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Antioxidant and Anti-Inflammatory Properties of Alpha Lipoic Acid Protect Against Valproic Acid Induced Liver Injury

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Antioxidant and Anti-Inflammatory Properties of Alpha Lipoic Acid Protect Against Valproic Acid Induced Liver Injury Canadian Journal of Physiology and Pharmacology Antioxidant and anti-inflammatory properties of alpha lipoic acid protect against valproic acid induced liver injury Journal: Canadian Journal of Physiology and Pharmacology Manuscript ID cjpp-2019-0456.R4 Manuscript Type: Article Date Submitted by the 20-Nov-2020 Author: Complete List of Authors: Mohammed, Marwa; Beni-Suef University; Beni-Suef University Gharib, Doaa; Cairo University Reyad, Hoda; Beni-Suef University Mohamed, Alaa; Beni-Suef University Elroby, Fadwa;Draft Beni-Suef University Mahmoud, Hoda; Beni-Suef University Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue: Keyword: ALA, VPA, Nrf2, Sirt1, SOD © The Author(s) or their Institution(s) Page 1 of 40 Canadian Journal of Physiology and Pharmacology Antioxidant and anti-inflammatory properties of alpha lipoic acid protect against valproic acid induced liver injury Marwa Abdeltawab Mohammed1, Doaa Mostafa Gharib2, Hoda Ramadan Reyad3, Alaa Aboud Mohamed4, Fadwa A Elroby5, Hoda Sayed Mahmoud5. l Department of Physiology, Faculty of Medicine, Beni-Suef University, Egypt. 2 Department of Biochemistry, FacultyDraft of Medicine, Cairo University, Egypt. 3Department of Biochemistry, Faculty of Medicine, Beni-Suef University, Egypt. 4Department of Tropical, Faculty of Medicine, Beni-Suef University, Egypt. 5 Department of Forensic Medicine & Toxicology, Faculty of Medicine, Beni-Suef University, Egypt. Corresponding author: Marwa Abdeltawab Mohammed Email: [email protected] Telephone: 002/01100616755 1 © The Author(s) or their Institution(s) Canadian Journal of Physiology and Pharmacology Page 2 of 40 Abstract: Valproic acid (VPA) is one of the most used anti-epileptic drugs inspite of its many adverse effects as anemia, leucopenia, thrombocytopenia, and liver toxicity. The hepatoprotective effect of alpha-lipoic acid (ALA) was confirmed. Aim of the study: The aim of this study was to detect the protective effect of ALA against the adverse effects of VPA. Materials& Methods: Thirty white albino Wistar male rats were divided into 4 groups. Group (1) was the control group; Group (2) included rats that received ALA (100mg/kg/day) orally for 14 days;Draft Group (3) and Group (4) included rats that received VPA (500 mg/kg/day) for 15 days intraperitoneal, but group 4 rats received ALA (100mg/kg/day) orally for 14 days prior to VPA. Blood samples were collected and livers were excised from rats for colorimetric analysis and rt-PCR. Results: Rats that received VPA showed leucopenia, thrombocytopenia, a significant decrease of SOD, glutathione, Nrf2, and Sirt1, besides a significant increase of MDA and TNF alpha. Prior treatment with ALA prevented all these results. Conclusion: ALA protected against VPA-induced liver damage and hematological disturbance via anti-oxidant and anti-inflammatory properties. Keywords: ALA, VPA, Nrf2, Sirt1, TNF-ɑ, SOD, glutathione. 2 © The Author(s) or their Institution(s) Page 3 of 40 Canadian Journal of Physiology and Pharmacology 1. Introduction: Valproic acid (VPA) is commonly used in the treatment of not only epilepsy but also bipolar disorders (Johannessen and Johannessen, 2003). With a chronic use of VPA, there is a high incidence to develop various side effects such as hemorrhagic pancreatitis, bone marrow suppression, and more frequently hepatic damage (Bedry et al., 2004). It was found that 44% of patients receiving VPA chronically are susceptible to an elevation of serum liver enzymes and lipid peroxidation during the first months of therapy (Powell et al., 1984). Draft Moreover, hepatotoxicity mediated by VPA is found to be characterized by deposition of fat with depletion of antioxidants, and induction of mitochondrial membrane disturbances as well (Spiller et al., 2000). Furthermore, VPA is documented to be teratogenic with the induction of heart abnormalities, craniosynostosis and neural tube defects (Ornoy, 2006). In addition, about 40% of patients who received VPA are more liable for the development of obesity and fatty liver (Amin et al., 2012), besides kidney disturbances (Chang et al., 2016). VPA is metabolized mainly in the liver by glucuronide conjugation and, to a lower extent, by mitochondrial β -oxidation and cytosolic ω -oxidation. The latter is involved in the production of toxic metabolites such as 4-en-valproate (Sztajnkrycer, 2002) and about 3% of the 3 © The Author(s) or their Institution(s) Canadian Journal of Physiology and Pharmacology Page 4 of 40 VPA is excreted unchanged in the urine (Thanacoody, 2009). The main mechanism of VPA toxicity is interference with mitochondrial beta-oxidation. This is due to its structural similarity to simple fatty acid, and its ability to cause exacerbation of an underlying mitochondrial cytopathy (Chen et al., 2007). Alpha-lipoic acid (ALA), a natural product, is known to be rapidly absorbed and converted to its reduced form; the dithiol compound dihydrolipoic acid by NAD (P)H-dependent enzymes (Packer et al., 1995; Smith et al., 2004). ALA actsDraft as a cofactor involved in the regulation of energy metabolism and redox status of the cell, as established by previous studies (Shay et al., 2009). ALA not only has a role in mitochondrial energy metabolism but also neutralizes oxygen species, helping the regeneration of antioxidants and stimulation of insulin signaling (Packer et al., 1995; Biewenga et al., 1997). Inside the mitochondria, lipoamide dehydrogenase or the thioredoxin/thioredoxin reductase system converts ALA into dihydro-lipoate which modulates the conversion of NADH into NAD+ and increases the NAD+/NADH ratio which in turn increases Sirt1 activity (Chen et al., 2012). 4 © The Author(s) or their Institution(s) Page 5 of 40 Canadian Journal of Physiology and Pharmacology Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that enhances transcription of multiple protective genes in response to oxidative stress conditions (Klaassen and Reisman, 2010). The sequestration of Nrf2 by kelch-like ECH associating protein 1 (Keap1) under the resting condition is released on exposure to oxidative stress, which helps Nrf2 to translocate into the nucleus to induce its response (Kensler et al., 2007). The protective effect of Nrf2 against drug-induced hepatotoxicity has been confirmed (Wu et al., 2012). While the mechanisms byDraft which VPA works are still not clear, it has been proposed to inhibit histone deacetylases (Pheil et al., 2001; Kanai et al., 2004). Sirt1, an NAD+-dependent protein deacetylase, regulates many cellular processes (Ghisays et al., 2015). Sirt1 was found to be involved in the activation of different antioxidant genes such as manganese superoxide dismutase (SOD) and catalase (Danz et al., 2009) through regulation of P53 (Budanov et al., 2005). Studies have recognized that Sirt1 can suppress cardiomyocyte inflammation and apoptosis through down-regulation of NF- κB (Han et al., 2008; Yang et al., 2015). The role of Sirt1 in the protection against drugs that induce liver toxicity has been investigated (Nicoletti et al., 2014; Yang et al., 2017). For example, up-regulation of Sirt1 was found to protect livers exposed to doxorubicin toxicity (Chen et al., 2019) through the 5 © The Author(s) or their Institution(s) Canadian Journal of Physiology and Pharmacology Page 6 of 40 effects on the expression of the antioxidants (Danz et al., 2009; Yang et al., 2015). Hence, we propose that Sirt1 may produce vital roles in the protection against hepatotoxicity induced by VPA. TNF-α is a cytokine responsible for numerous cellular activities, such as inflammatory mediators formation and enhancement of adhesion molecules up-regulation. Macrophages are the main source providing TNF-α; however, other cell types can produce TNF-α, like mast cells and hepatic stellate cells (Wajant et al., 2003). Shimizu et al. (2005) analyzed the link between TNF-α development of chemicals inducedDraft liver injury. TNF-α was found to be involved in lipopolysaccharide induced hepatic injury, which was abolished by TNF receptor 1 knockout (Shimizu et al., 2005). Tukov et al. (2007) demonstrated the role of TNF-α in lipopolysaccharide-induced liver injury by means of increasing levels of several inflammatory mediators and cytokines like IL-6 and MIP-2. The aim of the present study was to estimate the protective effect of ALA against hematological disorders and hepatic injury induced by the administration of VPA and to clarify the molecular mechanisms for this protection. 6 © The Author(s) or their Institution(s) Page 7 of 40 Canadian Journal of Physiology and Pharmacology 2. Materials and Methods: 2.1. Materials: ALA was purchased from Eva pharm, Egypt. VPA was purchased from Sanofi, France. Alanine transferase (ALT), aspartate transferase (AST), albumin, bilirubin kits were obtained from Bio-Diagnostic, Egypt. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were obtained from Bio-Diagnostic, Egypt. rt- PCR kits were purchased from Bio-System, USA.Draft 2.2 Experimental design: Thirty male white albino wistar rats with an average weight between 180-200 gm were used in this study (6 rats/control group, 8 rats/other groups). Rats were randomly divided into four groups. Doses and duration of the used drugs were determined according to previous studies. Group I includes control animals that received normal saline intraperitoneal all the duration of the study. Group II (ALA group) includes rats that received ALA (100 mg/kg/day) orally for 14 days.
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