Dehydrocrenatidine Inhibits Voltage-Gated Sodium Channels and Ameliorates Mechanic Allodia in a Rat Model of Neuropathic Pain

Abstract

Picrasma quassioides (D. Don) Benn, a medical plant, is used in clinic to treat inflammation, pain, sore throat, and eczema. The alkaloids are the main active components in P. quassioides. In this study, we examined the analgesic effect of dehydrocrenatidine (DHCT), a β-carboline alkaloid abundantly found in P. quassioides in a neuropathic pain rat model of a sciatic nerve chronic constriction injury. DHCT dose-dependently attenuated the mechanic allodynia. In acutely isolated dorsal root ganglion, DHCT completely suppressed the action potential firing. Further electrophysiological characterization demonstrated that DHCT suppressed both tetrodotoxin-resistant (TTX-R) and sensitive (TTX-S) voltage-gated sodium channel (VGSC) currents with IC₅₀ values of 12.36 μM and 4.87 µM, respectively. DHCT shifted half-maximal voltage (V_1/2) of inactivation to hyperpolarizing direction by ~16.7 mV in TTX-S VGSCs. In TTX-R VGSCs, DHCT shifted V_1/2 of inactivation voltage to hyperpolarizing direction and V_1/2 of activation voltage to more depolarizing potential by ~23.9 mV and ~12.2 mV, respectively. DHCT preferred to interact with an inactivated state of VGSCs and prolonged the repriming time in both TTX-S and TTX-R VGSCs, transiting the channels into a slow inactivated state from a fast inactivated state. Considered together, these data demonstrated that the analgesic effect of DHCT was likely though the inhibition of neuronal excitability.

Keywords: dehydrocrenatidine; neuropathic pain; voltage-gated sodium channels.

Figure 1

Dehydrocrenatidine (DHCT) attenuated sciatic nerve partial ligation-induced mechanical allodynia. (A) Chemical structure of DHCT. (B) Mechanical stimuli thresholds recorded after administration of vehicle (5% DMSO, 5% Tween-80, and 90% normal saline), DHCT (50, 150, and 250 μg/kg) or morphine (50 μg/kg) every 1.5 h, respectively. (C) Quantification of DHCT analgesic effects by using area under the curve (the area of paw withdrawal thresholds during 0–4.5 h after intrathecal administration). DHCT displayed significant analgesic effects in the rat chronic constriction neuropathic pain model. Each data point represents the mean ± SEM (n = 9). ** p < 0.01, model vs. sham group; # p < 0.05; ## p < 0.01, drugs vs. model group.

Figure 2

DHCT suppressed current-evoked action potential in dorsal root ganglion (DRG) neurons. (A) Representative traces of action potential (APs) evoked by an injection of a 30-pA (1 s) current in the absence and presence of 10 µM of DHCT. (B) Quantification of DHCT (10 μM) suppressed 30-pA induced APs in acutely dissociated rat DRG neurons. (C) Representative traces of APs evoked by an injection of a 200-pA (1 s) current in the absence and presence of 10 µM of DHCT. (D) Quantification of DHCT (10 μM) suppressed 200-pA induced APs in acutely dissociated rat DRG neurons. Each data point represents mean ± SEM. T-test was used to compared the statistical significance between Veh (0.1% DMSO) and DHCT (10 µM) groups. ** p < 0.01, n = 13.

Figure 3

DHCT suppressed tetrodotoxin-sensitive (TTX-S) Na⁺ currents in medium- and large-diameter -diameter DRG neurons. (A) Representative traces of DHCT suppressing TTX-S Na⁺ currents in medium- and large-diameter DRG neurons. (B) Concentration–response curve of DHCT suppressed TTX-S Na⁺ currents. Sodium currents were evoked by a 50-ms depolarization pulse to 0 mV from a holding potential of −100 mV. (C,D) Representative TTX-S Na⁺ currents evoked by different depolarizing voltages from −90 to +50 mV in a 5-mV step in the absence (C) and presence (D) of DHCT (30 µM). (E) Current–voltage (I-V) relationships of TTX-S VGSCs in the presence or absence of DHCT. (F) Steady-state activation and inactivation curves of TTX-S Na⁺ currents in the absence and presence of DHCT (30 µM). Each data point represents mean ± SEM (n = 5).

Figure 4

DHCT suppressed tetrodotoxin-resistant (TTX-R) Na⁺ currents in small-diameter DRG neurons in the presence of 300 nM TTX. (A) Representative traces of DHCT suppressing TTX-R Na⁺ currents. (B) Concentration–response curve of DHCT suppressed TTX-R Na⁺ currents. Sodium currents were evoked by a 50-ms depolarization pulse to 0 mV from a holding potential of −100 mV. (C,D) Representative TTX-R Na⁺ currents evoked by different depolarizing voltages from −90 to +50 mV in a 5-mV step in the absence (C) and presence (D) of DHCT (30 µM). (E) Current–voltage (I-V) relationships of TTX-R VGSCs in the presence or absence of 30 µM DHCT. (F) Steady-state activation and inactivation curves of TTX-R Na⁺ currents in the absence and presence of DHCT (30 µM). Each data point depicts mean ± SEM (n = 5).

Figure 5

DHCT preferentially interacted with an inactivated state of TTX-S and TTX-R VGSCs in DRG neurons. (A) Representative TTX-S Na⁺ current traces in the absence and presence of different concentrations of DHCT evoked by a depolarization potential to 0 mV from a holding potential of −120 mV. (B) Representative TTX-S Na⁺ current traces in the absence and presence of different concentrations of DHCT evoked by a depolarization potential to 0 mV from a holding potential of −60 mV. (C) Concentration–response curves of DHCT suppressing TTX-S Na⁺ currents at the holding potentials of −120 mV and −60 mV, respectively. (D) Representative TTX-R Na⁺ current traces in the absence and presence of different concentrations of DHCT evoked by a depolarization potential to 0 mV from a holding potential of −120 mV. (E) Representative TTX-R Na⁺ current traces in the absence and presence of different concentrations of DHCT evoked by a depolarization potential to 0 mV from a holding potential of −60 mV. (F) Concentration–response curves of DHCT suppressing TTX-R Na⁺ currents in DRG neurons at the holding potentials of −120 mV and −60 mV, respectively. Each data point represents mean ± SEM (n = 5–9).

Figure 6

DHCT exposure transited both TTX-S and TTX-R Na⁺ currents from a fast-inactivated state to a slow-inactivated state. (A,B) Representative traces of TTX-S Na⁺ currents when the holding potential was set at 0 mV for 100 ms, followed by a recovery interval of variable duration to −120 mV before testing for an active current with a 20-ms pulse to 0 mV in the absence (A) and presence (B) of DHCT (30 µM). (C) Recovery curves of TTX-S Na⁺ currents from fast-inactivation in the absence and presence of DHCT. (D,E) Representative traces of TTX-R Na⁺ currents when the holding potential was set at 0 mV for 100 ms, followed by a recovery interval of variable duration to −120 mV before testing for an active current with a 20-ms pulse to 0 mV in the absence (D) and presence (E) of DHCT (30 µM). (F) Recovery curves of TTX-R Na⁺ currents from fast-inactivation in the absence and presence of DHCT. (G,H) Representative traces of TTX-S Na⁺ currents when the cells were pre-conditioned at 0 mV for 10 s, followed by a recovery interval of variable duration to −120 mV before testing for an active current with a 20-ms pulse to 0 mV in the absence (G) and presence (H) of DHCT (30 µM). (I) Recovery curves of TTX-S Na⁺ currents from slow-inactivation in the absence and presence of DHCT. (J,K) Representative traces of TTX-R Na⁺ currents when the cells were pre-conditioned at 0 mV for 10 s, followed by a recovery interval of variable duration to −120 mV before testing for an active current with a 20-ms pulse to 0 mV in the absence (J) and presence (K) of DHCT (30 µM). (L) Recovery curves of TTX-R Na⁺ currents from slow-inactivation in the absence and presence of DHCT. Each data point represents mean ± SEM (n = 6–8).