Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

Abstract

Aims:

The precise mechanisms underlying the pathogenesis of opioid-induced thermal hyperalgesia and tolerance are not yet fully understood.

Methods:

In adult CD-1 mice, repeated morphine treatment was used to examine the expression of the non-canonical pathway of sonic hedgehog signaling, behavioral changes, and neurochemical alterations induced by morphine in the spinal cord and DRG. Additionally, to delve into the underlying mechanisms of the non-canonical pathway of Shh signaling in morphine-induced thermal hyperalgesia (MITH) and tolerance, we utilize the brain-derived neurotrophic factor (BDNF) inhibitor.

Results:

Morphine administration repeatedly resulted in apparent thermal hyperalgesia and tolerance. The initiation and maintenance of MITH and tolerance, as well as related neurochemical alterations, were greatly inhibited by pharmacological and genetic suppression of the mTOR. By blocking the mTOR/p70 ribosomal S6 protein kinase 1 (S6K1)/Gli1 signaling, the morphine-induced increase in BDNF was considerably inhibited. Moreover, mTOR activator injection in naive mice resulted in significant heat hyperalgesia and BDNF upregulation. Suppression of BDNF effectively mitigated the development of thermal hyperalgesia induced by the mTOR activator.

Conclusion:

These findings indicate that the non-canonical pathway of Shh signaling might serve as a crucial mediator in the development of MITH and tolerance through the regulation of BDNF expression.

Introduction

Opioids, including morphine, have long been used in clinics for treating acute and chronic pain. However, chronic usage of morphine undoubtedly results in various side effects including chronic morphine tolerance, dependence and addiction, which further limits its use. ¹ Morphine-induced thermal hyperalgesia (MITH) refers to a phenomenon wherein long-term morphine use paradoxically leads to an increase in pain perception, and tolerance is defined as a progressive decline in the effectiveness and duration of the drug’s action.^2,3 The development of morphine tolerance has been linked to many mechanisms, such as desensitization, phosphorylation and, internalization of the opioid receptor, ⁴ alterations in the glutamate receptor, ⁵ and activation of glial cells. ⁶ Despite decades of research and considerable advance that have been implicated, the precise cellular and molecular mechanisms underlying the pathogenesis of MITH remain unknown. Therefore, further research is required to explore the detailed mechanisms of morphine tolerance to investigate prospective therapeutic targets.

The sonic hedgehog (Shh) pathway is a complex and tightly-regulated signal transduction pathway that can be simplified into 4 main components: Shh ligand, patched (Ptch), smoothened (Smo), and Gli transcription factors.^7,8 Shh signaling is essential for cell fate specification, ⁹ axon guidance,^10,11 growth and differentiation. ¹² Shh can signal through a canonical and non-canonical way. The “canonical” pathway of Shh signaling transduction includes (i) binding of Shh to Ptch. In the absence of the ligand Shh, the activity of the Smo is repressed by the receptor Ptch1 and restricted to intracellular endocytic vesicles; (ii) activation of Smo; (iii) activation of Gli transcription factors; (iv) and regulation of cellular responses to Shh. Ultimately, this results in Gli-mediated transcriptional regulation of genes producing Shh pathway components as well as molecules outside the Shh pathway, which regulate a number of cellular functions. ¹³ Typically, the “non-canonical” pathway of Shh signaling happens via Gli-independent pathways. ¹⁴ For the non-canonical Shh signaling, the mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase expressed on mammalian neurons and is widely expressed in a variety of biological cells. ¹⁵ Moreover, it encourages nuclear translocation and increases Gli1 activity via the mTOR/p70 ribosomal S6 protein kinase 1 (S6K1) signaling cascade. ¹⁶ Emerging evidence has demonstrated the significant involvement of mTOR activation in various chronic pain conditions.^17–19 Moreover, mTOR plays an important role as a translation initiation regulator by regulating the growth and regeneration of pain-related axons, local translation of mRNA in dendrites, and local protein translation at synaptic sites in the nervous system. ²⁰

Our previous study provided for the first time that the “canonical” pathway of the Shh signaling pathway was involved in the development of cancer-induced bone pain. ²¹ Moreover, our previous study and experiments in other laboratories illustrated the critical role of the “canonical” pathway of Shh signaling in the development of opioid-induced hyperalgesia and tolerance.^22,23 We found that the administration of cyclopamine, an inhibitor of the hedgehog signaling pathway that inhibits Smo activation, resulted in a delay in the development of morphine tolerance. However, it did not exert a significant effect on already-established morphine tolerance. Conversely, when mTOR inhibitor KU-0063794, another inhibitor targeting S6K1 activation, was administered, it not only suppressed the development of morphine tolerance but also effectively suppressed the maintenance of morphine tolerance. ²² This phenomenon suggested that there may be other factors contributing to S6K1/Gli1 activation. Thus, we hypothesized that the non-canonical Shh signaling, especially mTOR/S6K1/Gli1 signaling, is also involved in the regulation of morphine tolerance. However, the role of the “non-canonical” pathway of Shh signaling in nociceptive regulation remains unclear.

Materials and methods

Experimental design

A total of 211 mice has been used in this study. Ten experiments were performed. (Experiment 1): Twenty-four mice have been randomized to control and MITH groups. Firstly, the MPE% and thermal withdrawal latency was measured (n = 6 in control for MPE%; n = 6 in control for thermal thresholds; (n = 6 in MITH for MPE%; n = 6 in MITH for thermal thresholds). MITH model was induced as previously described. ²² Briefly, morphine (10 mg/kg, i.p.) was injected twice a day for 7 consecutive days. The sham mice were injected with sterile saline (1 mL, i.p.) at the same time points as the control. (Experiment 2): Then, the expression of p-mTOR/p-S6K1/Gli1 signaling in the spinal and DRG after chronic morphine injection was investigated, separately. Twelve mice were randomized to control and MITH groups (n = 6 each group). (Experiment 3): As with western blot experiments, immunofluorescence staining studies were used to access the cellular localization of p-mTOR/p-S6K1/Gli1 signaling in the spinal cord dorsal horn (n = 4 each group). (Experiment 4) To assess the effect of mTOR inhibitor on MITH-induced pain behaviors, ninety-six mice have been randomized to control, morphine, morphine +DMSO and morphine+KU groups. We explored the effect of mTOR inhibitor KU-0063794 at the early stage and the late stage on MITH-induced pain behaviors (n = 6 per group). (Experiment 5) We assess the effect of KU-0063794 on the expression and activation of p-mTOR, p-S6K1, Gli1 and BDNF in the spinal cord. Besides, the expression of c-fos and CGRP in the spinal cord of mice after injection of KU-0063794 has been investigated (n = 6 per group). (Experiment 6) We further determined the effect of mTOR siRNA on MITH-induced pain behaviors. Thirty-six mice have been randomized to Sham+siRNA, morphine+con-siRNA and morphine+ siRNA groups. (Experiment 7) We assess the effect of mTOR siRNA on the expression and activation of p-mTOR, p-S6K1, Gli1 and BDNF in the spinal cord (n = 6 per group). (Experiment 8) To assess the effect of mTOR agonist L-leucine on the effect of KU-0063794, eighteen mice have been randomized to Vehicle, Leu and Leu+KU. The thermal withdrawal latency was measured (n = 6 per group). (Experiment 9) To assess the effect of mTOR/S6K1/Gli1 signaling pathway are involved in MITH and tolerance, thirty-six mice was used to test the effect of S6K1 or Gli1 inhibitors on L-leucine-induced MITH and tolerance. (Experiment 10) To assess the effect of BDNF inhibitor K252 on the effect of L-leucine, eighteen mice have been randomized to Vehicle, Leu and Leu+K252. The thermal withdrawal latency was measured (n = 6 per group). (Experiment 11) To assess the effect of BDNF inhibitor K252 on the effect of morphine, thirty-six mice have been randomized to Vehicle, morphine+DMSO and morphine+K252. The mechanical and thermal withdrawal latency was measured (n = 6 per group). In experiment 11, one mouse was excluded from the experiment because of unsuccessful catheterization.

Animals and ethical statement

Adult male CD-1 mice (23–25 g) were purchased from Xuzhou Medical College, Jiangsu, China. All experimental procedures were approved by the Institutional Animal Care and Use Committees of Xuzhou Medical University and performed in accordance with the ethics committee of the International Association for the Study of Pain. Researchers responsible for behavioral testing were blinded to both the treatment and group allocations.

Behavioral tests

Pain-related behaviors were determined by measurement of thermal withdrawal latency (TWL) using hot plate test as previously described. ²² To assess thermal hyperalgesia, Hargreaves tests were performed following established protocols. ²⁴ In brief, prior to the test, mice were individually placed in transparent compartments with a glass floor and allowed to acclimate for 30 min. A radiant heat source set to 50% intensity was then positioned beneath the hindpaw. Once the mice’s hind paw movement was detected, the stimulation promptly ceased, and the corresponding data were recorded.

Drug injection

The mTOR inhibitor KU-0063794 purchased from MedChemExpress (Shanghai, China) was diluted in 5% dimethyl sulfoxide (DMSO) at the concentration of 1mg/mL. KU-0063794 was administered intraperitoneally (i.p.) at 8mg/kg. The mTOR activator L-leucine purchased from MedChemExpress (Shanghai, China) was diluted in saline at the concentration of 2 mg/mL. L-leucine was administered i.p. at 50 mg/kg. The selective S6K1 inhibitor PF-4708671 and GANT-61 was purchased from MedChemExpress (Shanghai, China). 50 mM PF-4708671 (0.5 mL) was injected i.p. at 40 mg/kg per mouse as previous reported.^25,26 1 mM GANT-61 (10 μL) was injected intrathecally (i.t.) per mouse.

The BDNF inhibitor K252 purchased from MedChemExpress (HY-N6732, Shanghai, China) was diluted in 5% DMSO at a concentration of 0.4 mg/mL. The mTOR siRNA was synthesized by GenePharma (Suzhou, China). siRNA duplexes that specifically targeted mTOR were: sense 5′-CCACCAGAAUUGGCAGAUUTT-3′, and anti-sense 5′-AAUCUGCCAAUUCUGGUGGTT-3′ As a control siRNA, scramble siRNA was manufactured by a scrambled sequence of nucleotides. Scramble siRNA was: sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′. The siRNA was dissolved in 25 μL DEPC water at 1 μg/μL before injection. K252 and the mTOR siRNA were injected i.t. with 5 μg per mouse.

Western blot

To obtain the supernatant, the L4-6 spinal cord section and DRG of mice were immediately removed and homogenized. A total of 40 μg protein extractions were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (0. 2 μm, Millipore, USA). The primary antibodies used were as follows: anti-mTOR (1:1000; #2983, Cell Signaling Technology, MA, USA), anti-p-mTOR (1:500; #5536, Cell Signaling Technology, MA, USA), anti-S6K1 (1:1000; ab32529, Abcam, Cambridge, UK), anti-p-S6K1 (1:1000; #9204, Cell Signaling Technology, MA, USA), anti-Gli1 (1:1000; ab49314, Abcam, Cambridge, UK), BDNF (1:1000; ab108319, Abcam, Cambridge, UK), anti-c-fos (1:1000; ab222699, Abcam, Cambridge, UK), anti-CGRP (1:1000; #14959, Cell Signaling Technology, MA, USA), anti-Histone H3 (17168-1-AP, Proteintech, Wuhan, China), and anti-β-actin(1:10000; Abcam). The blots were incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for a duration of 2 h. Detection of the protein bands was carried out using an electrochemiluminescence (ECL) solution obtained from MedChemExpress (Monmouth Junction, NJ, USA). Subsequently, the blots were scanned and analyzed using the Molecular Imager (Gel DocTM XR, Bio-Rad Laboratories Inc., CA, USA) System for further analysis. For western blot analysis, proteins were normalized to β-actin, which was used as a loading control.

Immunofluorescence staining

Under isoflurane (2.5%) anesthesia, the mice were perfused intracardially with 60 mL 0.1 M PBS followed by 60 mL 4% ice-cold paraformaldehyde (PFA) in PBS. The L4-6 spinal cord segments and DRG were extracted and fixed in 4% PFA overnight at 4°C before being equilibrated in 30% sucrose for 2 d. The collected spinal cord and DRG sections were cryosectioned at 20 μm on a cryostat (Leica, Wetzlar, Germany). After washing for 5 min with PBS 3 times, the slides were penetrated with 0.3% TritonX-100 for 15 min before being blocked with 10% donkey serum for 2 h at room temperature. The slices were then treated with the following antibodies for 24 h at 4°C: p-mTOR (1:100, #5536, Cell Signaling Technology, MA, USA), p-S6K1 (1:100, #9204, Cell Signaling Technology, MA, USA), Gli1 (1:200, ab49314, Abcam, Cambridge, UK), anti-c-fos (1:500; ab222699, Abcam, Cambridge, UK), anti-CGRP (1:500; #14959, Cell Signaling Technology, MA, USA), anti-neuronal nuclei antibody (NeuN; 1:100, ab104224, Abcam, Cambridge, UK), anti-glial fibrillary acidic protein antibody (GFAP; 1:300, #3670, Cell Signaling Technology, MA, USA) and anti-Iba1 antibody (1:100, ab5076, Abcam, Cambridge, UK). After washing 3 times with PBST, the sections were incubated with the secondary antibodies for 2 h at room temperature. After the sections were rinsed with PBST for 30 min (10 min each time), the sections were scanned by laser confocal microscope (FV1000, Olympus, Japan).

Statistical analysis

All data are presented as means ± SEM. GraphPad Prism version 8.0 was used for all of the statistical analyses. Continuous outcomes were analyzed with a one-way ANOVA test. Categorical variables were tested using the χ2 test or Fisher’s exact test. Western blot and qPCR results were conducted using repeated-measure of one-way ANOVA. The thermal withdrawal latency was analyzed by two-way repeated-measure ANOVA. P values less than 0.05 were considered to be statistically significant.

Results

mTOR/S6K1/Gli1 signaling is upregulated and activated in the spinal cord and DRG of MITH mice

The pain-related behaviors were assessed following chronic morphine treatment. The MPE% was significantly down-regulated from d 3 to d 7 compared with the sham group. Moreover, mice developed apparent thermal hyperalgesia on d 5 after morphine injection compared with the sham group, which lasted at least until d 11 (Supplementary Figure 1). To assess the role of non-canonical Shh signaling, we began by investigating the mRNA and protein level of mTOR, S6K1 and Gli1 in the spinal cord and DRG. RT-PCR and western blot results showed that the mRNA expression of p-mTOR, p-S6K1 and Gli1 genes in the spinal cord was significantly increased in the morphine-treated group. Parallel to the elevated protein levels, immunofluorescence results showed that p-mTOR, p-S6K1, and Gli1 were scarcely detectable in sham mice but were induced in morphine-treated mice in the spinal cord dorsal horn (Figure 1). We also examined the expression of p-mTOR, p-S6K1 and Gli1 in the DRG following morphine injection. Interestingly, p-mTOR and p-S6K1 were significantly upregulated from d 1 and maintained a high level until d 7 after morphine administration. Gli1 was significantly increased from d 3 and maintained at a high level from d 3 to d 7 after morphine administration in the DRG. In consistent with the elevated protein levels, immunofluorescence results revealed that p-mTOR, p-S6K1, and Gli1 were induced in morphine-treated mice in the DRG compared with the sham-treated mice (Figure 2).

Figure 1.

The expression of p-mTOR/p-S6K1-Gli1 signaling in the spinal cord after chronic morphine injection. (a) Western blotting showed the time course of mTOR, p-mTOR, S6K1, p-S6K1 and nucleus Gli1 after chronic morphine injection in the spinal cord (n = 6 per group, ^*p < 0.05, ^**p < 0.01 compared with the Sham group). (b) Immunofluorescence staining results showed the expression of p-mTOR, p-S6K1 and Gli1 after chronic morphine injection in the spinal cord. (n = 6 per group, Scale bar = 100 μm).

Figure 2.

The expression of p-mTOR/p-S6K1/Gli1 signaling in the DRG after chronic morphine injection. (a) Western blotting showed the time course of mTOR, p-mTOR, S6K1, p-S6K1 and nucleus Gli1 after chronic morphine injection in the DRG. (n = 6 per group, ^*p < 0.05, ^**p < 0.01 compared with the Sham group). (b) Immunofluorescence staining results showed the expression of p-mTOR, p-S6K1 and Gli1 after chronic morphine injection in the DRG. (n = 6 per group, Scale bar = 50 μm).

Previous studies demonstrated that Gli1 is a transcriptional effector of Shh signaling, which transfers to the nucleus to mediate Shh signaling transduction.^7,8 To assess the activation of non-canonical Shh signaling, the expression of Gli1 in the nuclear extract was examined. Western blotting data showed that the nuclear translocation of Gli1 in the spinal cord and DRG significantly increased from d 3 to d 7 after morphine injection (Figures 1 and 2). These results showed that the non-canonical Shh signaling pathway (mTOR/S6K1/Gli1 signaling) was significantly increased and activated both in the spinal cord and DRG after chronic morphine injection.

Localization of p-mTOR, p-S6K1 and Gli1 in the spinal cord dorsal horn

To explore the location of p-mTOR, p-S6K1 and Gli1 after chronic morphine treatment, double labelling immunofluorescence with neurons, astrocytes and microglia was performed in the spinal cord dorsal horn, respectively. Results showed that p-mTOR was mainly colocalized with neurons and barely colocalized with astrocytes and microglia in the spinal cord dorsal horn. Consistently, p-S6K1 and Gli1 were mainly colocalized with neurons and a minority with astrocytes and microglia in the spinal cord dorsal horn after chronic morphine treatment (Figure 3).

Figure 3.

Cellular localization of p-mTOR/p-S6K1/Gli1 signaling in the spinal cord dorsal horn after chronic morphine treatment. p-mTOR/p-S6K1/Gli1 immunoreactivity in the ipsilateral dorsal horn was mostly detected in neurons (NeuN) after chronic morphine treatment. (n = 4 per group, Scale bar = 100 μm).

mTOR inhibitor suppressed MITH and tolerance

To determine the role of non-canonical Shh signaling in the initiation of morphine-induced tolerance and MIH, the mTOR selective inhibitor KU-0063794 (4 mg/kg, 8 mg/kg, 16 mg/kg, i.p., twice a day) was injected 30 min before morphine injection at d 1, d 2, and d 3. The behavioral tests showed that treatment with 8 mg/kg and 16 mg/kg KU-0063794 significantly delayed the decrease of MPE% and attenuated thermal hyperalgesia compared with the morphine-injected group. However, treatment with 4 mg/kg KU-0063794 has no significant effect on MPE% and thermal hyperalgesia compared with the morphine-injected group (Figure 4(a) and (c)). These results showed that mTOR inhibitors markedly delayed and inhibited the generation of MITH and tolerance.

Figure 4.

Treatment with mTOR inhibitor significantly attenuated chronic morphine injection-induced pain behaviors. (a) Treatment with 8 mg/kg and 16 mg/kg mTOR inhibitor KU-0063794 at the early stage (from d 1 to d 3) effectively delayed the decrease of MPE%. (b) Treatment with 8 mg/kg and 16 mg/kg mTOR inhibitor KU-0063794 at the late stage (from d 4 to d 6) significantly delayed the decrease of MPE%. (c) Early treatment with 8 mg/kg and 16 mg/kg KU-0063794 at the early stage (from d 1 to d 3) effectively mitigated thermal withdrawal latency induced by repeated morphine injections. (d) Treatment with 8 mg/kg and 16 mg/kg KU-0063794 at the late stage (from d 4 to d 6) significantly mitigated thermal withdrawal latency induced by repeated morphine injections. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the sham group, ^#p < 0.05, ^##p < 0.01 compared with the morphine group). (e) Treatment with 8mg/kg KU-0063794 significantly suppressed the expression and activation of p-mTOR, p-S6K1 and Gli1 in the spinal cord by mTOR agonist L-leucine. (n = 6 per group, **p < 0.01 compared with the morphine + DMSO group, ^#p < 0.05, ^##p < 0.01 compared with the group treated with L-leucine).

To determine the role of non-canonical Shh signaling in the maintenance of MITH and tolerance, KU-0063794 (4 mg/kg, 8 mg/kg, 16 mg/kg, i.p., twice a day) was injected 30 min before morphine injection on d 5, d 6, and d 7. The behavioral tests showed that treatment with 8 mg/kg and 16 mg/kg KU-0063794 significantly inhibited and reversed the decrease of MPE%. Moreover, thermal hyperalgesia was effectively inhibited compared with the morphine-injected group (Figures 4(b) and (d)). These results showed that mTOR inhibition significantly inhibited the maintenance of MITH and tolerance. We further examined the expression of p-mTOR, p-S6K1 and Gli1 in the spinal cord, results showed that treatment with KU-0063794 significantly decreased the expression of p-S6K1 and Gli1 in the spinal cord. However, there was no significant change in p-mTOR and S6K1 expression compared with the morphine-treated group. These results showed that mTOR inhibitors significantly inhibited the maintenance of MITH and tolerance.

mTOR inhibition suppressed neurochemical signs in the spinal cord after chronic morphine injection

Our previous study and studies from other laboratories showed chronic morphine exposure significantly increased the expression of neurochemical signs in the spinal cord, including c-fos and CGRP.^22,27,28 To determine the role of mTOR inhibitor in MITH and tolerance, KU-0063794 was injected from d 5 to d 7. The expression of p-mTOR, mTOR, p-S6K1, S6K1 and nucleus Gli1 in the spinal cord was examined. These results showed that p-mTOR, p-S6K1 and nucleus Gli1 in the spinal cord were significantly inhibited following the KU-0063794 injection. In contrast, there is no significant difference in the expression of mTOR and S6K1 in the spinal cord (Figure 4(e)). Consistently, the present study showed that repeated morphine injection significantly increased the expression of c-fos and CGRP compared with the sham group. However, pretreatment with the mTOR selective inhibitor KU-0063794 significantly inhibited the upregulation of c-fos and CGRP in the spinal cord (Supplemental Figure 2). These results showed that non-canonical Shh signaling inhibition prevented the upregulation of neurochemical signs.

mTOR siRNA suppressed MITH and tolerance

To further confirm the critical role of the mTOR/S6K1/Gli1 signaling pathway in MITH and tolerance, mTOR siRNA was used to knockdown mTOR genes in naïve mice. The knockdown effect of mTOR siRNA was examined by western blot after 3 d of injection (1 μg/5μL, i.t., from d 1 to d 3). Results showed that the knockdown of mTOR significantly inhibited the expression of mTOR (Figure 5(a)). Moreover, pretreatment with mTOR siRNA significantly prevented morphine-induced MPE% decrease compared with the scramble siRNA + morphine group. In contrast, the normal pain sensation was not changed in sham mice treated with mTOR siRNA (Figure 5(b) and (c)). These results showed that mTOR inhibition significantly suppressed the initiation and maintenance of MITH and tolerance.

Figure 5.

Treatment with mTOR siRNA significantly attenuated chronic morphine injection-induced pain behaviors. (a) Western blot results showed the expression of mTOR in the spinal cord of mice after injection of mTOR siRNA. (n = 6 per group, **p < 0.01 compared with the con-siRNA group). (b) Treatment with mTOR siRNA from d 1 to d 3 before morphine injection significantly reduced the decline in morphine’s maximum possible analgesic effect. (c) Treatment with mTOR siRNA from d 1 to d 3 before morphine injection significantly attenuated thermal withdrawal latency induced by repeated morphine injections. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Morphine + con-siRNA group).

Activation of mTOR/S6K1/Gli1 signaling contributing to MITH and tolerance by regulating BDNF in the spinal cord

Consistent with our previous study, the present study showed that BDNF significantly increased from d 3 and was maintained at least 5 d after repeated morphine injections in the spinal cord (Figure 6(a)). Treatment with KU-0063794 significantly inhibited the upregulation of BDNF following morphine injection in the spinal cord (Figure 6(b)). Moreover, compared with the control siRNA-treated mice, the delivery of mTOR siRNA significantly inhibits the upregulation of BDNF following morphine injection in the spinal cord (Figure 6(c)). These results illustrated that BDNF was involved in mTOR-mediated MITH and tolerance.

Figure 6.

The expression of BDNF in the spinal cord of mice following chronic morphine injection. (a) Western blotting showed the time course of BDNF after chronic morphine injection in the spinal cord. (b) Western blotting showed the time course of BDNF after chronic morphine injection in the DRG. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Sham group). (c) Treatment with mTOR inhibitor KU-0063794 significantly reduced morphine injection-induced BDNF upregulation. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Sham group, ^##p < 0.01 compared with the morphine + DMSO group). (d) Treatment with mTOR siRNA significantly reduced morphine injection-induced BDNF upregulation. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Sham group, ^#p < 0.05 compared with the morphine + con-si-RNA group). (e) Injection of mTOR agonist L-leucine induced an upregulation of the protein expression of BDNF. (n = 6 per group, **p < 0.01 compared with the Vehicle group, ^##p < 0.01 compared with the L-leucine-treated group). (f) Pharmacological S6K1 inhibition by PF-4708671 significantly reversed L-leucine-induced thermal hyperalgesia. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Vehicle group, ^#p < 0.05, ^##p < 0.01 compared with the L-leucine-treated group). (g) Pharmacological Gli1 inhibition by GANT-67 significantly reversed L-leucine-induced thermal hyperalgesia. (n = 6 per group, *p < 0.05, **p < 0.01 compared with the Vehicle group, ^#p < 0.05, ^##p < 0.05 compared with the L-leucine-treated group). (h) Treatment with mTOR inhibitor KU-0063794 significantly reversed L-leucine-induced BDNF upregulation. (n = 6 per group, **p < 0.01 compared with the Vehicle group, ^##p < 0.01 compared with the L-leucine group). (i) Treatment with BDNF inhibitor K252 significantly reduced the decline in morphine’s maximum possible analgesic effect. (n = 6 per group, **p < 0.01 compared with the Vehicle group). (j-k) Treatment with BDNF inhibitor K252 significantly delayed the decrease of MPE% and suppressed thermal withdrawal latency induced by mTOR agonist L-leucine. (n = 6 per group, **p < 0.01 compared with the morphine + DMSO group, ^#P < 0.05, ^##p < 0.01 compared with the group treated with L-leucine).

To further investigate the role of mTOR in MITH and tolerance, mTOR agonist L-leucine was injected in naïve mice. Results showed that the L-leucine induced thermal hyperalgesia and caused a significant increase of BDNF in the spinal cord of naïve mice, which was reversed by mTOR inhibitor KU-0063794 (Figure 6(d)–(e)). These results illustrated that mTOR plays an essential role in MITH and tolerance by regulating BDNF in the spinal cord.

To further investigate the processes by which MITH and tolerance are induced by mTOR/S6K1/Gli1 signaling, mTOR agonist L-leucine was injected in naïve mice. To evaluate the role of S6K1 in L-leucine-induced thermal hyperalgesia, ²⁹ the selective inhibitor PF-4708671 was administrated 1h after L-leucine injection. Results showed that pharmacological S6K1 inhibition by PF-4708671 significantly reversed L-leucine-induced thermal hyperalgesia (Figure 6(f)). Similarly, to evaluate the role of Gli1 in L-leucine-induced thermal hyperalgesia, the selective inhibitor GANT-67 was administrated 1h after L-leucine injection. Results showed that pharmacological Gli1 inhibition by GANT-67 significantly reversed L-leucine-induced thermal hyperalgesia (Figure 6(g)). Moreover, mTOR agonist L-leucine induced thermal hyperalgesia and caused a significant increase of BDNF in the spinal cord of naïve mice, which was reversed by BDNF antagonist K252 (Figure 6(h) and (i)). Moreover, K252 significantly inhibited thermal hyperalgesia and the reduction of morphine-induced MPE% (Figure 6(j) and (k)). These results illustrated that activation of mTOR signaling contributes to MITH and tolerance by regulating BDNF in the spinal cord.

Discussion

In this study, we suggest that activation of mTOR/S6K1/Gli1 contributes to MITH and tolerance via both peripheral and central mechanisms. In the present study, p-mTOR, p-S6K1 and nuclear Gli1 were significantly elevated and activated after chronic morphine injection both in the DRG and spinal cord. Pharmacological and genetic suppression of the mTOR significantly suppressed the initiation and maintenance of MITH and tolerance, as well as related neurochemical alterations. Moreover, chronic morphine use resulted in BDNF upregulation. By blocking the non-canonical pathway of Shh signaling, the morphine-induced increase in BDNF was considerably downregulated. Additionally, mTOR activator injection in naive mice resulted in significant heat hyperalgesia and BDNF upregulation. Suppression of BDNF effectively mitigated the development of hyperalgesia induced by the mTOR activator. These results provide a novel understanding of the pathogenesis of MITH and tolerance and suggested that non-canonical Shh signaling may be a potential target for alleviating MITH and tolerance.

In several areas of the nervous system, Shh signaling is recognized to be crucial for synaptic plasticity and neuronal excitability.^8,30 Our previous study showed that canonical Shh/Smo/Gli1 signaling in the spinal cord is involved in the generation of morphine tolerance. ²² However, the role of the non-canonical Shh signaling pathway, mTOR/S6K1/Gli1, in the modulation of morphine tolerance has not been investigated. For the non-canonical Shh signaling, mTOR is a serine/threonine protein kinase which encourages nuclear translocation and increases Gli1 activity via mTOR/S6K1 signaling cascade. ¹⁶ mTOR signaling is involved in the initiation and maintenance of pain hypersensitivity induced by bone cancer.^19,31 Moreover, a previous study demonstrated significant activation of mTOR and S6K1 in the spinal cord in the context of chronic inflammatory pain but not spinal nerve ligation (SNL)-induced neuropathic pain. ³² In contrast, Zhang et al. employed the chronic constriction injury (CCI) model to investigate the involvement of mTOR in the development of neuropathic pain. They observed an upregulation of phosphorylated mTOR and S6K in the spinal cord at both 7 and 14 d following CCI. ³³ The findings of these studies indicate that various types of peripheral nerve injuries may exhibit differential regulation in terms of activating the mechanistic target of mTOR and its downstream effectors within the spinal cord and DRG.

In the present study, we first examined the expression and activity of mTOR and its downstream effectors p-S6K1 and nucleus Gli1 following chronic morphine injection in the spinal cord and DRG. We observed that repeated administration of morphine led to the activation of mTOR and its downstream effectors in the spinal dorsal horn and DRG. Moreover, nuclear expression of Gli1 also exhibited a time-dependent increase during the development of morphine tolerance in the spinal dorsal horn and DRG. Immunofluorescence staining revealed that the increased expression of p-mTOR, p-S6K1, and Gli1 was mainly observed in laminae I and II, as well as IV and V, of the spinal dorsal horn. Laminae I and II are primarily associated with the projection of C-fibers, while laminae IV and V are mainly associated with the projection of Aδ fibers, both of which are involved in the transmission of nociceptive information. ³⁴ Moreover, our immunofluorescence experiments further confirmed the predominant expression of non-canonical Shh signaling pathway-related proteins in spinal cord dorsal horn neurons, suggesting a possible mechanism by which this pathway regulates morphine tolerance through the modulation of neuronal excitability and synaptic plasticity. ³⁵ Notably, it has been demonstrated that mTOR and S6K1 contribute to the remodeling of dendritic spines and synapses by regulating local mRNA translation, potentially enhancing the sensitivity and responsiveness of nociceptive transmission neurons in the post-synaptic dorsal horn of the spinal cord,^36–39 thereby reducing the analgesic effects of morphine. Furthermore, activated Gli1 translocated to the nucleus, binding to its DNA binding site and synergistically regulating synaptic plasticity by promoting the expression of BDNF, collectively contributing to morphine tolerance.

Our previous research has demonstrated that the Shh signaling pathway is activated and contributed significantly to the development of morphine tolerance and chronic pain.^21,22 Specifically, in bone cancer pain, there is an upregulation in the expression and activation of the Shh canonical signaling pathway. Inhibiting this pathway with cyclopamine effectively reduces the production and maintenance of bone cancer pain in the spinal cord dorsal horn. ²¹ The Shh signaling pathway modulates bone cancer pain by regulating neuronal excitability, with inhibition of the pathway leading to a reduction in intracellular Ca²⁺ concentrations and downregulation of neuronal excitability. ²¹ Similarly, in a chronic morphine tolerance model, we observed an upregulation in the expression and activation of the Shh canonical pathway in both the peripheral and central nervous systems as morphine tolerance developed. Inhibiting this pathway effectively suppressed the development of morphine tolerance and prevented the altered neurochemistry observed in the dorsal horn of the spinal cord. ²² In this study, a specific inhibitor of mTOR, KU-0063794, was injected into MITH and tolerance model. KU0063794 is a second-generation mTOR inhibitor that primarily targets the phosphorylation of S6K1, an mTOR downstream substrate. ⁴⁰ The findings from this study demonstrate that the administration of KU0063794 effectively suppressed the initiation and maintenance of MITH and tolerance. Additionally, we utilized mTOR siRNA to achieve knockdown of the mTOR gene. Consistent with a previous study, our present study showed that mTOR inhibition effectively reduced the decline in morphine’s maximum possible analgesic effect and mitigated mechanical threshold and thermal latency induced by repeated morphine injections, no matter at the early stage or the late stage. ¹⁸ Our findings, combined with previous research, highlight the pivotal role of the Shh signaling pathway in neuropathic pain, morphine tolerance, and related processes. Through our previous study and the support of other scholars, we confirmed that the Shh signaling pathway plays a pivotal role in morphine tolerance and chronic pain status.

We further investigated the role of the non-canonical Shh signaling pathway in the regulation of morphine tolerance. BDNF is a neurotrophic factor that plays a critical role in modulating neuronal function and synaptic plasticity. ⁴¹ Moreover, our previous study showed that BDNF is an important mediator for canonical Shh signaling in MITH and tolerance. ²² Furthermore, recent studies have revealed that BDNF can activate mTOR-mediated signaling pathways. Activation of the mTOR signaling pathway by BDNF has been implicated in various physiological processes, including pain perception, memory formation, and depression.^42,43 In our current study, we observed a significant increase in BDNF protein expression in the spinal cord following repeated morphine administration. However, the upregulation of BDNF expression induced by repeated morphine injections was markedly suppressed by the early administration of KU-0063794, an inhibitor of the non-canonical Shh signaling pathway. Similarly, the knockdown of the mTOR gene with siRNA significantly attenuated the upregulation of BDNF expression caused by repeated morphine use.

Additionally, we found that in naive mice, administration of L-leucine, an mTOR agonist, resulted in a significant elevation in BDNF expression. Conversely, pre-administration of KU-0063794 followed by L-leucine injection effectively hindered the upregulation of BDNF expression induced by L-leucine. Moreover, the BDNF inhibitor substantially alleviated nociceptive hypersensitivity resulting from repeated morphine injections. Collectively, these results suggest that the non-canonical Shh signaling pathway modulates BDNF expression.

In conclusion, this study unveils the significant role of the non-canonical Shh signaling pathway in the development and maintenance of morphine tolerance, thereby contributing to a novel understanding of its pathogenesis. Subcutaneous administration of morphine repeatedly triggers the activation of the non-canonical Shh signaling pathway, resulting in enhanced activation and upregulation of non-canonical Shh signaling pathway-associated proteins. Conversely, inhibition of this pathway markedly delays and diminishes the development of morphine tolerance and nociceptive hypersensitivity. Furthermore, our previous investigation identified the involvement of the non-canonical Shh pathway in morphine tolerance, specifically through the induction of BDNF expression. In contrast, the present experiment reveals the engagement of the non-canonical Shh pathway in both the production and maintenance of morphine tolerance via BDNF induction. Thus, we hypothesize that BDNF acts as a mediator bridging the interaction between the canonical and non-canonical Shh pathways, with the canonical Shh signaling pathway further augmenting the activation of the non-canonical Shh pathway through BDNF, thereby amplifying the signals of both pathways. This intricate interplay leads to the persistence and sustained development of morphine tolerance, warranting further exploration. Our study has some limitations, we have observed that the mechanical hyperalgesia after chronic morphine exposure was decreased, however, we did not explore the role of mTOR/S6K1/Gli1 signaling in the morphine-induced mechanical hyperalgesia. Nevertheless, two methods were used to strongly indicated that the mTOR/S6K1/Gli1 pathway plays a key role in morphine-induced heat hyperalgesia.

Supplemental Material

sj-pdf-1-mpx-10.1177_17448069251376198 – Supplemental material for Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

Supplemental material, sj-pdf-1-mpx-10.1177_17448069251376198 for Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance by Xing-He Wang, Long Wang, Long Yang, Yang Bai, Ling-Fei Xu, Miao-Miao Li, Yu-Cheng Liu, Jia Sun and Su Liu in Molecular Pain

Supplemental Material

sj-pdf-2-mpx-10.1177_17448069251376198 – Supplemental material for Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance

Supplemental material, sj-pdf-2-mpx-10.1177_17448069251376198 for Inhibition of mTOR/S6K1/Gli1 signaling alleviates morphine-induced thermal hyperalgesia and tolerance by Xing-He Wang, Long Wang, Long Yang, Yang Bai, Ling-Fei Xu, Miao-Miao Li, Yu-Cheng Liu, Jia Sun and Su Liu in Molecular Pain