Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

Abstract

The mammalian target of rapamycin (mTOR) is known to regulate cell proliferation and growth by controlling protein translation. Recently, it has been shown that mTOR signaling pathway is involved in long-term synaptic plasticity. However, the role of mTOR under different pain conditions is less clear. In this study, the spatiotemporal activation of mTOR that contributes to pain-related behaviors was investigated using a novel animal inflammatory pain model induced by BmK I, a sodium channel-specific modulator purified from scorpion venom. In this study, intraplantar injections of BmK I were found to induce the activation of mTOR, p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in rat L5-L6 spinal neurons. In the spinal cord, mTOR, p70 S6K and 4E-BP1 were observed to be activated in the ipsilateral and contralateral regions, peaking at 1-2 h and recovery at 24 h post-intraplantar (i.pl.) BmK I administration. In addition, intrathecal (i.t.) injection of rapamycin - a specific inhibitor of mTOR - was observed to result in the reduction of spontaneous pain responses and the attenuation of unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I. Thus, these results indicate that the mTOR signaling pathway is mobilized in the induction and maintenance of pain-activated hypersensitivity.

Figure 1

Immunoreactivity of p-mTOR in the spinal cord dorsal horn after intraplantar BmK I injection. Compared with the saline group (A, B), BmK I treated groups (C-F) showed obvious immunoreactivity of p-mTOR in both ipsi- and contralateral spinal cord dorsal horn.

Figure 2

4E-BP1 is phosphorylated in the spinal cord dorsal horn after intraplantar BmK I injection. Compared with the saline group (A, B), BmK I treated groups (C-F) showed obvious immunoreactivity of p-4E-BP1 in both ipsi- and contralateral spinal cord dorsal horn.

Figure 3

Immunoreactivity of p-p70 S6K in the spinal cord dorsal horn after intraplantar BmK I injection. Compared with the saline group (A, B), the immunoreactivity of p-p70 S6K in both sides of spinal cord dorsal horn increased significantly in BmK I treated groups (C-F).

Figure 4

BmK I-induced changes in spinal levels of p-mTOR, p-4E-BP1 and p-p70 S6K assessed by Western blot. (A) &(F), representative western blots showing levels of p-mTOR, p-4E-BP, p-p70 S6K, total mTOR and β-actin in both ipsi- (A) and contralateral (B) side of the spinal cord. (B-E) &(G-J), histograms represent the mean levels with respect to each control group at different time points after intraplantar BmK I injection. The data are presented as mean ± S.E.M. of three rats per group. *p<0.05, **p<0.01, ***p<0.001, compared with control group by One-way ANOVA, followed by Bonferroni's post hoc test.

Figure 5

Cellular localization of p-mTOR immunoreactivity in the spinal cord dorsal horn. (A-I) Cell-type-specific immunolabeling of p-mTOR in the ipsilateral dorsal horn at 2 h after intraplantar BmK I injection. Arrows indicate colocalization of the p-mTOR (red) with the respective cell markers (green). (J) Histogram of the cellular distribution of p-mTOR.

Figure 6

Cellular localization of p-4E-BP1 immunoreactivity in the spinal cord dorsal horn. (A-I) Cell-type-specific immunolabeling of p-4E-BP1 in the ipsilateral dorsal horn at 2 h after intraplantar BmK I. Arrows indicate colocalization (yellow) of the p-4E-BP1 (red) with the respective cell markers (green). (J) Statistical histogram of the cellular distribution of p-4E-BP1.

Figure 7

Cellular localization of p-p70 S6K immunoreactivity in the ipsilateral spinal cord dorsal horn. (A-I) Cell-type-specific immunolabeling of p-p70 S6K in the ipsilateral dorsal horn at 2 h after intraplantar BmK I. Arrows indicate colocalization (yellow) of the p-p70 S6K (red) with the respective cell markers (green). (J) Statistical histogram of the cellular distribution of p-p70 S6K.

Figure 8

Suppression effect of rapamycin on BmK I-induced rat pain behaviors. (A) Rat flinch behavior was attenuated when rats were pre-treated with rapamycin (20 μM, 200 μM and 2 mM) 30 min before BmK I administration. Suppression of total number of the rat paw flinches (B) and total number of paroxysmal pain-like behaviors (C) by rapamycin (20 μM, 200 μM and 2 mM) during 2 h after BmK I injection. Suppression effect on ipsilateral (E) and contralateral (F) mechanical hypersensitivity, and ipsilateral (G) thermal hypersensitivity by different doses of rapamycin pre-treatment (20 μM, 200 μM and 2 mM). However, rapamycin did NOT affect the lifting and licking behaviors (D) and the contralateral basal thermal threshold value (H). *p<0.05, **p<0.01, ***p<0.001, compared with BmK I group; #p<0.05, ##p<0.01, ###p<0.001, compared with DMSO vehicle group. n=8 for each group.

Figure 9

200 μM rapamycin inhibited the activation of spinal mTOR, 4E-BP1 and p70 S6K assessed by Western blot. (A) &(F), representative western blots showing levels of p-mTOR, p-4E-BP, p-p70 S6K, total mTOR and β-actin in ipsi- (A) and contralateral (B) side of the spinal cord. (B-E) &(G-J), histograms represent the mean levels with respect to each saline-treated group at 2 h after i.pl. BmK I injection. The data are presented as mean ± S.E.M. of three rats per group. N.s., **p<0.01, ***p<0.001, compared with saline-treated group by One-way ANOVA, followed by Bonferroni's post hoc test.

Figure 10

Suppression effect of CCI-779 on BmK I-induced rat pain behaviors. (A) Rat flinch behavior was attenuated when rats were pre-treated with CCI-779 (250 μM) 30 min before BmK I administration. Suppression of total number of the rat paw flinches (B) and total number of paroxysmal pain-like behaviors (C) by CCI-779 during 2 h after BmK I injection. (D) No effects on lifting and licking behaviors. Suppression on ipsilateral (E) and contralateral (F) mechanical, and ipsilateral thermal (G) hypersensitivity by CCI-779, but no effect on contralateral basal thermal threshold value (H). *p<0.05, **p<0.01, ***p<0.001, compared with BmK I group. #p<0.05, ##p<0.01, ###p<0.001, compared with vehicle group. n=8 for each group.

Figure 11

Post-treatment with rapamycin suppressed unilateral thermal hypersensitivity and bilateral mechanical hypersensitivity induced by intraplantar BmK I injection. (A and B) showed the suppressive effects on BmK I-induced the ipsilateral (A) and contralateral (B) mechanical hypersensitivity by different doses of rapamycin post-treatment (20 μM, 200 μM and 2 mM). (C and D) showed that different doses of rapamycin post-treatment (20 μM, 200 μM and 2 mM) suppressed ipsilateral (C) thermal hypersensitivity but did not affect the contralateral basal thermal threshold value (D). **P<0.01, ***P<0.001, compared with relevant control groups treated with DMSO. n=8 for each group.