Activation of GABAB Receptor Suppresses Diabetic Neuropathic Pain through Toll-Like Receptor 4 Signaling Pathway in the Spinal Dorsal Horn

Abstract

Diabetic neuropathic pain (DNP) is a prevalent complication in diabetes patients. Neuronal inflammation and activation of Toll-like receptor 4 (TLR4) are involved in the occurrence of DNP. However, the underlying mechanisms remain unclear. Downregulation of gamma-aminobutyric acid B (GABA_B) receptor contributes to the DNP. GABA_B receptor interacts with NF-κB, a downstream signaling factor of TLR4, in a neuropathic pain induced by chemotherapy. In this study, we determined the role of TLR4/Myd88/NF-κB signaling pathways coupled to GABA_B receptors in the generation of DNP. Intrathecal injection of baclofen (GABA_B receptor agonist), LPS-RS ultrapure (TLR4 antagonist), MIP (MyD88 antagonist), or SN50 (NF-κB inhibitor) significantly increased paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWTL) in DNP rats, while intrathecal injection of saclofen (GABA_B receptor blocker) decreased PWT and PWTL in DNP rats. The expression of TLR4, Myd88, NF-κBp65, and their downstream components IL-1 and TNF-α was significantly higher in the spinal cord tissue in DNP rats compared to control rats. Following inhibition of TLR4, Myd88, and NF-κB, the expression of IL-1 and TNF-α decreased. Activation of GABA_B receptors downregulated the expression of TLR4, Myd88, NF-κBp65, IL-1, and TNF-α. Blockade of GABA_B receptors significantly upregulated expression of TLR4, Myd88, NF-κBp65, IL-1, and TNF-α. These data suggest that activation of the TLR4/Myd88/NF-κB signaling pathway is involved in the occurrence of DNP in rats. Activation of GABA_B receptor in the spinal cord may suppress the TLR4/Myd88/NF-κB signaling pathway and alleviate the DNP.

Figure 1

Figure 2

Neuropathic pain behavior in the control, diabetic neuropathic pain (DNP), DNP + LPS-RS (TLR4 antagonist), DNP + MIP (Myd88 antagonist), and DNP + SN50 (NF-κB inhibitor) groups at 0, 1, 2, and 3 days after intrathecal administration. (a, c, e). The mechanical paw withdrawal threshold (PWT) (g). (b, d, f). The paw withdrawal thermal latency (PWTL) (s). Bar graphs represent mean ± SEM. ^∗p < 0.05, compared with the DNP group (n = 12).

Figure 3

The development of mechanical allodynia and thermal hyperalgesia in the control, diabetic neuropathic pain (DNP), DNP + baclofen (GABA_B receptor agonist), and DNP + saclofen (GABA_B receptor blocker) groups at 0, 1, 2, and 3 days after intrathecal administration. (a) The mechanical paw withdrawal threshold (PWT). (b) The paw withdrawal thermal latency (PWTL). Bar graphs represent mean ± SEM. ^∗p < 0.05, compared with the DNP group (n = 12).

Figure 4

The protein expression of Myd88, TLR4, and NF-κBp65 in each group. (a) Myd88 in the control (n = 4), DNP (n = 4), DNP + LPS-RS (TLR4 antagonist, n = 6), DNP + baclofen (GABA_B receptor agonist, n = 4), and DNP + saclofen (GABA_B receptor blocker, n = 4) groups. (b) NF-κBp65 in the control (n = 4), DNP (n = 4), DNP + LPS-RS (TLR4 antagonist, n = 6), DNP + MIP (Myd88 antagonist, n = 6), DNP + baclofen (GABA_B receptor agonist, n = 4), and DNP + saclofen (GABA_B receptor blocker, n = 4) groups. (c) TLR4 in the control, DNP, DNP + baclofen (GABA_B receptor agonist), and DNP + saclofen (GABA_B receptor blocker) groups. ^∗p < 0.05; ^∗∗p < 0.01.

Figure 5

Single fluorescence labeling of TLR4, Myd88, and NF-κBp65 (nucleus stained with DAPI) in the spinal cord of the control, DNP, DNP + baclofen, and DNP + saclofen (n = 4) groups. Quantification of the percentage of TLR4, Myd88, and NF-κBp65-positive cells relative to all cells. Bar graphs represent mean ± SD. Scale bars are 100 μm in (a) and 50 μm in (b). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 6

The expression of TNF-α and IL-1 in the spinal cord of control (n = 4), DNP (n = 4), DNP + baclofen (GABAB receptor agonist, n = 4), DNP + saclofen (GABAB receptor blocker, n = 4), DNP + LPS-RS (TLR4 antagonist, n = 6), DNP + MIP (Myd88 antagonist, n = 6), and DNP + SN50 (NF-κB inhibitor, n = 12) groups by ELISA. (a) TNF-α. (b) IL-1. ^∗∗∗p < 0.001.