Protective Effects of Myricetin Against Valproic Acid-Induced Endoplasmic Reticulum Stress, Apoptosis and Inflammation in Rats: Molecular Docking Analysis of PERK and IRE1

Technical Summary: Protective Effects of Myricetin Against Valproic Acid-Induced Toxicity

Problem Statement
Valproic acid (VPA) is a first-line broad-spectrum antiepileptic drug widely used for epilepsy, bipolar disorder, and neuropathic pain. However, its long-term or high-dose administration is associated with significant hepatotoxicity and nephrotoxicity. The mechanisms underlying this toxicity involve the induction of Endoplasmic Reticulum (ER) stress, inflammation, and apoptosis. Specifically, VPA exposure triggers the Unfolded Protein Response (UPR) via sensors such as Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1), leading to the activation of inflammatory pathways (e.g., p38 MAPK, TNF-α, IL-1β) and oxidative stress (indicated by Malondialdehyde/MDA levels). While synthetic inhibitors exist for ER stress pathways, there is a need to investigate natural compounds that might mitigate these adverse effects without introducing new toxicities. Myricetin (MYR), a flavonol with known antioxidant properties, was hypothesized to protect against VPA-induced tissue damage by modulating these specific molecular pathways.

Methodology
The study employed a multi-faceted approach combining in vivo animal experimentation with in silico molecular modeling:

• Animal Model: Thirty-six male Wistar-Albino rats were divided into four groups (n=9): Control (saline), VPA (500 mg/kg, intraperitoneal), VPA + MYR50 (50 mg/kg oral), and VPA + MYR100 (100 mg/kg oral). Treatments were administered for 15 days.
• Biochemical Analysis: Serum levels of liver enzymes (AST, ALT, ALP), renal function markers (BUN, Creatinine), and lipid profiles were measured. Tissue homogenates from the liver and kidney were analyzed for oxidative stress (MDA), inflammatory cytokines (TNF-α, IL-1β), and ER stress/apoptosis markers (PERK, IRE1, p38 MAPK, JNK, Bcl-2) using ELISA.
• Histopathology: Liver and kidney tissues were fixed, sectioned, and stained with Hematoxylin–Eosin (H&E). Lesions were graded semi-quantitatively based on degeneration, inflammation, and necrosis.
• Molecular Docking and Dynamics:
  • PERK: Docking simulations (AutoDock 4.2) were performed using the crystal structure (PDB: 4G31) to compare the binding affinity of MYR against ATP and synthetic inhibitors (AMG44, GSK derivatives).
  • IRE1: Molecular dynamics (MD) simulations (500 ns) were conducted using Amber20 on the IRE1α kinase domain (PDB: 6W3B). The study analyzed Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and critical salt bridge distances (Lys599–Glu612) to assess structural stability and conformational changes upon MYR binding compared to ATP.

Key Results

• Biochemical Protection: VPA administration significantly elevated serum AST, ALT, ALP, BUN, and lipid profiles (TC, TG, LDL), indicating liver and kidney dysfunction. MYR treatment at both 50 and 100 mg/kg significantly attenuated these elevations. Regarding plasma glucose, the 50 mg/kg dose produced a statistically significant reduction compared to the VPA group, whereas the reduction observed in the 100 mg/kg group did not reach statistical significance.
• Modulation of ER Stress and Inflammation: VPA significantly increased hepatic and renal levels of PERK, IRE1, p38 MAPK, and pro-inflammatory cytokines (TNF-α, IL-1β). MYR treatment significantly reduced these levels, suggesting the suppression of the ER stress-UPR axis and downstream inflammatory cascades. While the text notes that the 50 mg/kg dose produced a greater reduction in TNF-α and IL-1β levels than the 100 mg/kg dose in some instances, the overall histological data indicates a dose-dependent protective effect where the 100 mg/kg dose provided superior histological improvement.
• Oxidative Stress and Apoptosis Markers: VPA induced significant lipid peroxidation (elevated MDA) in both tissues. MYR significantly reduced MDA levels at both doses. Regarding Bcl-2, VPA increased levels in both liver and kidney (interpreted by the authors as a compensatory anti-apoptotic response to stress). MYR treatment significantly reduced renal Bcl-2 levels toward control values, while hepatic reductions were not statistically significant. JNK levels showed no significant changes across groups.
• Histopathological Findings: VPA caused severe hepatocellular degeneration, necrosis, and inflammatory infiltration, as well as renal tubular necrosis and glomerular atrophy. MYR treatment markedly reversed these structural damages, with the 100 mg/kg dose showing a more prominent histological improvement in the kidney, suggesting a dose-dependent protective effect.
• Molecular Interactions:
  • PERK: MYR exhibited a binding free energy (ΔG) of −7.52 kcal/mol, slightly more favorable than ATP (−7.20 kcal/mol), suggesting it can compete for the ATP-binding pocket, though with lower affinity than synthetic inhibitors like AMG44 (−14.47 kcal/mol).
  • IRE1: MD simulations revealed that the MYR-bound complex was more structurally stable (lower RMSD) than the ATP-bound complex. MYR restricted the flexibility of the αC-helix and activation loop and "locked" the catalytic Lys599–Asp711 distance, suggesting it acts as a competitive inhibitor that stabilizes the active conformation while preventing the dynamic transitions required for catalysis.

Significance and Claims
The authors conclude that Myricetin possesses therapeutic potential in preventing or reducing VPA-associated hepatic and renal toxicity. The study posits that MYR exerts its protective effects through a multi-target mechanism:

1. Attenuation of Oxidative Stress: By reducing lipid peroxidation (MDA).
2. Suppression of Inflammation: By lowering TNF-α and IL-1β levels.
3. Modulation of ER Stress: By downregulating the expression of PERK and IRE1 and inhibiting the p38 MAPK pathway.
4. Direct Molecular Interaction: The in silico data supports the hypothesis that MYR can directly interact with the kinase domains of PERK and IRE1, potentially acting as an ATP-competitive inhibitor.

The paper maintains a modest tone regarding these molecular claims, acknowledging that while docking and MD simulations suggest direct binding and inhibition, further experimental studies are required to confirm the direct inhibitory effects of MYR on PERK and IRE1 in vivo. The study highlights the potential of natural flavonoids as adjuvants to mitigate the side effects of essential but toxic pharmaceutical agents like Valproic Acid.