Dihydromyricetin As a Novel Anti-Alcohol Intoxication Medication

DHM ameliorates EtOH withdrawal symptoms in rats. Four groups of rats were injected intraperitoneally with single-dose vehicle, EtOH (3 g/kg), EtOH plus DHM (1 mg/kg), or DHM alone. A, Anxiety was measured by elevated plus maze. The E/W group spent a shorter time in the open arms and longer time in the closed arms compared to the V/W group. The E+D/W group spent a similar amount of time in each arm as the V/W group (n = 5–6 rats per group). B, Tolerance was measured by LORR. The E/W group showed a significantly shorter duration of acute EtOH-induced LORR. The E+D/W group showed no different in LORR compared with the V/W group (n = 5 rats per group). C, The E/W group increased PTZ-induced seizure duration. The E+D/W group showed similar PTZ-induced seizures as the V/W group. The D/W group showed no difference compared with the V/W group in all three assays (n = 6–13 rats per group). *Statistical significance versus the V/W group.

DHM prevents the escalation of EtOH consumption in the voluntary intermittent two-bottle choice paradigm in rats. A, EtOH consumption gradually escalated in the group of intermittent access to two-bottle choice of 20% EtOH/water. Coadministration of DHM (0.05 mg/ml) with EtOH (E+D/water) counteracted this increase (*, Statistical significance vs E/water group). The symbols are mean EtOH intake (grams per kilogram per day) ± SEM. After 6 weeks, rats in the E/water group were separated into two subgroups: one continuing intermittent access EtOH and one with intermittent access to E+D. Whereas the E/water group kept a high level of EtOH consumption, the E+D/water group showed a great reduction in EtOH consumption within three doses of DHM (†, statistical significance between two subgroups), and became similar in EtOH consumption by the fourth dose of DHM. B, Fluid intake preference in the four groups at the third and the seventh weeks of two-bottle choice paradigm. The bottle containing EtOH or EtOH–DHM were marked as “drug” bottles. Fluid intake preference (percentage) was calculated: B/(B + V) * 100%. B is fluid intake volume from the drug bottle; V is fluid intake from the water bottle (n = 6–8 rats per group; *, statistical significance vs E 3rd week TBC). C, Plasma [EtOH] was measured at the end of sixth week of two-bottle choice. Blood samples were collected from the lateral tail vein after 30, 45, 60, and 100 min from E/water and E+D/water group rats (n = 2 rats per group; *, statistical significance between E/water and E+D/water).

DHM antagonizes EtOH-induced GABA_AR potentiation and the effects are blocked by flumazenil. All recordings were whole-cell voltage-clamped at −70 mV. A, Recording from DGCs in hippocampus slices. The gray dashed lines represent the mean currents after complete blockade of all GABA_AR currents by picrotoxin (PTX; a GABA_AR antagonist; 100 μm) as a baseline to calculate the magnitude of GABA_AR-mediated extrasynaptic tonic currents (I_tonic). Bath application of EtOH (60 mm, E 60) increased I_tonic and mIPSCs. DHM (0.3 and 1.0 μm) antagonized these EtOH effects. B, C, Summary of I_tonic and mIPSC area in response to EtOH and DHM (n = 8 neurons/3 rats). D, DHM (3 μm) antagonism on acute EtOH-induced GABA_AR potentiation was reversed by 10 μm flumazenil. E, F, Summary of I_tonic and mIPSC area in response to EtOH, DHM, and flumazenil (n = 5 neurons/3 rats). G, Quercetin (0.3, 1 μm) did not affect EtOH-induced enhancement of GABA_AR-mediated currents. H, I, Summary of I_tonic and mIPSC area in response to EtOH and quercetin (n = 5 neurons/3 rats). *Statistical significance versus drug 0.

DHM is a positive modulator of GABA_ARs at BZ sites. All recordings were whole-cell voltage clamped at −70 mV. A, Recording from DGCs (left) in hippocampal slices and superimposed averaged mIPSCs (right). B, C, Summary of I_tonic and mIPSC area potentiated by DHM from 0.1 to 30 μm (n = 8 neurons/3 rats). *Statistical significance versus drug 0 (A, C). D, Recording from a cultured hippocampal neuron at DIV14. DHM (1 μm; D1) enhancement of GABA_AR-mediated I_tonic and mIPSCs were reversed by flumazenil (F; 10 and 100 μm). All GABA_AR currents were blocked by bicuculline (GABA_AR antagonist; Bic; 10 μm; gray dashed line). E, F, Summary (% of DHM-induced current) of D1 enhancements of I_tonic and mIPSCs, which were inhibited by flumazenil (n = 8 neurons/3 rats). G, DHM inhibited [³H]flunitrazepam (flu) binding in rat cortex membrane homogenates. Increasing the final concentrations of DHM (0.03–100 μm) results in displacement of [³H]flunitrazepam (final concentration of 1 nm) at cortical binding sites. Results are graphed by GraphPad Prism 4.0 and presented as the average of two experiments with each point done in triplicate (n = 2). *Statistical significance versus flumazenil 0 (D–F).

DHM potentiates GABA_AR-mediated inhibition in a concentration-dependent manner in primary cultured hippocampal neurons (DIV14). Neurons were whole-cell voltage clamped at −70 mV. A, B, Dose–response curves of DHM on I_tonic and mIPSCs (n = 9–10 neurons). *Statistical significance versus drug 0. C, DHM (1 μm) enhanced GABA_AR currents evoked by focal puffs of 10 and 300 μm GABA. D, The concentration–response curve of GABA_AR currents induced by focal puffs of GABA was left shifted by DHM (0.3 and 1 μm). Data (mean ± SEM) were obtained from the average of GABA-induced currents normalized to the peak currents induced by 300 μm GABA in neurons (n = 6–8 neurons).

DHM prevents EtOH exposure/withdrawal-induced alteration in GABA_AR α4 subunit expression in rat hippocampus. Four groups of rats were injected (intraperitoneally) with single-dose vehicle (V/W), EtOH (3 g/kg; E/W), EtOH plus DHM (1 mg/kg; E+D/W), or DHM alone (D/W). A, Western blots of hippocampal tissue GABA_AR α4 subunit after 48 h withdrawal from rats injected with vehicle, EtOH, E+D, or DHM. β-actin is shown as a loading control. B, Quantification of total α4 subunit protein from A. EtOH-withdrawal induced an increase in α4 GABA_AR subunit, whereas E+D/W treatment prevented this increase. DHM did not produce changes in α4 GABA_AR subunit protein (n = 3/group). *Significant difference versus the V/W group.

DHM prevents EtOH exposure/withdrawal-induced GABA_AR plasticity. Rats were divided into four groups and gavaged with vehicle (V/W), EtOH (5 g/kg; E/W), EtOH combined with DHM (1 mg/kg; E+D/W) or DHM (D/W). Then whole-cell patch-clamp recordings at −70 mV were performed on DGCs in hippocampal slices. A, Acute EtOH (60 mm) enhanced I_tonic and mIPSCs in vehicle-treated rats. B, In the E/W group, EtOH did not increase I_tonic, but greatly enhanced mIPSC area. C, In the E+D/W group, EtOH increased I_tonic and mIPSCs similar to those of the V/W group. D, The responses of I_tonic and mIPSCs to EtOH from the D/W group were similar to those of the V/W group. E, F, Summary of EtOH effects on I_tonic and mIPSCs in the four groups (n = 4–7 neurons per group). *Significant difference between 60 mm and 0 EtOH; ^†significant difference versus the V/W group.