SHOTGUN PROTEOMICS ANALYSIS OF PROTEINS RESPONDING TO DRUG ADDICTION IN FRONTAL CORTEX

Abstract

Drug addiction is regarded as a major public health problem in Thailand. The most common addictive drugs are methamphetamine (METH) and over the counter medication such as dextromethorphan (DXM). Drug addiction has been reported as a behavioral abnormality, through the alteration of molecular and cellular levels in the brain, such as the frontal cortex. METH is an addictive psychostimulant with potent effects on the central nervous system (CNS), prolonged use can impair brain structures and functions. As well, DXM is an antitussive agent which is included in several over-the- counter cough and cold medications, prolonged use can cause psychoactive effects and hallucinogen-like addiction. Several studies have shown drug addiction's effect on the neurotransmitters including glutamate, GABA, dopamine and associated protein in the frontal cortex, resulting in cognitive, behavioral, and physiological abnormalities. So, the therapeutic approaches for drug addiction have been involved in these neurotransmitter systems, such diazepam have anxiolytics that are specific to the GABAergic system, but chronic use can develop into addiction. Nutraceuticals are an alternative abundant interest, that is pre-germinated brown rice (PGBR), high GABA contents. However, little is known about the underlying mechanisms of PGBR effects on neurobiological proteins. Therefore, the aim is to investigate the protein expression profiling in the rat frontal cortex after exposure to addictive drugs and addiction treatments. Male Sprague-Dawley rats were divided into 2 groups of addiction models, including METH and DXM addiction. In METH, the rats were treated with saline (CM) and escalating binge dose of METH (0.1 to 4 mg/kg of METH (3 times /day), for 14 days and binge dose, 6 mg/kg (4 times /day) at day 15), respectively. In DXM, The animals were divided into six groups including control (CD), dextromethorphan (D), withdrawal (DW), diazepam (DD), synthetic GABA (DS), and pre-germinated brown rice (DR). The animals in the control group received saline i.p. for 15 days and treated with distilled water by oral administration for 60 days. Animals in the DW group were drug-withdrawal by receiving oral administration of distilled water. Animals in the DD group received 10 mg/kg diazepam. Animals in the DS group were administered with 0.8 mg/kg synthetic GABA. Animals in the DR group were treated with 5 mg/kg PGBR. After the last administration of the subgroup, animals were sacrificed, and the brain was collected. The alteration proteins in rat frontal cortex were investigated by proteomic technique (LC-MS/MS). An expression level of some protein was validated by western blotting technique. The proteomic result shows the alteration of neuronal signaling proteins expression in rat frontal cortex after drug addiction, drug withdrawal and drug treatment (diazepam, GABA and PGBR). Several biochemical functions appear to be enriched among these proteins, the glutamatergic and GABAergic pathway. These pathways were enriched in receptor, transporter, enzyme and associated proteins. Moreover, western blotting confirmed the associated candidate proteins, which refer to glutamatergic and GABAergic dysfunction after METH addiction. These findings highlight the identification of glutamatergic and GABAergic neurotransmitter systems and provide insight into the biological function involved in drug addiction and its treatment.

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