Dihydromyricetin as a novel anti-alcohol intoxication medication

Abstract

Alcohol use disorders (AUDs) constitute the most common form of substance abuse. The development of AUDs involves repeated alcohol use leading to tolerance, alcohol withdrawal syndrome, and physical and psychological dependence, with loss of ability to control excessive drinking. Currently there is no effective therapeutic agent for AUDs without major side effects. Dihydromyricetin (DHM; 1 mg/kg, i.p. injection), a flavonoid component of herbal medicines, counteracted acute alcohol (EtOH) intoxication, and also withdrawal signs in rats including tolerance, increased anxiety, and seizure susceptibility; DHM greatly reduced EtOH consumption in an intermittent voluntary EtOH intake paradigm in rats. GABA(A) receptors (GABA(A)Rs) are major targets of acute and chronic EtOH actions on the brain. At the cellular levels, DHM (1 μM) antagonized both acute EtOH-induced potentiation of GABA(A)Rs and EtOH exposure/withdrawal-induced GABA(A)R plasticity, including alterations in responsiveness of extrasynaptic and postsynaptic GABA(A)Rs to acute EtOH and, most importantly, increases in GABA(A)R α4 subunit expression in hippocampus and cultured neurons. DHM anti-alcohol effects on both behavior and CNS neurons were antagonized by flumazenil (10 mg/kg in vivo; 10 μM in vitro), the benzodiazepine (BZ) antagonist. DHM competitively inhibited BZ-site [(3)H]flunitrazepam binding (IC(50), 4.36 μM), suggesting DHM interaction with EtOH involves the BZ sites on GABA(A)Rs. In summary, we determined DHM anti-alcoholic effects on animal models and determined a major molecular target and cellular mechanism of DHM for counteracting alcohol intoxication and dependence. We demonstrated pharmacological properties of DHM consistent with those expected to underlie successful medical treatment of AUDs; therefore DHM is a therapeutic candidate.

Figures

Figure 1.

DHM counteracts EtOH intoxication and the effect of DHM is antagonized by flumazenil. A, Chemical structure of DHM. B, Top, Plasma [EtOH] assay associated with EtOH-induced LORR. The x-axis shows time points after intraperitoneal injection of EtOH (3 g/kg) or coapplication of DHM (0.3, 0.5, 1, and 10 mg/kg) with EtOH (n = 3–4 rats per group). Zero represents the time from intraperitoneal injection of EtOH or E+D to complete sample venous blood collection (usually it took 0 to 3 min). Bottom, E3 induced LORR, while concurrent injection of DHM with EtOH (E3+D0.3, E3+D0.5, E3+D1, and E3+D10) increased LORR onset time and greatly reduced the duration of LORR dose dependently. DHM (D1) as the saline control did not induce LORR (n = 4–12 rats per group). *Statistical significance versus the E3 group. C, Coinjection of EtOH and DHM (3 mg/kg; E3+D3) greatly reduced the E3-induced LORR. Concurrent injection of flumazenil (10 mg/kg; F10) with EtOH and DHM (E3+D3+F10) reversed the DHM effect. When we increased the dose of DHM to 10 mg/kg (E3+D10+F10), flumazenil partially reversed the effect of DHM. When we increased the dose of flumazenil to 30 mg/kg (E3+D3+F30), stronger antagonism of DHM was observed. Coinjection of flumazenil with EtOH (E3+F10) did not alter LORR duration (n = 5–13 rats per group). D, DHM application 30 min before EtOH injection counteracted EtOH-induced LORR, whereas 30 min after EtOH injection (indicated as gray lines), DHM injection reduced the residue of LORR (n = 5 rats per group).

Figure 2.

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.

Figure 3.

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).

Figure 4.

DHM antagonizes EtOH-induced GABAAR 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 GABAAR currents by picrotoxin (PTX; a GABAAR antagonist; 100 μm) as a baseline to calculate the magnitude of GABAAR-mediated extrasynaptic tonic currents (Itonic). Bath application of EtOH (60 mm, E 60) increased Itonic and mIPSCs. DHM (0.3 and 1.0 μm) antagonized these EtOH effects. B, C, Summary of Itonic and mIPSC area in response to EtOH and DHM (n = 8 neurons/3 rats). D, DHM (3 μm) antagonism on acute EtOH-induced GABAAR potentiation was reversed by 10 μm flumazenil. E, F, Summary of Itonic 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 GABAAR-mediated currents. H, I, Summary of Itonic and mIPSC area in response to EtOH and quercetin (n = 5 neurons/3 rats). *Statistical significance versus drug 0.

Figure 5.

DHM is a positive modulator of GABAARs 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 Itonic 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 GABAAR-mediated Itonic and mIPSCs were reversed by flumazenil (F; 10 and 100 μm). All GABAAR currents were blocked by bicuculline (GABAAR antagonist; Bic; 10 μm; gray dashed line). E, F, Summary (% of DHM-induced current) of D1 enhancements of Itonic 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).

Figure 6.

DHM potentiates GABAAR-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 Itonic and mIPSCs (n = 9–10 neurons). *Statistical significance versus drug 0. C, DHM (1 μm) enhanced GABAAR currents evoked by focal puffs of 10 and 300 μm GABA. D, The concentration–response curve of GABAAR 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).

Figure 7.

DHM prevents EtOH exposure/withdrawal-induced alteration in GABAAR 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 GABAAR 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 GABAAR subunit, whereas E+D/W treatment prevented this increase. DHM did not produce changes in α4 GABAAR subunit protein (n = 3/group). *Significant difference versus the V/W group.

Figure 8.

DHM prevents EtOH exposure/withdrawal-induced GABAAR 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 Itonic and mIPSCs in vehicle-treated rats. B, In the E/W group, EtOH did not increase Itonic, but greatly enhanced mIPSC area. C, In the E+D/W group, EtOH increased Itonic and mIPSCs similar to those of the V/W group. D, The responses of Itonic 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 Itonic 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.

Figure 9.

DHM potentiates GABAAR-mediated inhibition in EtOH pre-exposed cultured neurons. Coadministration of DHM with EtOH prevents EtOH exposure/removal-induced GABAAR plasticity in vitro. A, B, In cultured hippocampal neurons (DIV13–DIV14) 24 h after EtOH exposure (60 mm, 30 min), DHM still enhanced both GABAAR-mediated Itonic (A) and mIPSC area (B) concentration dependently without tolerance (compare Fig. 6A,B; n = 8–9 neurons per group). *Significant difference versus drug 0 (n = 8–9 neurons per group). C, Coadministration of EtOH with DHM prevents EtOH exposure/removal-induced GABAAR plasticity. Representative Western blot shows cell-surface expression (sur) versus total (tot) expression of GABAAR α4 subunit in cultured hippocampal neurons (DIV13–DIV14) detected 24 h after the four treatments of V/W, E/W, E+D/W, or D/W, respectively. β-Actin is shown as a loading control and was not detectable on cell surfaces. D, Quantification of surface GABAAR α4 protein (% V/W). Surface signal was normalized to the respective β-actin signal (vehicle, 100%). EtOH induced a 1.5-fold increase in surface expression of GABAAR α4 protein, while E+D prevented this increase (n = 5/group). *Significant difference versus the V/W group.