Schisandrin B, a dual positive allosteric modulator of GABAA and glycine receptors, alleviates seizures in multiple mouse models

Abstract

Epilepsy is a prevalent and severe neurological disorder and approximately 30% of patients are resistant to existing medications. It is of utmost importance to develop alternative therapies to treat epilepsy. Schisandrin B (SchB) is a major bioactive constituent of Schisandra chinensis (Turcz.) Baill and has multiple neuroprotective effects, sedative and hypnotic activities. In this study, we investigated the antiseizure effect of SchB in various mouse models of seizure and explored the underlying mechanisms. Pentylenetetrazole (PTZ), strychnine (STR), and pilocarpine-induced mouse seizure models were established. We showed that injection of SchB (10, 30, 60 mg/kg, i.p.) dose-dependently delayed the onset of generalized tonic-clonic seizures (GTCS), reduced the incidence of GTCS and mortality in PTZ and STR models. Meanwhile, injection of SchB (30 mg/kg, i.p.) exhibited therapeutic potential in pilocarpine-induced status epilepticus model, which was considered as a drug-resistant model. In whole-cell recording from CHO/HEK-239 cells stably expressing recombinant human GABA_A receptors (GABA_ARs) and glycine receptors (GlyRs) and cultured hippocampal neurons, co-application of SchB dose-dependently enhanced GABA or glycine-induced current with EC₅₀ values at around 5 μM, and application of SchB (10 μM) alone did not activate the channels in the absence of GABA or glycine. Furthermore, SchB (10 μM) eliminated both PTZ-induced inhibition on GABA-induced current (I_GABA) and strychnine (STR)-induced inhibition on glycine-induced current (I_glycine). Moreover, SchB (10 μM) efficiently rescued the impaired GABA_ARs associated with genetic epilepsies. In addition, the homologous mutants in both GlyRs-α1(S267Q) and GABA_ARs-α1(S297Q)β2(N289S)γ2L receptors by site-directed mutagenesis tests abolished SchB-induced potentiation of I_GABA and I_glycine. In conclusion, we have identified SchB as a natural positive allosteric modulator of GABA_ARs and GlyRs, supporting its potential as alternative therapies for epilepsy.

Keywords: GABAA receptors; Schisandrin B; epilepsy; glycine receptors.

Fig. 1. SchB induced potentiation of glycine-elicited currents (I_glycine) in the recombinant α1, α2, and α3 glycine receptors.

a Left, chemical structure of SchB; right, schematic diagram for patch-clamp recordings in GlyR-CHO cells. b Bar graphs illustrating SchB-induced potentiating effect on I_glycine. c Representative current traces for SchB-induced potentiation of I_glycine activated by EC_10-20 concentrations of glycine (40 μM for α1 receptor, 80 μM for α2 receptor, and 80 μM for α3 receptor). d The concentration-response curves for SchB-induced potentiation of I_glycine in cells expressing different α subunits. e Strychnine (STR)-induced inhibition of I_glycine was reversed by SchB 10 μM in recombinant α1 glycine receptors.***P < 0.001, ns not significant (P > 0.05), Student’s paired t-test. All data are expressed as the mean ± SD, n = 5-6.

Fig. 2. SchB induced potentiation of GABA-elicited currents (I_GABA) in recombinant GABA_A receptors (α1β2γ2L, α2β2γ2L, α4β3δ, and α6β3δ).

a Schematic diagram for patch-clamp recordings in GABA_AR-CHO/HEK-293 cells. b Bar graphs depicting the potentiating effect of SchB on I_GABA. c Representative current traces for SchB-induced potentiation of GABA receptors. d Concentration-response curves illustrating the enhancement of I_GABA by SchB on α1β2γ2L, α2β2γ2L, α4β3δ, and α6β3δ GABA_ARs. e SchB (10 μM) eliminated the pentylenetetrazole (PTZ)-induced inhibition of I_GABA in recombinant α1β2γ2L GABA_ARs. ***P < 0.001, ns not significant (P > 0.05), Student’s paired t-test. All data are expressed as the mean ± SD, n = 5–6.

Fig. 3. Effects of SchB on GABA- and glycine-evoked currents in cultured hippocampal neurons.

a The schematic diagram depicts the process of primary culture and patch-clamp recordings in hippocampal neurons. b, d Representative current traces for GABA_A receptors (GABA_ARs) and glycine receptors (GlyRs) recorded in cultured hippocampal neurons in the absence and presence of SchB (pre-incubated for 1 min). c, e Concentration–response curves of SchB for GABA_A and glycine receptors. Each data point represents the mean ± SD, n = 5. f SchB 10 μM reversed the inhibition effect of pentylenetetrazole (PTZ) (0.5 mM) on GABA-induced currents, and (g) of strychnine (STR) (30 nM) on glycine-induced currents. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s paired t test. Data are represented as mean ± SD, n = 5.

Fig. 4. Effects of SchB on seizure models in mice.

a Schematic diagram illustrating PTZ-induced seizures in mice. b The statistical results for latency to the onset of generalized tonic-clonic seizures (GTCS) in the PTZ-induced seizure model as a result of SchB (30 and 60 mg·kg⁻¹, i.p.) and positive drug sodium valproate (VPA, 300 mg·kg⁻¹, i.p.) treatment. c Pie charts for the reduction in incidence of GTCS and mortality rates in the PTZ-induced seizure model after the treatment of SchB and VPA. d Schematic diagram illustrating STR-induced seizures in mice. e The statistical results for latency to onset of GTCS in the STR (0.75 mg·kg⁻¹, s.c.)-induced seizure model in the absence and presence of SchB (30, 60 mg·kg⁻¹, i.p.) and VPA (300 mg·kg⁻¹, i.p.). f Pie charts for the reduction in mortality rates in the STR-induced seizure model in the absence and presence of SchB (10, 30, and 60 mg·kg⁻¹, i.p.) and VPA (300 mg·kg⁻¹, i.p.). g Schematic diagram for pilocarpine-induced seizures in mice. h Effects of SchB (30 mg·kg⁻¹, i.p.) on Racine score after pilocarpine administration (360 mg·kg⁻¹, s.c.). i Effects of SchB (30 mg·kg⁻¹, i.p.) on the intensity of the seizure attack in mice. *P < 0.05, **P < 0.01 compared with the vehicle control group using one-way ANOVA followed by Dunnett’s multiple comparisons test. ^#P < 0.05, ^##P < 0.01 compared with the vehicle control group using Wilcoxon rank-sum test. All data are expressed as the mean ± SEM, n = 8.

Fig. 5. Effects of SchB on mutations in GABA_A receptors associated with genetic epilepsies.

a Topology of GABA_AR α1 and γ2. This diagram displays the four transmembrane segments, as well as the N-terminal and C-terminal domains. The solid circles highlighted in various colors indicate the locations of the mutations under investigation. b Representative traces for the responses to the application of 1 mM GABA were recorded from CHO cells expressing mutant α1β2γ2L GABA_A receptors (α1: p.D219N, p.G251S; γ2L: p.R177G, p.P322A). c The average current density was obtained from CHO cells expressing wild-type α1β2γ2L (n = 8), p.D219N (n = 7), p.G251S (n = 7), p.R177G (n = 6), and p.P322A (n = 6) mutations. ***P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test, compared to the wild type. The data are expressed as mean ± SD. d The dose-response curve for GABA in wild-type α1β2γ2L (n = 8), p.D219N (n = 7), p.G251S (n = 7), p.R177G (n = 6), and p.P322A (n = 6) receptors was recorded. Statistically significant differences in the EC₅₀ values were verified by ANOVA followed by Dunnett’s multiple comparisons test. **P < 0.01 for p.D219N, ***P < 0.001 for p.G251S, p.R177G, and p.P322A (in Table 1). e Representative current traces for GABA (EC_10-20)-induced GABA currents in mutant α1β2γ2L GABA_A receptors in the presence and absence of 10 μM SchB (pre-incubation for 1 min). f Summary data for the effects of SchB on wild-type and mutant α1β2γ2 GABA_A receptors. All data are expressed as the mean ± SD, n = 5–6.

Fig. 6. Effects of mutations at sites important for other positive allosteric modulators of α1 GlyR on the activity of SchB.

a Topology of GlyR α1 subunit showing the four transmembrane segments, N-terminal domain, and C-terminal domain. The solid circles highlighted in different colors indicate the location of the investigated mutations. b Concentration-response curves for glycine in wild-type and mutant α1 GlyRs. Responses at indicated concentrations in each cell were normalized to the maximum glycine-evoked peak current. Each data point represents the mean ± SD, n = 5–6. c Representative current traces activated by EC_10-20 glycine of wild type and mutant α1 GlyRs before and after application of 10 μM SchB (pre-incubation 1 min). d Summary data for the effects of SchB on wild-type and mutant α1 GlyRs. ***P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test, compared to wild type. All data are expressed as the mean ± SD, n = 5.

Fig. 7. SchB-induced potentiation of GABA-evoked currents (I_GABA) in recombinant α1β2γ2L is mediated through sites independent of those of diazepam (DZP) and etomidate (ETO).

a Left, representative current traces for the potentiation of I_GABA by SchB in the absence and presence of flumazenil (Flu) (10 μM) in α1β2γ2L. Right, summary for SchB-induced potentiation in the absence and presence of Flu; ns not significant (P > 0.05), Student’s paired t-test. b Left, examples of current traces for the potentiation of I_GABA by SchB (10 μM) and DZP (1 μM) in the mutant channel α1(H129R)β2γ2L. Right, summary for the potentiation induced by SchB and DZP. ***P < 0.001, ns = not significant (P > 0.05), Student’s paired t-test. c Current traces for the potentiation of EC_10-20 GABA-elicited currents by SchB (10 μM) and ETO (10 μM) in the wild-type (WT) and mutant channel α1β2(N289S)γ2L. d Summary data for the potentiation induced by SchB (10 μM) and ETO (10 μM) in the WT and mutant channel α1β2(N289S)γ2L; ***P < 0.001, ns not significant (P > 0.05), Student’s unpaired t-test, compared to WT; **P < 0.01, Student’s paired t-test, compared to WT. All data are expressed as the mean ± SD, n = 5.

Fig. 8. The TM2 residues α1S297 and β2N289, located in the β+/α- subunit interfaces, are crucial for SchB-induced potentiation of GABA-elicited currents in recombinant α1β2γ2L.

a Sequence alignment of the transmembrane domains 2 (TM2) of human GlyR α1, GABA_AR α1, and β2 subunits. b Representative traces depicting EC_10-20-induced GABA currents in the absence and presence of 10 μM SchB, 100 μM pentobarbital (PB) or the co-application of the two compounds in wild type (WT), α1(S297Q)β2γ2L, α1β2(N289S)γ2L, and α1(S297Q)β2(N289S)γ2L GABA_ARs. c Summary data for the effects of SchB and PB on wild-type and mutant α1β2γ2 GABA_ARs. ***P < 0.001, ^###P < 0.001, ns = not significant (P > 0.05), one-way ANOVA followed by Dunnett’s multiple comparisons test, compared to the wild type. All the data are expressed as the mean ± SD, n = 5. d α1β2γ2L GABA_A receptor is composed of two α, two β and one γ2L subunit. Two GABA-binding sites are located at the interfaces of the two β+/α- subunits. The scheme illustrates the localization of the point mutations α1 S297 and β2 N289 in TM2. “+“ and “−“ indicate the positive and negative sides of the subunits. The red stars represent possible binding sites for SchB and PB.