Effect of Riluzole on the Expression of HCN2 in Dorsal Root Ganglion Neurons of Diabetic Neuropathic Pain Rats

Abstract

Neuropathic pain since early diabetes swamps patients' lives, and diabetes mellitus has become an increasingly worldwide epidemic. No agent, so far, can terminate the ongoing diabetes. Therefore, strategies that delay the process and the further complications are preferred, such as diabetic neuropathic pain (DNP). Dysfunction of ion channels is generally accepted as the central mechanism of diabetic associated neuropathy, of which hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channel has been verified the involvement of neuropathic pain in dorsal root ganglion (DRG) neurons. Riluzole is a benzothiazole compound with neuroprotective properties on intervention to various ion channels, including hyperpolarization-activated voltage-dependent channels. To investigate the effect of riluzole within lumbar (L3-5) DRG neurons from DNP models, streptozocin (STZ, 70 mg/kg) injection was recruited subcutaneously followed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), which both show significant reduction, whilst relieved by riluzole (4 mg/kg/d) administration, which was performed once daily for 7 consecutive days for 14 days. HCN2 expression was also decreased in line with alleviated behavioral tests. Our results indicate riluzole as the alleviator to STZ-induced DNP with involvement of downregulated HCN2 in lumbar DRG by continual systemic administration in rats.

Figure 1

Effects of riluzole on PWMT and PWTL in DNP rats. (a) Time course of the paw withdrawal thermal latency in STZ-treated rats.  ^∗(p) < 0.05 vs. control; #(p) < 0.05 vs. DMSO. (b) Time course of sensitivity to mechanical stimuli, shown as the von Frey force threshold for withdrawal, in STZ-treated rats.  ^∗(p) < 0.05 vs. control; #(p) < 0.05 vs. DMSO.

Figure 2

Changes of HCN2 level in DRG detected by western blotting. (a) Western blotting analysis of HCN2 from L3-5 DRG neurons derived of rats in group control, DNP, DMSO, and riluzole. (b) Quantitative analysis showed that riluzole significantly decreased the expression of HCN2 protein in diabetic rats compared to group DMSO.  ^∗(p) < 0.05 vs. control; #(p) < 0.05 vs. DMSO.

Figure 3

Changes of HCN2 level in DRG detected by immunofluorescence analysis. (a) Representative images of the immunofluorescent analysis showing HCN2 (green) expression of L3-5 DRG from control, DNP, DMSO, and riluzole. Scale bar, 100 μm. (b) Quantification of HCN2-positive neurons in the four different groups.  ^∗(p) < 0.05 vs. control; #(p) < 0.05 vs. DMSO.