This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

• Klíčová slova
• chronic pain treatment, label-free quantification, morphine, multifunctional enkephalin analogs, proteomic analysis, rat brain, rat spleen lymphocytes,
• Publikační typ
• časopisecké články MeSH

Chronic pain is associated with time-dependent structural and functional reorganization of the prefrontal cortex that may reflect adaptive pain compensatory and/or maladaptive pain-promoting mechanisms. However, the molecular underpinnings of these changes and whether there are time-dependent relationships to pain progression are not well characterized. In this study, we analyzed protein composition in the medial prefrontal cortex (mPFC) of rats at two timepoints after spinal nerve ligation (SNL) using two-dimensional gel electrophoresis (2D-ELFO) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). SNL, but not sham-operated, rats developed persistent tactile allodynia and thermal hyperalgesia, confirming the presence of experimental neuropathic pain. Two weeks after SNL (early timepoint), we identified 11 proteins involved in signal transduction, protein transport, cell homeostasis, metabolism, and apoptosis, as well as heat-shock proteins and chaperones that were upregulated by more than 1.5-fold compared to the sham-operated rats. Interestingly, there were only four significantly altered proteins identified at 8 weeks after SNL (late timepoint). These findings demonstrate extensive time-dependent modifications of protein expression in the rat mPFC under a chronic neuropathic pain state that might underlie the evolution of chronic pain characterized by early pain-compensatory and later aberrant mechanisms.

• Klíčová slova
• affective dimension of pain, neuropathic pain, pain chronification, prefrontal cortex, proteomics,
• MeSH
• časové faktory MeSH
• chromatografie kapalinová MeSH
• hyperalgezie etiologie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• měření bolesti MeSH
• míšní nervy zranění   MeSH
• neuralgie etiologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• prefrontální mozková kůra metabolismus   MeSH
• proteomika metody   MeSH
• regulace genové exprese MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

• Klíčová slova
• energy metabolism, gel-based proteomics, nLC-MS/MS, oxidative stress, protracted morphine withdrawal, rat brain cortex, rat hippocampus,
• Publikační typ
• časopisecké články MeSH

The observation of the immunomodulatory effects of opioid drugs opened the discussion about possible mechanisms of action and led researchers to consider the presence of opioid receptors (OR) in cells of the immune system. To date, numerous studies analyzing the expression of OR subtypes in animal and human immune cells have been performed. Some of them confirmed the expression of OR at both the mRNA and protein level, while others did not detect the receptor mRNA either. Although this topic remains controversial, further studies are constantly being published. The most recent articles suggested that the expression level of OR in human peripheral blood lymphocytes could help to evaluate the success of methadone maintenance therapy in former opioid addicts, or could serve as a biomarker for chronic pain diagnosis. However, the applicability of these findings to clinical practice needs to be verified by further investigations.

• Klíčová slova
• addiction, chronic pain, immune cells, opioid drugs, opioid receptors, stem cells,
• MeSH
• biologické markery MeSH
• chronická bolest farmakoterapie   etiologie   metabolismus   MeSH
• imunitní systém účinky léků   imunologie   metabolismus   MeSH
• kmenové buňky účinky léků   metabolismus   MeSH
• lidé MeSH
• opioidní analgetika farmakologie   MeSH
• receptory opiátové genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• zánět komplikace   etiologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH
• opioidní analgetika MeSH
• receptory opiátové MeSH

Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and -M10) or 20 days after the last dose of morphine (groups +M10/-M20 and -M10/-M20). The six altered proteins (≥2-fold) were identified in group (+M10) when compared with group (-M10) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, β-synuclein, α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/-M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/β-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (-M10). After 20 days of morphine withdrawal (±M10/-M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.

• MeSH
• abstinenční syndrom patologie   MeSH
• časové faktory MeSH
• hipokampus účinky léků   patologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• morfin škodlivé účinky   MeSH
• mozková kůra účinky léků   patologie   MeSH
• opioidní analgetika škodlivé účinky   MeSH
• poruchy spojené s užíváním opiátů patologie   MeSH
• potkani Wistar MeSH
• proteomika MeSH
• regulace genové exprese účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• morfin MeSH
• opioidní analgetika MeSH

4,5-Dihydroxyanthraquinone-2-carboxylic acid (Rhein) has been shown to have various physiological and pharmacological properties including anticancer activity and modulatory effects on bioenergetics. In this study, we explored the impact of rhein on protein profiling of undifferentiated (UC) and differentiated (DC) SH-SY5Y cells. Besides that, the cellular morphology and expression of differentiation markers were investigated to determine the effect of rhein on retinoic acidinduced neuronal cell differentiation. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry we evaluated the changes in the proteome of both UC and DC SH-SY5Y cells after 24 h treatment with rhein. Validation of selected differentially expressed proteins and the assessment of neuronal differentiation markers were performed by western blotting. Proteomic analysis revealed significant changes in the abundance of 15 proteins linked to specific cellular processes such as cytoskeleton structure and regulation, mitochondrial function, energy metabolism, protein synthesis and neuronal plasticity. We also observed that the addition of rhein to the cultured cells during differentiation resulted in a significantly reduced neurite outgrowth and decreased expression of neuronal markers. These results indicate that rhein may strongly interfere with the differentiation process of SH-SY5Y neuroblastoma cells and is capable of inducing marked proteomic changes in these cells.

• MeSH
• anthrachinony farmakologie   MeSH
• buněčná diferenciace účinky léků   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nervové kmenové buňky účinky léků   MeSH
• neurity účinky léků   patologie   MeSH
• neuroblastom farmakoterapie   genetika   patologie   MeSH
• neuronální růst účinky léků   MeSH
• neurony účinky léků   MeSH
• proteomika * MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anthrachinony MeSH
• rhein MeSH Prohlížeč

BACKGROUND: Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC. METHODS: Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies. RESULTS: Significant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/-M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats. CONCLUSIONS: In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal. GENERAL SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.

• MeSH
• 2D gelová elektroforéza MeSH
• abstinenční syndrom * MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• krysa rodu Rattus MeSH
• morfin aplikace a dávkování   škodlivé účinky   MeSH
• potkani Wistar MeSH
• přední mozek metabolismus   MeSH
• receptory opiátové delta metabolismus   MeSH
• receptory opiátové kappa metabolismus   MeSH
• receptory opiátové mu metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• morfin MeSH
• receptory opiátové delta MeSH
• receptory opiátové kappa MeSH
• receptory opiátové mu MeSH