jin et al. (2014) who reported that VPA induced a rise in nuclear factor-kappa B (NF-jB) level as well as stimulation of IL-1b and IL-6 expression. It appears that oxidative stress associated with VPA triggers transcription factors, as NF-jB, leading to oxidative damage and inflammation (Hamza et al., 2016). SO showed a protective effect against these inflammatory reactions via lowering the serum levels of both IL-1b and IL-6 in accordance with Hsu et al. (2013) who found that SO diminished the levels of some inflammatory mediators (tumor necrosis factor-a, IL-1b and IL-6) via suppression of inducible nitric oxide synthase (iNOS) production and inhibition of neutrophil infiltration. Sesaminol, one of SO active constituent, decreased the inflammatory cytokine expression in mice's brains (Katayama et al., 2016). Previously, it was reported that SO inhibited NF-jB that had an anti-inflammatory impact in cultured rat astrocytes. SO inhibited translocation of NF-jB from cytoplasm in to the nucleus thus, it is clear that SO has components that inhibit inflammation through blocking NF-jB activation (Selvarajan al., 2015). These data suggested that suppression of inflammatory cytokine production could be part of the mechanism behind SO's hepato-protective impact against VPA-induced liver damage.
PXR is a well-known regulator of drug and xenobiotic metabolism. It is engaged in medication-drug interactions, which impact drug metabolism and excretion, lowering efficacy or raising toxicity (Hogle et al., 2018). In our study, VPA significantly decreased the expression level of PXR gene in liver tissue as a result of inflammatory reactions associated with its administration in line with Pascussi et al. (2000) who reported that the levels of hepatic PXR mRNA have been downregulated in response to inflammatory signals. He found that PXR mRNA is downregulated by the proinflammatory cytokine IL-6 in primary human hepatocytes. PXR regulates the expression of several drug metabolizing enzymes. It defends the body against dangerous outside toxins as well as endogenous toxins (Kodama et al., 2017). The daily administration of SO significantly increased the gene expression level of PXR in agreement with accumulating data which revealed that PXR is a biological target of a variety of herbal extracts or active substances found in herbal medicine (Lazar, 2004). Natural products have been discovered to interact with PXR in some way, causing enzymes and transporters to upregulate to speed up their own metabolism and, as a result, the metabolism of concomitant medications that are substrates for the same enzymes and transporters. PXR is participating in the adjustment of immunological and inflammatory reactions (Kodama et al., 2017). Therefore, PXR activators can be used for the remedy of liver inflammatory disorders. The benefits of SO and its components, such as sesaminol, can modify the amount of gene transcription in diverse targets, such as hepatocytes, endothelial cells and macrophages, according to the majority of the research discussed (Sankar et al., 2006).
We suggest that the protective effect of SO might be a result of its anti-inflammatory activity in harmony with Pavek (2016) who reported that proinflammatory stimuli decrease the liver drug-metabolizing activities by inhibiting PXR activation. Sesame lignans or antioxidants such as sesamin, episesamin, sesaminol and sesamolin are responsible for many of SO's distinctive chemical and physiological qualities. Sesamin improves liver detoxification, lowers the risk of chemically generated cancers, protects neuronal cells from oxidative stress and has anti-inflammatory and anti-allergic properties. In addition, the interaction of natural compounds with distinct cytochrome isoforms contributes to their protective potential (Yarnell & Abascal, 2007). After the in vivo experiment's procedure and analysis, it was essential to predict the changes during the inhibition process in different measured parameters. This prediction was carried out by the in silico docking (Ghalla et al., 2022). Beginning with the binding energy, in the Gpx docked complexes, sesamin, and sesaminol showed the best results of -7.1, in the IL-1b docked complexes, sesaminol showed the best results of -7.9, in the IL-6 docked complexes, sesamin, and sesaminol showed the best results of -8.6; in the PXR, sesamin showed the best results of -8.4. The sesamin component generally offered the best results among all the docked complexes against most targeted proteins. Second, the 2D chemical interaction analysis, which indicates the strongest of the binding interactions among each complex, showed many powerful interactions. In Gpx docked complexes with: sodium valproate, there were two conventional hydrogen bonds in the LEU A: 22 and THR A: 23 residues, with sesamin there was one conventional hydrogen bond in the ARG A: 34, with sesaminol, there was one conventional hydrogen link in the ASP A:169 and one carbon-hydrogen link in the GLY A: 31, with sesamol there were three conventional hydrogen bonds in the LEU A: 22, THR A: 23 and PRO A: 104 residues and there was one pi-donor hydrogen bond in the LEU A: 108 residue, with sesamolin there were three conventional hydrogen bonds in the MET A: 1, CYS A: 2 and ARG A: 174 residues and there was one carbon hydrogen bond in the ASN A: 199 residue. Also, in IL-1b docked complexes with sodium valproate, there was one carbon hydrogen bond in the SER A: 65, with, with sesamol there were two conventional hydrogen bonds in the ALA A: 2 and ASN A: 8 residues, and with sesamolin, there was one conventional hydrogen bond in the ASN A: 8 residue. In IL-6 docked complexes with: sodium valproate, there was one conventional hydrogen bond in the GLN A: 94, with sesaminol, there was one carbon hydrogen bond in the SER A: 115 residue, with sesamol, there was one carbon hydrogen bond in the MET A: 127 residue, with sesamolin there was one carbon hydrogen bond in the SER A: 115 residue. And finally, in the 2D chemical interactions of PXR complexes with: sodium valproate there were two conventional hydrogen links in the GLN A: 92 and ASN A: 69 residues, with sesamin, there were two conventional hydrogen links in the LEU A: 332 and TYR A: 337 and there was one carbon hydrogen bonds in the LEU A: 327 residue, with sesaminol there were two conventional hydrogen bonds in the LEU A: 332 and TYR A: 337 and there was one carbon hydrogen bonds in the LEU A: 327 residue, with sesamolin there was one conventional hydrogen bonds in the LEU A: 332 and there were four carbon hydrogen bonds in the GLN A: 331, THR A: 287, TYR A: 337 and LEU A: 190 residues. In the drug design strategies of inhibitors, hydrophobicity is one of the most critical parameters of the success of the inhibition procedure. Especially examining how inhibitors interact with the hydrophobic and hydrophilic regions of the enzyme active site (Ashraf et al., 2021). During the analysis of the most hydrophobic areas: Gpx-sesaminol, IL1b-sesamin, IL6-sesaminol and PXR-sesamol docked complexes were the most in the brown color (hydrophobicity). On the other hand, during the changes in the binding site interactions, water may hinder the formation of solute intermolecular interactions by forming hydrogen bonds with proton donors and acceptors in the solute. So, H-bond as acceptor and donors is an essential parameter in the inhibition process. Among the areas of H-bonds as donors (pink color) and acceptors (green color) in the all docked complexes: Gpx-sesamolin, IL1b-sesamol, IL6-sesamin and PXR-sesamin were the most. SAS is defined as the location of the solvent molecule's center as it rolls over the protein's van der Waals surface. The SAS interactions areas analysis (blue color) among all docked complexes showed that Gpx-sesamin, IL1b-sodium valproate, IL6-sesaminol and PXR-sesamin were the most.

Conclusion
In conclusion, in vivo and in silico findings revealed that SO has the potential capacity to alleviate VPA-induced hepatotoxicity due to the antioxidant and the anti-inflammatory properties that are attributed to radical scavenging activities of its components. Therefore, SO can be considered as a promising compound to prevent liver toxicity induced by VPA.