Activation of NR2A-Wnt-TLR2 Signaling Axis in Satellite Glial Cells of the Dorsal Root Ganglion Contributes to Neuropathic Pain Induced by Nerve Injury in Diabetic Mice

Diabetic peripheral neuropathic pain (DPNP), a common diabetic mellitus (DM) complication, may result from the activation of satellite glial cells (SGCs) in the dorsal root ganglion (DRG), potentially enhancing peripheral sensitization. The N-methyl-D-aspartate receptor (NMDAR) subtype NR2A and Toll-like Receptor (TLR)2 play key roles in neuroimmune interactions. However, their roles in SGCs of DRG and the precise mechanisms mediating peripheral sensitization in DPNP remain unclear. Here, we found that the expression of glial fibrillary acidic protein (GFAP), NR2A, and TLR2 in SGCs from DRG significantly increased under increased glucose and NMDA stimulation in vitro. Additionally, upregulation of interleukin (IL)-6 and nerve growth factor (NGF) was observed. Notably, lentivirus-induced NR2A knockdown (KD) and C29 (TLR2 Inhibitor) significantly blocked the above SGCs changes induced by NMDA and increased glucose. Behavior tests showed mechanical and thermal sensitivities induced by sciatic nerve ligation (SNL) were more obvious in DM background related to streptozotocin (STZ) injection than non-DM background mice, which were significantly alleviated by NR2A conditional knockout (CKO) in SGCs and TLR2 KO. Moreover, immunofluorescence (IF) results revealed the co-expression of NR2A and TLR2 in neurons and SGCs in the DRG. Following SNL in DM mice, the upregulation of NR2A, TLR2, GFAP, β-catenin, p-GSK-3β, p-nuclear factor kappa (NF-κ)-B, IL-6, NGF, Bcl-2-associated X protein (Bax), and Caspase 3, and the significant downregulation of Bcl-2 were consistent with the changes observed after increased glucose and NMDA treatment. The upregulation of TLR2 was blocked by NR2A CKO and Wnt signal pathway inhibition. Additionally, the activation of SGCs, upregulated IL-6 as well as NGF secretion and increased Apoptosis, associated with nerve injury in DM background were altered by TLR2 KO and NF-κB pathway inhibition. In conclusion, the activation of the NR2A-Wnt-TLR2 signaling axis mediated peripheral sensitization in the DRG by influencing SGCs' activation, and the synthesis and secretion of pro-inflammatory cytokines and NGF, promoting SGCs' Apoptosis, thus exacerbating a peripheral nerve injury related-NP in DM background. Our study provided insights into the role of NR2A-Wnt-TLR2 signaling axis of SGCs in mediating the generation and maintenance of DPNP and suggested targeting this signaling axis may be a promising therapeutic approach for DPNP.

Apoptosis; Diabetic peripheral neuropathic pain; NR2A-Wnt-TLR2 signaling axis; Nerve growth factor; Nerve injury; Peripheral sensitization; Pro-inflammatory cytokines; Satellite glial cells activation.