PKM2 is involved in neuropathic pain by regulating ERK and STAT3 activation in rat spinal cord

Abstract

Background: Pyruvate kinase isozymes M2 (PKM2), as a member of pyruvate kinase family, plays a role of glycolytic enzyme in glucose metabolism. It also functions as protein kinase in cell proliferation, signaling, immunity, and gene transcription. In this study, the role of PKM2 in neuropathic pain induced by chronic constriction injury (CCI) was investigated.

Methods: Rats were randomly grouped to establish CCI models. PKM2, extracellular regulated protein kinases (EKR), p-ERK, signal transducers and activators of transcription (STAT3), p-STAT3, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and p-PI3K/AKT proteins expression in spinal cord was examined by Western blot analysis. Cellular location of PKM2 was examined by immunofluorescence. Knockdown of PKM2 was achieved by intrathecal injection of specific small interfering RNA (siRNA). Von Frey filaments and radiant heat tests were performed to determine mechanical allodynia and thermal hyperalgesia respectively. Lactate and adenosine triphosphate (ATP) contents were measured by specific kits. Tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels were detected by ELISA kits.

Results: CCI markedly increased PKM2 level in rat spinal cord. Double immunofluorescent staining showed that PKM2 co-localized with neuron, astrocyte, and microglia. Intrathecal injection of PKM2 siRNA not only attenuated CCI-induced ERK and STAT3 activation, but also attenuated mechanical allodynia and thermal hyperalgesia induced by CCI. However, PKM2 siRNA failed to inhibit the activation of AKT. In addition, PKM2 siRNA significantly suppressed the production of lactate and pro-inflammatory mediators.

Conclusion: Our findings demonstrate that inhibiting PKM2 expression effectively attenuates CCI-induced neuropathic pain and inflammatory responses in rats, possibly through regulating ERK and STAT3 signaling pathway.

Keywords: Chronic constriction injury; Lactate; Neuropathic pain; PKM2; p-ERK; p-STAT.

Fig. 1

Changes of mechanical allodynia, heat hyperalgesia and PKM2 expression in rats after CCI. a, b CCI induced a significant decrease in PWT (a) and PWL (b). *P < 0.05 versus sham group. Five rats per group. c, d Western blot was performed to detect PKM2 expression in spinal cord. Each group comprised four corresponding spinal cord segments. Representative PKM2 protein bands were exhibited on the left (c), and statistical analysis histogram was shown on the right (d). *P < 0.05 versus sham group

Fig. 2

Expression and distribution of PKM2 in spinal cord dorsal horn after CCI. A Immunofluorescence showed that expression of PKM2 (red) was increased at day 7 and day 10. B Double staining showed that PKM2 co-localized with neuron marker NeuN (a-d), microglia marker Iba-1 (e-h), and astrocyte marker GFAP (i-l)

Fig. 3

Intrathecal PKM2 siRNA treatment attenuated pain hypersensitivity and the production of lactate and pro-inflammatory transmitter induced by CCI. a The expression of PKM2 following intrathecal injection of siRNAs after CCI; PKM2 siRNA and NT siRNA (daily for 3 consecutive days, 15 μl each time, 0.75 μg/μl) were intrathecally injected. Tissues were harvested at day 3, 6 h after the last injection. *P < 0.05 versus NT siRNA group, ^#P < 0.05 versus naïve group. Four rats per group. b Intrathecal injection of PKM2 siRNA for three consecutive days (day 1, 2, 3) increased paw withdraw threshold and latency for almost 7 days (day 3 to day 10). c PKM2 siRNA significantly attenuated lactate production, while ATP production were not influenced. d Meanwhile, both TNF-α and IL-1β production were also inhibited by PKM2 siRNA. *P < 0.05 versus NT siRNA group. ^#P < 0.05 versus naïve group. Four rats per group

Fig. 4

PKM2 siRNA reversed the phosphorylation of ERK and STAT3 in the spinal cord L4–5 segments induced by CCI. Western blot was performed to evaluate the levels of phosphorylated ERK, STAT3, and AKT in protein extracted from the L4–5 segment of spinal cord in each group. GAPDH was used as an internal control. a Treatment with PKM2 siRNA significantly reduced the pERK/ERK and pSTAT3/STAT3 ratios. *P < 0.05 versus NT siRNA group. ^#P < 0.05 versus naïve group. Four rats per group. b PKM2 siRNA failed to inhibit the phosphorylation of AKT. ^#P < 0.05 versus naïve group. Four rats per group