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, 17 (4), 5229-5237

Effects of Metformin on the Expression of AMPK and STAT3 in the Spinal Dorsal Horn of Rats With Neuropathic Pain

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Effects of Metformin on the Expression of AMPK and STAT3 in the Spinal Dorsal Horn of Rats With Neuropathic Pain

Anqi Ge et al. Mol Med Rep.

Abstract

Neuropathic pain (NP) is a frustrating and burdensome problem. Current treatments for NP have unendurable side effects and/or questionable efficacy, and once these therapies are stopped, the symptoms often return. Thus, novel drugs are needed to enhance the effectiveness of treatments for NP. One novel target for pain treatments is adenosine monophosphate‑activated protein kinase (AMPK), which regulates a variety of cellular processes, including protein translation, which is considered to be affected in NP. Metformin is a widely available drug that possesses the ability to activate AMPK. The signal transducer and activator of transcription 3 (STAT3) pathway plays an important role in neuroinflammation. The present study investigated the analgesic effect of metformin on NP induced by chronic constriction injury (CCI), and the influence of metformin on the expression of AMPK and STAT3 in the spinal dorsal horn (SDH). In CCI rats, paw withdrawal latencies in response to thermal hyperalgesia were significantly shorter, while phosphorylated (p)‑AMPK was expressed at lower levels and p‑STAT3 was expressed at higher levels in the SDH. Administering intraperitoneal injections of metformin (200 mg/kg) for 6 successive days activated AMPK and suppressed the expression of p‑STAT3, in addition to reversing hyperalgesia. Finally, metformin inhibited the activation of microglia and astrocytes in the SDH, which may explain how it alleviates NP.

Figures

Figure 1.
Figure 1.
Intraperitoneal administration of metformin attenuates neuropathic pain in CCI rats. (A) CCI rats exhibited a significant decrease in the PWLs 3–14 days post-surgery. The intraperitoneal administration of metformin (200 mg/kg, once per day on days 5–10) attenuated thermal hyperalgesia (n=8; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS). (B) The intrathecal administration of compound C (30 µg) on day 10 could reverse the analgesic effect of metformin (n=8; ##P<0.01 vs. CCI + NS, ^^P<0.01 vs. CCI + metformin). NS, normal saline; PWL, paw withdrawal latency; CCI, chronic constriction injury.
Figure 2.
Figure 2.
Fasting blood glucose levels in the sham + NS, sham + metformin, CCI + NS and CCI + metformin groups. Metformin had no effect on fasting blood glucose levels in rats in the 4 groups (n=8; P>0.05). NS, normal saline; CCI, chronic constriction injury.
Figure 3.
Figure 3.
Metformin activated AMPK in the spinal cords of CCI rats. (A) Metformin increased AMPK phosphorylation levels in the spinal cord after CCI (n=4; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS). (B) There was no significant difference in the expression of AMPK among the four groups (n=4; P>0.05). Tissues were collected on day 10 after CCI. AMPK, adenosine monophosphate-activated protein kinase; NS, normal saline; CCI, chronic constriction injury.
Figure 4.
Figure 4.
Confocal images and immunofluorescence data showing p-AMPK expression in the spinaldorsal horn. The quantification of p-AMPK immunofluorescence is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar, 100 µm). Tissues were collected on day 10 after CCI. (p)-AMPK, phosphorylated adenosine monophosphate-activated protein kinase; NS, normal saline; CCI, chronic constriction injury.
Figure 5.
Figure 5.
Metformin inhibited the activation of STAT3 in the spinal cords of CCI rats. (A) Metformin decreased the level of phosphorylated STAT3 in the spinal cord after CCI. (n=4; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS) (B) There was no significant difference in the expression of STAT3 among the four groups (n=4; P>0.05). STAT3, signal transducer and activator of transcription 3; NS, normal saline; CCI, chronic constriction injury.
Figure 6.
Figure 6.
Confocal images and immunofluorescence analysis data showing p-STAT3 expression in the spinal dorsal horn. The quantification of p-STAT3 immunofluorescence is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar, 100 µm). Tissues were collected on day 10 after CCI. p-STAT3, phosphorylated signal transducer and activator of transcription 3; NS, normal saline; CCI, chronic constriction injury.
Figure 7.
Figure 7.
Confocal images and immunofluorescence staining for Iba-1 and GFAP expression in rat spinal dorsal horns. (A) The quantification of Iba-1 immunofluorescence staining is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar, 100 µm). (B) The quantification of GFAP immunofluorescence staining is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar,100 µm). Tissues were collected on day 10 after CCI. Iba-1, ionized calcium binding adaptor molecule 1; GFAP, glial fiber acidic protein; NS, normal saline; CCI, chronic constriction injury.
Figure 7.
Figure 7.
Confocal images and immunofluorescence staining for Iba-1 and GFAP expression in rat spinal dorsal horns. (A) The quantification of Iba-1 immunofluorescence staining is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar, 100 µm). (B) The quantification of GFAP immunofluorescence staining is presented as the mean fluorescence intensity and the number of positively stained cells (n=6; **P<0.01 vs. sham + NS; ##P<0.01 vs. CCI + NS; scale bar,100 µm). Tissues were collected on day 10 after CCI. Iba-1, ionized calcium binding adaptor molecule 1; GFAP, glial fiber acidic protein; NS, normal saline; CCI, chronic constriction injury.

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