Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

Abstract

Background: Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.

Results: To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment.

Conclusions: These results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.

Figure 1

Resveratrol suppresses ERK and mTOR signaling in sensory neurons in a concentration-dependent manner. Treatment of TG neurons with resveratrol (10, 30, and 100 μM) for 1 h induces a concentration-dependent increase in phosphorylation of AMPK (A). Resveratrol treatment significantly decreases the phosphorylation of ERK (B), eIF4E (C), AKT (D), mTOR (E), TSC2 (F) 4EBP (G) and rS6p (I) but increases eIF4G phosphorylation (H).

Figure 2

Suppression of ERK and mTOR signaling by resveratrol is time dependent. TG neurons were treated with 100 μM resveratrol for 0, 10, 30, and 100 min. Resveratrol induces an increase in phosphorylation of AMPK (A) maximally with 10 and 30 min treatment. Resveratrol decreases the phosphorylation of ERK (B), eIF4E (C), AKT (D), mTOR (E), TSC2 (F) 4EBP (G) and rS6 (I) and this effect is time-dependent. Resveratrol increased eIF4G phosphorylation (H).

Figure 3

Suppression of ERK and mTOR signaling by resveratrol is reversible. TG neurons were treated with 100 μM resveratrol for 1 h followed by 1 or 2 h washout periods. Effects of resveratrol were reversible in all cases upon 1 h washout.

Figure 4

Resveratrol suppresses eIF4F complex formation in sensory neurons. TG neurons were treated with resveratrol (10, 30 and 100 μM) for 1 h. A) Western blot for eIF4G, 4EBP and eIF4E from trigeminal neurons co-precipitated using 7-methyl-GTP conjugated beads. B) Resveratrol induces a significant increase in 4EBP (negative regulator of translation) association with the cap-binding protein eIF4E in a dose-dependent manner. C) Resveratrol induces a significant decrease in eIF4G association with the cap-binding protein eIF4E (a component of eIF4F complex) in a dose-dependent manner.

Figure 5

Resveratrol blocks IL-6 induced signaling in sensory neurons. TG neuron cultures were pre-treated with resveratrol (100 μM, 15 min) followed by subsequent co-treatment with IL-6 (50 ng/ml, 15 min). Western blot for eIF4E (A) and ERK (B) from TG neurons treated with IL6 and/or resveratrol. Resveratrol blocked IL-6 mediated phosphorylation of eIF4E and ERK in TG cultures.

Figure 6

Local resveratrol blocks IL-6 induced acute allodynia in a dose-related manner. A) Intraplantar injection of IL-6 (0.1 ng) and co-treatment with resveratrol (0.1, 1 and 10 μg) blocks IL-6 induced allodynia. B) Area under the curve (AUC) analysis shows that resveratrol reduces IL-6 induced allodynia in a dose-related manner.

Figure 7

Local resveratrol blocks plantar incision-induced allodynia in a dose-related manner. Animals received a plantar incision on the left hindpaw. Resveratrol (1 μg or 10 μg) or vehicle was injected into the paw around the incision either immediately following incision and 1 day post incision (A, B) or 1 and 3 days following incision (C). A) Resveratrol injection (1 μg or 10 μg) immediately following incision and 1 day post incision significantly blocked plantar incision induced allodynia in a dose-dependent manner. B) Area under the curve (AUC) analysis showing dose-related effects in A. C) Resveratrol injection (10 μg) on day 1 and 3 following incision significantly blocks plantar incision induced allodynia. Red arrows show times of resveratrol injection.

Figure 8

Resveratrol blocks IL-6- and plantar incision-induced persistent sensitization. A) Intraplantar injection of IL-6 (0.1 ng) with resveratrol (10 μg) co-treatment on Day 1 abolished the IL-6 induced persistent sensitization precipitated by PGE₂ injection on day 6. B) Intraplantar injection of resveratrol (10 μg) at the time of incision and 1 day post incision abolished plantar incision-induced persistent sensitization precipitated by PGE₂ injection on day 14 after incision. C) Intraplantar injection of resveratrol (10 μg) on day 1 and 3 post incision abolished plantar incision induced persistent sensitization precipitated by PGE₂ injection on day 14 after incision.