separation-rat-epidermis-dermis-with-thermolysin-to-detect-site

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...

Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, Î²-funaltrexamine (Î²-FNA). Interferon (IFN)Î³+HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with Î²-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH(2) (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNÎ³+HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent Î²-FNA mediated inhibition of IFNÎ³+HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-ÎºB activation (SN50) to demonstrate that IFNÎ³+HIV-1 Tat-induced CXCL10 expression is NF-ÎºB-dependent in NHA. Subsequent experiments revealed that Î²-FNA did not significantly affect NF-ÎºB activation. Interestingly, we discovered that Î²-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of Î²-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by Î²-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation.

Î²-Funaltrexamine inhibits chemokine (CXCL10) expression in normal human astrocytes.

Neuroinflammation ist integraler Bestandteil der neurodegenerativen Erkrankungen, CNS Infektion und Trauma. Astroglial Chemokine, wie CXCL10, sind ma&szlig;geblich an neuroinflammatorische Signalisierung sowie Neurotoxizit&auml;t. Wir haben Proinflammatory induziert CXCL10 Ausdruck im normalen menschlichen Astrozyten (NHA) genutzt, wie ein Modell, in dem die entz&uuml;ndungshemmenden Aktionen des Rezeptorantagonisten selektive, Mu-Opioid-(MOR), &beta;-Funaltrexamine (&beta;-FNA) zu beurteilen. Interferon (IFN) &gamma; + induziert durch HIV-1 Tat CXCL10 Ausdruck (sekretierten Proteine und mRNA) durch Co-treatment mit &beta;-FNA gehemmt wurde. Weder der MOR-selektiver Antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH(2) (CTAP) oder der unselektiv opioid-Rezeptor-Antagonist, hemmte Naltrexon IFN&gamma; + HIV-1 Tat-induzierte CXCL10 Ausdruck. Dar&uuml;ber hinaus hat Co-treatment mit &uuml;bersch&uuml;ssigen CTAP oder Naltrexon &beta;-FNA-vermittelte Hemmung der CXCL10 IFN&gamma; + HIV-1 Tat-induzierte Expression nicht verhindert. Dar&uuml;ber hinaus verwendet wir einen Inhibitor der NF-&kappa;B Aktivierung (SN50) zeigen, dass CXCL10 IFN&gamma; + HIV-1 Tat-induzierte Expression NF-&kappa;B-abh&auml;ngigen in NHA ist. Nachfolgende Experimente ergeben, dass &beta;-FNA NF-&kappa;B Aktivierung nicht wesentlich beeinflussten. Interessant ist, entdeckten wir, dass &beta;-FNA gehemmt-p38-Aktivierung, wie durch verminderte Expression von Phospho-p38. Zusammen, empfehlen diese Erkenntnisse die hemmenden T&auml;tigkeiten von &beta;-FNA sind MOR-unabh&auml;ngig und vermittelt, teilweise &uuml;ber einen transkriptionelle Mechanismus. Diese Erkenntnisse hinzuf&uuml;gen zu unserem Verst&auml;ndnis des Mechanismus durch den Chemokine Ausdruck von &beta;-FNA gehemmt wird. In Verbindung mit zuk&uuml;nftigen Untersuchungen d&uuml;rften diese neuartige Erkenntnisse bieten Einblicke in die Entwicklung von sicheren und wirksamen Behandlungen f&uuml;r Neuroinflammation.

&Beta;-Funaltrexamine hemmt Chemokine (CXCL10) Ausdruck im normalen menschlichen Astrozyten.

Opioid-immune crosstalk occurs when opioid drugs alter the activity of the immune system. In this study, the opioid antagonist β-funaltrexamine (β-FNA) decreases the expression and release of an inflammatory chemokine, interferon-γ inducible protein-10 (CXCL10) from normal human astrocytes stimulated by interleukin 1β (IL-1β). β-FNA decreased CXCL10 by an unknown action that did not involve the mu opioid receptor (MOR). As IL-1β acts through its receptor to activate NF-κB/MAPK signaling pathways which leads to CXCL10 expression and release, key steps in the IL-1β signaling pathways were examined following β-FNA treatment. IL-1β-induced activation of p38 mitogen-activated protein kinases (p38 MAPK) was inhibited by β-FNA as shown by decreased p38 MAPK phosphorylation in treated cells. β-FNA also decreased the levels of activated subunits of NF-κB (p50 and p65) in treated astrocytes. The impact of β-FNA was also observed in proteins that act to negatively regulate NF-κB signaling. IL-1β upregulated the expression of A20, a ubiquitin (Ub)-editing enzyme that dampens NF-κB signaling by altering ubiquination patterns on IL-1 receptor second messengers, and the increase in A20 was significantly inhibited by β-FNA treatment. Inhibition of the Ub-activating enzyme E1 by the inhibitor PYR41 also decreased CXCL10 release, like β-FNA, and concurrent treatment with both PYR41 and β-FNA inhibited CXCL10 more than did either agent alone. In mice, lipopolysaccharide-induced CXCL10 expression in the brain was inhibited by treatment with β-FNA. These findings suggest that β-FNA exerts an anti-inflammatory action in vitro and in vivo that is MOR-independent and possibly due to the alkylating ability of β-FNA. 

The opioid antagonist, β-funaltrexamine, inhibits NF-κB signaling and chemokine expression in human astrocytes and in mice.

Pseudo-unipolar cell bodies of somatosensory primary neurons are located in the dorsal root ganglia (DRG). The somatic and peripheral domains of DRG neurons are often studied in sensory pain research to understand molecular mechanisms involved in the activation of pain and maintenance of inflammation. Adjuvant-induced arthritis (AIA) is an inflammatory model that elicits a robust and rapid onset immune response with a maximal swelling period of 24-48 h and persisting for several weeks. The AIA model in the hind paw of the rat elicits a potent inflammatory response of the dermis and epidermis, leading to protein expression changes for sensitization of many DRG neurons; however, it is unknown if the AIA model in the hind paw of the rat induces DRG neuronal injury, necrosis, or apoptosis at the somatic level. Neuronal nuclei (NeuN) antigen is a biomarker for post-mitotic neurons, neuronal identification, protein alterations, injury, and loss. Calcitonin gene-related peptide (CGRP) is expressed in C and Aδ DRG neurons, a subset of DRG neurons known to play a role in peripheral sensitization. The focus of this research was to evaluate the expression pattern of NeuN immunoreactivity, in size (soma) and CGRP subpopulations of DRG neurons in naïve and inflamed groups. Confirmed by both immunofluorescence and immunoprecipitation, DRG neuronal expression of NeuN was localized to nuclear and cytoplasmic subcellular compartments. NeuN increased within the nucleus of small CGRP positive DRG neurons during inflammation, indicating a potential role for NeuN in a subset of nociceptive neurons.

Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflammation.

Article	Year
Î²-Funaltrexamine inhibits chemokine (CXCL10) expression in normal human astrocytes. Neurochemistry international\| PubMed ID: 23376103	2013
The opioid antagonist, β-funaltrexamine, inhibits NF-κB signaling and chemokine expression in human astrocytes and in mice. European journal of pharmacology\| PubMed ID: 26007645	2015
Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflam... Neuroscience letters\| PubMed ID: 34146639	2021

Subhas Das

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