RUTIN ATTENUATES ALCOHOL WITHDRAWAL INDUCED ANXIETY IN RATS VIA NMDA RECEPTOR MODULATION, ANTIOXIDANT DEFENSE, AND MULTI TARGET NEUROINFLAMMATORY PATHWAY REGULATION: AN INTEGRATED NETWORK PHARMACOLOGY AND PRECLINICAL STUDY

Abstract

Background: Alcohol withdrawal syndrome (AWS) is a clinically relevant syndrome associated with anxiety, neuroinflammation and glutamatergic excitotoxicity, in which there is a limited number of safe, effective and non addictive treatments available. There is no systematic study on the effect of the bioflavonoid glycoside rutin on the anxiety induced by AWS using an integrated in silico and in vivo approach. Objective: To explore the anti-anxiety, neuroprotective and anti-inflammatory activity of rutin and its combination with the NMDA receptor antagonist memantine in ethanol withdrawal-induced anxiety in rats by network pharmacology and molecular docking studies. Materials and Methods: A network pharmacology approach was carried out with the GeneCards, OMIM, Swiss Target Prediction, STRING, Cytoscape and GeneCodis databases. Molecular docking was done using PyRx and the visualization presented in Biovia Discovery Studio. Male Wistar rats were made alcohol dependent via oral ethanol administration for a period of 21 days (2.4% v/v to 4.8% v/v to 7.2% v/v). Seven experimental groups were treated for 7 days post withdrawal – Normal control, Ethanol control, Rutin 50 mg/kg p.o., Rutin 100 mg/kg p.o., Rutin 200 mg/kg p.o., Memantine (20 mg/kg p.o.), and Rutin + Memantine (50 + 20 mg/kg p.o.). The Elevated Plus Maze (EPM), Light/Dark Box Test (LDT), and Open Field Test (OFT) were used to assess anxiety. Oxidative stress markers (GSH, SOD, CAT, MDA, NO), neurotransmitters (GABA, serotonin, dopamine, glutamate, norepinephrine), TNF-α and NMDA receptor expression were measured in brain tissue. Histopathological analyses of the hippocampal tissue were conducted by H&E staining. Results: Network pharmacology revealed that there were 69 common targets between rutin and AW-anxiety, including the hub protein of TNF, TP53, EGFR, PTGS2 and GSK3B. The molecular docking studies showed that rutin has good binding affinity with PTGS2, TNF, HSP90AB1, and GSK3B with −10.1 kcal/mol, −9.1 kcal/mol, −9.1 kcal/mol, and −8.4 kcal/mol binding energy respectively. In vivo, rutin dose-dependently reduced anxiety like behavior in all three behavioral paradigms, significantly increased depleted GABA, serotonin and dopamine, decreased elevated glutamate and NE, decreased MDA and NO, increased GSH, SOD and CAT activity and decreased elevated TNF-α, and decreased NMDA receptor expression. The rutin + memantine association (50 + 20 mg/kg) was the most effective treatment, almost normalizing all parameters. The simultaneous neuroprotective effects were confirmed by hippocampal histopathology. Conclusion: Rutin has dose-dependent anxiolytic and neuroprotective activities in ethanol withdrawal, primarily via modulation of NMDA receptors, restoration of GABAergic/glutamatergic balance, antioxidant reinforcement, and suppression of neuroinflammation. The synergistic effects of the rutin–memantine combination indicate potential for translation in the management of AWS.