Background and purpose C5a is a crucial terminal component of the complement cascade. Several reports suggest that C5a can support nociceptive sensitization and inflammation. Within the central nervous system (CNS), C5aR is constitutively expressed not only in astrocytes and microglia (1) but also in neurons (2) of different brain regions. Several neuro-inflammatory conditions increased C5aR expression (3) and pain also accompanies many types of inflammation and injury. Moreover, it has been demonstrated that inappropriate activation of complement produces a local inflammatory reaction and neurodegenerative diseases (4). Thus C5aR may provide a novel target for the control of pain and inflammation. Herein, primary rat cortical neurons were exposed to oxygen-glucose deprivation-reoxygenation (OGD-R), an in vitro sub-lethal neuronal inflammatory model, in order to characterize the activity of DF3016A, a selective C5aR antagonist. Methods Primary cortical rat cells were exposed to OGD-R. DF3016A and OGD-R cytotoxicity was evaluated by MTS (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-assay. Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) was performed to analyze C5aR, BDNF, MeCP2, miR132 and miR181a gene expressions. Protein levels were measured by Western Blot analysis. Results Under OGD-R condition, DF3016A treatment leads to a significant decrease of C5aR gene expression and BDNF protein increase linked to ERK/CREB phosphorylation. BDNF neurotrophic pathway markedly increased the expression of miR132 which, in turn, is regulated by pCREB. Furthermore, in OGD-R neurons miR181 gene expression, a regulatory factor of pro inflammatory cytokines, is inverted by DF3016A treatment. Conclusions These data provide the first evidence that sub-lethal OGD-R is able to induce C5aR gene expression upregulation that is reversed by C5aR antagonist toward control levels. 500nM DF3016A activity can induce neurotrophic pathway that involves BDNF upregulation. Within CNS, the inhibition of a proinflammatory receptor-mediated function of the C5aR can interfere with neuronal stress suggesting a novel therapeutic target for reducing nociceptive inflammation of central neurons. References 1. Müüller-Ladner, et al. 1996. Enhanced expression of chemotactic receptors in multiple sclerosis lesions. J. Neurol. Sci. 144:135 2. O'Barr SA, et al. 2001.Neuronal expression of a functional receptor for the C5a complement activation fragment. J Immunol.;166(6):4154-62. 3. Gasque P., et al. 1997. Expression of the receptor for complement C5a (CD88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system. Am. J. Pathol. 150:31. 4. Zhou J, et al. 2008.Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease. J Neurochem.; 106(5):2080-92.

Role of the C5aR antagonist in nociceptive inflammation of cortical neurons

GRANNONICO, MARTA;Brandolini, Luca;PALADINI, ANTONELLA;PIROLI, ALBA;VARRASSI, Giustino;DI LORETO, SILVIA
2017-01-01

Abstract

Background and purpose C5a is a crucial terminal component of the complement cascade. Several reports suggest that C5a can support nociceptive sensitization and inflammation. Within the central nervous system (CNS), C5aR is constitutively expressed not only in astrocytes and microglia (1) but also in neurons (2) of different brain regions. Several neuro-inflammatory conditions increased C5aR expression (3) and pain also accompanies many types of inflammation and injury. Moreover, it has been demonstrated that inappropriate activation of complement produces a local inflammatory reaction and neurodegenerative diseases (4). Thus C5aR may provide a novel target for the control of pain and inflammation. Herein, primary rat cortical neurons were exposed to oxygen-glucose deprivation-reoxygenation (OGD-R), an in vitro sub-lethal neuronal inflammatory model, in order to characterize the activity of DF3016A, a selective C5aR antagonist. Methods Primary cortical rat cells were exposed to OGD-R. DF3016A and OGD-R cytotoxicity was evaluated by MTS (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-assay. Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) was performed to analyze C5aR, BDNF, MeCP2, miR132 and miR181a gene expressions. Protein levels were measured by Western Blot analysis. Results Under OGD-R condition, DF3016A treatment leads to a significant decrease of C5aR gene expression and BDNF protein increase linked to ERK/CREB phosphorylation. BDNF neurotrophic pathway markedly increased the expression of miR132 which, in turn, is regulated by pCREB. Furthermore, in OGD-R neurons miR181 gene expression, a regulatory factor of pro inflammatory cytokines, is inverted by DF3016A treatment. Conclusions These data provide the first evidence that sub-lethal OGD-R is able to induce C5aR gene expression upregulation that is reversed by C5aR antagonist toward control levels. 500nM DF3016A activity can induce neurotrophic pathway that involves BDNF upregulation. Within CNS, the inhibition of a proinflammatory receptor-mediated function of the C5aR can interfere with neuronal stress suggesting a novel therapeutic target for reducing nociceptive inflammation of central neurons. References 1. Müüller-Ladner, et al. 1996. Enhanced expression of chemotactic receptors in multiple sclerosis lesions. J. Neurol. Sci. 144:135 2. O'Barr SA, et al. 2001.Neuronal expression of a functional receptor for the C5a complement activation fragment. J Immunol.;166(6):4154-62. 3. Gasque P., et al. 1997. Expression of the receptor for complement C5a (CD88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system. Am. J. Pathol. 150:31. 4. Zhou J, et al. 2008.Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease. J Neurochem.; 106(5):2080-92.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/117415
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