Diabetic neuropathic pain (DNP) is one of the most common symptoms of diabetic peripheral neuropathy, characterized as spontaneous pain, hyperalgesia, and allodynia.1 DNP develops in approximately 30 % of patients with diabetes and impacts the patient's quality of life, such as insomnia, mood disorders, and reduction of employability at work.2., 3. Although several agents have been used for the treatment of DNP, the present treatment is purely symptomatic but do not alter the disease process, and has some intolerable adverse effects, such as dizziness, postural hypotension and nausea.4 Therefore, understanding the potential mechanism and exploring more effective treatments of DNP are important.

The microglia-mediated neuroinflammation is responsible for the development and maintenance of DNP.5 The pro-inflammatory cytokines released from activated microglia can consequently induce central sensitization of neurons by altering the excitatory or inhibitory synaptic transmission, contributing to pain facilitation.6 Activated microglia and increased pro-inflammatory cytokines have been found both in the spinal cord and brain of DNP rats.7., 8. And administration of minocycline, inhibitor of microglia, significantly attenuated neuropathic pain and decreased the level of pro-inflammatory cytokines in central nervous system of diabetic rats.8., 9. Therefore, microglia are the potential target for the treatment of DNP.

Glucagon-like peptide-1 (GLP-1) is an endogenous incretin hormone mainly secreted from L cells of the small intestine, and participates in diverse biological functions via activating GLP-1 receptor (GLP-1R).10., 11. GLP-1R agonists (GLP-1RAs) are a class of well-established antidiabetic drugs for type 2 diabetes.12 In addition to regulating the homeostasis of blood glucose, GLP-1RAs have been reported to attenuate DNP through activating microglial GLP-1R in the spinal cord.13 Furthermore, we previously were the first to show that intracerebroventricular administration of GLP-1RA liraglutide significantly alleviated neuropathic pain and inhibited the activation of microglia in the brain of DNP rats.8 However, the potential mechanism of GLP-1RA alleviating DNP through brain microglia is still unclear.

Glycogen synthesis kinase-3β (GSK3β), a serine/threonine kinase, is considered constitutively active and mainly regulated by phosphorylation at the serine-9 residue.14., 15. The activity of GSK3β increases in many types of neuroinflammation related diseases, such as Alzheimer's disease and experimental autoimmune encephalomyelitis.16., 17. In addition, GSK3β activity also increases in the spinal cord of animal model of neuropathic pain.18., 19. And pharmacological inhibition of GSK3β attenuates nociceptive responses induced by formalin injections or nerve injury,18., 20. and decreases the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the spinal cord.19 Furthermore, it has been showed that the suppression of GSK3β decreases the production of pro-inflammatory cytokines and augments the production of anti-inflammatory cytokines in lipopolysaccharide (LPS) triggered cortical microglia in vitro.21 However, it is unknown about the role and mechanism of GSK3β in DNP.

Our previous study showed NOD-like receptor protein 3 (NLRP3) inflammasome in the brain microglia was the key target of DNP. And GSK3β plays an important role in the activation of NLRP3 inflammasome. Ying et al. showed that liraglutide inhibited the activity of GSK3β through phosphorylating GSK3β at serine-9 in the retinae of mice with diabetic retinopathy. We hypothesized that GLP-1RA alleviated DNP through inhibiting GSK3β/NLRP3 inflammasome signaling pathway. In the present study, we aimed to investigate the role and mechanism of GSK3β involving in the analgesic effect of GLP-1RA on DNP using GSK3β(S9A) mice and the selective GLP-1RA liraglutide.