The P2X7 Receptor Is Involved in Diabetic Neuropathic Pain Hypersensitivity Mediated by TRPV1 in the Rat Dorsal Root Ganglion

Abstract

The purinergic 2X₇ (P2X₇) receptor expressed in satellite glial cells (SGCs) is involved in the inflammatory response, and transient receptor potential vanilloid 1 (TRPV1) participates in the process of neurogenic inflammation, such as that in diabetic neuropathic pain (DNP) and peripheral neuralgia. The main purpose of this study was to explore the role of the P2X₇ receptor in DNP hypersensitivity mediated by TRPV1 in the rat and its possible mechanism. A rat model of type 2 diabetes mellitus-related neuropathic pain (NPP) named the DNP rat model was established in this study. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of DNP rats were increased after intrathecal injection of the P2X₇ receptor antagonist A438079, and the mRNA and protein levels of TRPV1 in the dorsal root ganglion (DRG) were decreased in DNP rats treated with A438079 compared to untreated DNP rats; in addition, A438079 also decreased the phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) in the DNP group. Based on these results, the P2X₇ receptor might be involved in DNP mediated by TRPV1.

Keywords: P2X7 receptors; TRPV1; diabetic neuropathic pain; dorsal root ganglion; neurons; satellite glial cells.

FIGURE 1

The effect of A438079 on the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) of DNP rats. (A) Changes in the MWTs of rats from each group. (B) Changes in the TWLs of rats from each group. The MWTs and TWLs of rats from the DNP group were significantly lower than the values of rats from the control group (namely, the sensitivity was increased). At the end of the 8th week, the MWTs and TWLs of rats in the DNP group treated with an intrathecal injection of the P2X₇ receptor antagonist A438079 (DNP + A438079 group) were significantly higher than those of rats in the DNP group (i.e., decreased sensitivity). The data are presented as the mean ± SEM for the six animals in each group; compared with the control group, **P < 0.01; compared with the DNP group, ^#P < 0.05, ^##P < 0.01.

FIGURE 2

The levels of the TRPV1 mRNA and protein in the DRG were detected. (A) The level of the TRPV1 mRNA in the DRG from each group. (B) SDS-PAGE band of the TRPV1 protein in the DRG from each group of experimental rats. (C) Analysis of the relative level of the TRPV1 protein in the DRG from each group of experimental rats. Higher levels of the TRPV1 mRNA and protein were detected in the DRG in the DNP group than in the control group, while lower levels of the TRPV1 mRNA and protein were observed in the DRG in the DNP + A438079 group than in the DNP group. The data are presented as the mean ± SEM, n = 6; compared with the control group, **P < 0.01; compared with the DNP group, ^##P < 0.01.

FIGURE 3

The coexpression of TRPV1 with NeuN and P2X₇ receptor with GFAP was detected by immunofluorescence staining. (A) Levels of TRPV1 with NeuN coexpression in neurons in the rat DRG (n = 6 rats for each group). (B) The histogram shows the mean optical density of the coexpression of TRPV1 with NeuN. (C) Levels of P2X₇ with GFAP coexpression in SGCs in the rat DRG (n = 6 rats for each group). (D) The histogram shows the mean optical density of the coexpression of the P2X₇ receptor with GFAP. In the DNP group, the coexpression of TRPV1 with NeuN in the DRG was increased compared with that in the control group, while the coexpression of TRPV1 with NeuN in the DNP + A438079 group was decreased compared with that in the DNP group. Furthermore, the coexpression of P2X₇ receptor with GFAP in the DRG was consistent with the coexpression trend described above. The green signal is fluorescein isothiocyanate (FITC)-labeled NeuN and FITC-labeled GFAP; the red signal is tetramethylrhodamine isothiocyanate (TRITC)-labeled TRPV1 and TRITC-labeled P2X₇; and the yellow signal is the combination of the green and red signals. The arrow indicates activated neuronal cells and SGCs in the DRG. The scale bar represents 50 μm. The data are presented as the mean ± SEM; compared with the control group, **P < 0.01; compared with the DNP group, ^##P < 0.01.

FIGURE 4

The relative levels of IL-1β and IL-10 in the DRG were detected by Western blotting. (A) SDS-PAGE band of IL-1β in the DRG in each group of experimental rats. (B) The relative levels of IL-1β in the DRG from each group of experimental rats. (C) SDS-PAGE band of IL-10 in the DRG in each group of experimental rats. (D) The relative levels of IL-10 protein in the DRG from each group of experimental rats. The relative level of the IL-1β protein in the DRG from the DNP group was higher than that in the control group, while a lower level of IL-1β was observed in the DRG from the DNP + A438079 group than in the DNP group. In contrast, the relative level of the IL-10 protein in the DRG from the DNP group was lower than that in the control group, while a higher level of IL-10 was detected in the DRG in the DNP + A438079 group than in the DNP group. The data are presented as the mean ± SEM, n = 6; compared with the control group, *P < 0.05, **P < 0.01; compared with the DNP group, ^#P < 0.05, ^##P < 0.01.

FIGURE 5

Western blotting was performed to detect the levels of proteins related to the p38 and ERK1/2 signaling pathways in the DRG. (A) SDS-PAGE bands of the p-p38 and p38 proteins in each group. (B) The ratio of the optical density of p-p38 to that of the internal reference. (C) The ratio of the optical density of p38 to that of the internal reference. (D) SDS-PAGE bands of p-ERK1/2 and ERK1/2 in each group. (E) The ratio of the optical density of p-ERK1/2 to that of the internal reference. (F) The ratio of the optical density of ERK1/2 to that of the internal reference. The data are presented as the mean ± SEM, n = 6; compared with the control group, **P < 0.01; compared with the DNP group, ^#P < 0.05, ^##P < 0.01; ns indicates a non-significant difference.

FIGURE 6

Molecular docking of A438079 with the TRPV1. (A), top view. (B), the docking pocket between A438079 and TRPV1. (C), front view. (D), A438079 does not form a hydrogen bond with TRPV1, and A438079 is in the middle.