Intrathecal inhibition of activator protein 1 (AP-1) attenuates Spared Nerve Injury-induced hypernociception by regulating the expression of NO, IL-17A and MMP-2 and -9 in mice.

Rafael Poloni, Miriam Fonseca, Flávia Santa-Cecília, Ieda dos Santos, Rangel Silva, Fernando Cunha, Sérgio Ferreira. Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

Neuropathic pain results from nerve damage or dysfunction, which is associated to the painful process’ chronification (1). This process may include participation of the inducible genes, which may be modulated by transcription factors, including the activator protein-1 (AP-1). The activation of AP-1 induces the production of proinflammatory mediators and matrix metalloproteinases (MMP), which can trigger central and peripheral sensitization. Our hypothesis is that activation of transcription factor AP-1 would participate in the maintenance of neuropathic pain, by inducing the production/release of proinflammatory mediators, e.g. interleukin (IL)-17A, nitric oxide (NO), MMP-2 and -9 in mice spinal cord. Thus pharmacological inhibition of AP-1 may be a potential therapeutic strategy for treating neuropathic pain.

Female C57BL/6 mouse (20-30 g) were housed in temperature-controlled rooms (22-25 °C) with access to water and food ad libitum and 5-8 animals per experimental group. All experiments were conducted in accordance with the Ethics Committee of the Ribeirão Preto Medical School (070/2011, University of São Paulo, Brazil). The animals received inhalatory anesthesia (2% isoflurane) and were submitted to an experimental model of neuropathic pain Spared Nerve Injury (SNI) (2). The animals were treated intrathecally (i.t.) with AP-1 inhibitor SR11302 ((E,E,Z,E)-3-Methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid) in the dose of 3 µg/site or vehicle (DMSO, tween®20 and saline). The mechanical nociceptive evaluation was performed with von Frey filaments. After anesthesia, the spinal cord of the animals were exposed and the segment L4-L5 was removed for evaluating the expression of messenger RNA (mRNA) of proinflammatory mediators were measured by RT-PCR (3) and Griess reaction (4) was applied to determine nitrite/nitrate concentration. Spinal cord samples were also assayed by SDS-polyacrilamide gel electrophoresis gelatin zymography (5) to evaluate MMP-2 and MMP-9 activity. The results are presented as the means ± SEM. One-way ANOVA followed by Bonferroni’s t-test was performed and differences were considered to be statistically significant at P < 0.05. Intrathecal single injection of an inhibitor of AP-1 (SR11302) on day 7 after SNI is enough to attenuated mechanical hypernociception in 3-5 hours after injection (P < 0.05). On the seventh day after SNI, segment L4-L5 of the spinal cords of animals submitted or not to SNI were removed 3 hours after i.t. injection of the inhibitor of AP-1 (SR11302) for RT-PCR and Zymography. The PCR experiments using n = 5 animals per group, whereas the gel zymography used n = 8 animals per group. The results showed that the SNI causes an increase of mRNA expression of IL-17A (1.32 ± 0.30, P < 0.05), inducible NO sintase (1.66 ± 0.71, P < 0.05), MMP-2 (0.88 ± 0.09, P < 0.05) and MMP-9 (1.27 ± 0.17, P < 0.05), compared to the false-operated animals (0.47 ± 0.12; 0.69 ± 0.10; 0.59 ± 0.05; 0.87 ± 0.02) (P < 0.05), respectively. This increase was attenuated when animals were pretreated (3 hours before) with the AP-1 blocker, SR11302 (0.48 ± 0.10; 0.51 ± 0.09; 0.48 ± 0.08; 0.59 ± 0.06, respectively) (P < 0.05). This study shows that the inhibition of transcription factor AP-1 may be a potential analgesic/anti-inflammatory for treating neuropathic pain by reducing the hypernociception and the synthesis of proinflammatory mediators, e.g. IL-17A, NO, MMP-2 and MMP-9.

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(3) Vieira SM et al, J Immunol 188(10):5116, 2012.

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(5) Ceron CS et al, Matrix Biol 31(4):261, 2012.