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. 2022 Nov 17:2022:4661519.
doi: 10.1155/2022/4661519. eCollection 2022.

BHF177 Suppresses Diabetic Neuropathic Pain by Blocking PKC/CaMKII/ERK1/2/CREB Signaling Pathway through Activating GABAB Receptor

Boyu Liu  1 , Fengxi Guan  2 , Jiapeng Zhao  3 , Yao Niu  1 , Hongbo Jiang  4
Affiliations

BHF177 Suppresses Diabetic Neuropathic Pain by Blocking PKC/CaMKII/ERK1/2/CREB Signaling Pathway through Activating GABAB Receptor

Boyu Liu et al. Oxid Med Cell Longev. .

Abstract

The gamma-aminobutyric acid type B (GABAB) receptor may participate in the development of diabetic neuropathic pain (DNP). BHF177 serves as a positive allosteric modulator of the GABAB receptor. In the current study, we sought to study the role of the BHF177-GABAB receptor in DNP and its underlying mechanism. Streptozotocin was adopted to induce a rat model of DNP, followed by determination of the paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and glucose level. The effect of BHF177 on DNP by regulating the GABAB receptor in vivo was determined by the injection of BHF177 and/or CGP46381 (a GABAB receptor antagonist) into rat models of DNP. Hippocampal neuronal cells were isolated and cultured, and the neurons and DNP model rats were treated with activators of PKC (PMA), CaMKII (CaCl2), or ERK1/2 (EGF) to study the role of GABAB receptors in DNP via regulation of the NR2B-PKC-CaMKII-ERK-CREB pathway. BHF177 suppressed DNP symptoms by activating the GABAB receptors, as evidenced by increased PWT and PWL of DNP rats and the increased number of neurons expressing the GABAB receptor, but this effect was reversed by CGP46381 treatment. BHF177 treatment markedly repressed PKC, CaMKII, p-ERK1/2, and p-CREB expressions in the rat DNP model, but these suppressive effects were abrogated by treatments with PMA, CaCl2, or EGF treatment, respectively. To sum up, BHF177 suppresses DNP symptoms by blocking the PKC/CaMKII/ERK1/2/CREB signaling pathway to activate the GABAB receptors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
BHF177 confers protection against DNP by regulating GABAB receptor. (a) The expression heat map of the 250 genes with the most significantly low expression in the microarray GSE27382. (b) The intersection of 250 lowest-expressed genes from the microarray GSE27382 and diabetic neuralgia-related genes predicted by GeneCards, showing 19 intersection genes. (c) The expression box plot of GABBR1 in the microarray GSE27382; the blue box indicates the expression of normal samples, and the red box indicates the expression of diabetic samples. (d) The PWT and PWL of DNP rats treated with BHF177 and/or CGP46381. (e) The plasma glucose level of DNP rats treated with BHF177 and/or CGP46381. n = 10. ∗p < 0.05 vs. normal rats, #p < 0.05 vs. rat models of DNP, and &p < 0.05 vs. DNP rats treated with BHF177. Animal, n = 10.
Figure 2
Figure 2
BHF177 increases the number of neurons positive for the GABAB receptor in DNP rats. (a) The number of cells positive for the GABAB receptor in DNP rats determined using immunohistochemistry (×200). (b) Statistic analysis of (a). ∗p < 0.05 vs. normal rats, #p < 0.05 vs. DNP rats, and &p < 0.05 vs. DNP rats treated with BHF177. n = 10 animals per group.
Figure 3
Figure 3
BHF177 targets the GABAB receptor and NR2B NMDA subunit by inhibiting the PKC-CaMKII-ERK-CREB pathway in DNP rats. (a) The mRNA expression of GABAB receptors, NR2B, PKC, or CaMKII in spinal dorsal horn tissues of DNP rats determined using RT-qPCR. (b) The protein expression of GABAB receptors, NR2B, PKC, CaMKII, p-ERK1/2, or p-CREB in spinal dorsal horn tissues of DNP rats determined using immunoblotting. (c) Immunohistochemistry of the number of cells positive for related markers in rat spinal dorsal horn tissue. n = 10. ∗p < 0.05 vs. normal rats, #p < 0.05 vs. rat models of DNP, and &p < 0.05 vs. DNP model rats treated with BHF177. n = 10 rats per group.
Figure 4
Figure 4
BHF177 inactivates the NR2B-PKC-CaMKII-ERK-CREB pathway in vitro. The neurons were treated with BHF177, PMA, CaCl2, or EGF. (a) The mRNA expression of PKC and CaMKII in neurons determined using RT-qPCR. (b) The protein expression of PKC, CaMKII, p-ERK1/2, or p-CREB in neurons determined using immunoblotting. ∗p < 0.05 vs. normal neuronal cells, #p < 0.05 vs. neurons treated with BHF177.
Figure 5
Figure 5
BHF177 activates the GABAB receptor to attenuate DNP symptoms by inactivating the NR2B-PKC-CaMKII-ERK-CREB pathway. DNP rats were injected with BHF177+PMA, BHF177+CaCl2, and BHF177+EGF (n = 6/group). (a) The PWT and PWL of DNP rats after different treatments. (b) The mRNA expression of PKC or CaMKII in DNP rats determined using RT-qPCR. (c) The protein expression of GABAB receptors, NR2B, PKC, CaMKII, p-ERK1/2, or p-CREB in DNP rats determined using immunoblotting. n = 6. ∗p < 0.05 vs. DNP rats #p < 0.05 vs. DNP rats treated with BHF177.
Figure 6
Figure 6
Schematic map of the regulatory role of BHF177 in DNP. BHF177 suppresses NMDA receptor NR2B subunit expression by activating GABAB receptors to block the PKC/CaMKII/ERK1/2/CREB signaling pathway, thus inhibiting DNP.
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