Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.

• Klíčová slova
• Crude membrane fractions, KOR antagonism, Morphine, Multifunctional enkephalin analogs, Proteomic analysis, Rat brain,
• MeSH
• analgetika MeSH
• enkefaliny metabolismus   MeSH
• hipokampus metabolismus   MeSH
• krysa rodu Rattus MeSH
• morfin * farmakologie   MeSH
• mozek metabolismus   MeSH
• opioidní analgetika farmakologie   MeSH
• receptory opiátové mu * metabolismus   MeSH
• receptory opiátové metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• analgetika MeSH
• enkefaliny MeSH
• morfin * MeSH
• opioidní analgetika MeSH
• receptory opiátové mu * MeSH
• receptory opiátové MeSH

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