Activating Sirt1 by resveratrol suppresses Nav1.7 expression in DRG through miR-182 and alleviates neuropathic pain in rats

Abstract

Neuropathic pain is clinically unsatisfactorily treated because of unclear mechanisms. The present study aims to explore the concrete mechanisms underlying the alleviation of resveratrol-activated silent information regulator 1 (Sirt1) to chronic constriction injury (CCI)-induced neuropathic pain. CCI surgery was conducted to the unilateral sciatic nerve of male Sprague-Dawley rats to induce neuropathic pain experimentally. Resveratrol with or without miR-182 antagomir were administered to CCI rats via intrathecal catheter. Behavioral tests including paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were conducted to explore mechanical allodynia and thermal hyperalgesia. Western blot, qRT-PCR were used to detect the expression levels of Sirt1, miR-182, and Nav1.7 in CCI dorsal root ganglions (DRGs). CCI rats displayed lower PWT and PWL compared with the sham control. Also, the CCI DRGs displayed lower Sirt1 and miR-182 expression as well as higher Nav1.7 expression, which would be almost reversed by resveratrol treatment for 4 successive days. We also found that miR-182 expression inhibition erased the analgesia effect of resveratrol to CCI-induced neuropathic pain possibly through upregulating Nav1.7 expression. In summary, resveratrol alleviated CCI-induced neuropathic pain, possibly through activating Sirt1 to suppress Nav1.7 expression via upregulating miR-182 expression in CCI DRGs.

Keywords: Nav1.7; Neuropathic pain; Sirt1; miR-182; resveratrol.

Figure 1.

Sirt1 expression was significantly decreased in CCI–induced neuropathic pain model. (a-d) Ipsilateral and contralateral mechanical (a, c) and thermal (b, d) sensitivity in sham and CCI groups. (e) Sirt1 mRNA expression in L4-6 DRG in sham and CCI groups. (f) Sirt1 protein expression in L4-6 DRG in sham and CCI groups. *p < 0.05, **p < 0.01, ***p < 0.001 versus naïve group; repeated measured two-way ANOVA followed by Turkey’s post hoc test (a-d) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (e-f). n = 6 rats in each group.

Figure 2.

Activating Sirt1 alleviated CCI–induced pain hypersensitivity. (a) Sirt1 activity in L4-L6 DRG in sham, sham+EX527 (a Sirt1 inhibitor), CCI and CCI+SRT1720 (a Sirt1 activator) groups. (b-e) Ipsilateral and contralateral mechanical (b, c) and thermal (d, e) sensitivity; *p < 0.05, ***p < 0.001 sham+EX527 versus sham group. The time of drug injection is indicated as red lines. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 CCI+SRT1720 versus CCI group; by two-way (b-e) or one-way ANOVA followed by Turkey’s post hoc test (a). n = 6 in each group.

Figure 3.

Resveratrol alleviated CCI–induced neuropathic pain through activating DRG Sirt1 in rats. (a) Sirt1 activity in L4-L6 DRG of sham, CCI, and CCI+Resveratrol group. (b-e) Ipsilateral and contralateral mechanical (b, c) and thermal (d, e) sensitivity in sham, CCI and CCI+Resveratrol groups. The time of drug injection is indicated as red lines. *p < 0.05, ***p < 0.001 versus sham group; ^##p < 0.01, ^###p < 0.001 versus CCI group by one-way (a) or two-way (b-e) ANOVA followed by Turkey’s post hoc test. n = 6 in each group.

Figure 4.

Resveratrol up-regulated miR-182 expression in CCI rats. (a) Time course of miR-182 expression in DRGs of CCI rats. (b) DRG miR-182 mRNA level in sham, CCI, and CCI+Resveratrol group at day 11 after surgery. (c) DRG miR-182 mRNA level in sham, sham+EX527, CCI and CCI+SRT1720 groups. *p < 0.05, **p < 0.01 by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. n = 6 rats in each group.

Figure 5.

Alleviation of CCI–induced neuropathic pain by resveratrol was mediated by miR-182. (a) miR-182 expression level in L4-L6 DRGs. (b-e) Ipsilateral and contralateral mechanical (b, c) and thermal (d, e) sensitivity. The time of drug injection is indicated as red lines. *p < 0.05, **p < 0.01 versus sham group; ^†p < 0.05, ^††p < 0.05 and ^†††p < 0.001 CCI+Res.+miR-182antagomir group versus CCI+Resveratrol group; Kruskal-Wallis test followed by Dunn’s multiple comparisons test (a) or two-way ANOVA followed by Turkey’s post hoc test (b-e). n = 6 rats in each group.

Figure 6.

Resveratrol down-regulated Nav1.7 expression in CCI rats. (a) The expression of Nav1.7 protein in DRG from sham, CCI, CCI+Resveratrol, CCI+Res.+miR-182antagomir, and CCI+Res.+miR-182antagomir-N.C groups. (b) Expression of Nav1.7 protein in DRG from sham, sham+EX527, CCI and CCI+SRT1720 groups. *p < 0.05, **p < 0.01 versus sham group by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. n = 6 rats in each group.