| PurposeEpilepsy, one of the most common neurological disorders, is induced by abnormal electrical discharge, manifestes by recurrent seizure. According to the WHO statistics, there were about50million epilepsy patients worldwide. In2003, the national epidemiological survey of epilepsy demonstrated that the morbidity rate of epilepsy was7.0‰. Epilepsy duration long high recurrent disability seriously affects the patient’s quality of life. Clinical commonly used antiepileptic drug, cannot effectively control epileptic seizures and there were about40%of intractable epilepsy can not be controlled with drugs. Beside, most antiepileptic drugs have a variety of acute or chronic adverse reactions and side effects, and some drugs are expensive, long-term use to family and society bring heavy psychological pressure and great economic burden. Only clear the pathogenesis of epilepsy, and can guide high efficiency with good security antiepileptic drug research and development. Seizures are caused by the brain excitatory and inhibitory dynamic imbalance of neurons, and increased neuronal excitability caused abnormal synchronization discharge. Ion channels are responsible for the central nervous system excitability activity (that is, the neurons action potential conduction) and the formation of neural circuits (that is, synapses between neurons signal transmission) at the core of the artifacts. Any ion channel gene mutation may have alienated the normal function of the channel proteins, caused the central nervous system excitability-inhibitory dynamic imbalances, and triggered neurons abnormal synchronization discharge, eventually. Ion channels contain voltage-gated ion channels, such as Voltage-gated K+channel and voltage-gated Na+channels, and ligand-gated ion channel, such as NMDA receptor channel. Recently, With the development of molecular biology patch clamp techniques, ion channels has increasingly aroused people’s attention in the pathogenesis of epilepsy research.From the perspective of electrophysiology taking Experimental models simulating human epilepsy seizures to study the pathogenesis of epilepsy has become the research hotspot in the field. Low-Mg2+solution induced spontaneous recurrent epileptiform discharges (SREDs) in cultured hippocampal neurons model is the internationally recognized imitating humans acquired epilepsy cell model. And Low-Mg2+solution induced continuous epileptiform high-frequency bursts (SEs) model is imitating human status epilepticus cell model. The two cell models have been widely used in biochemical, electrophysiological and the molecular biology mechanism in epilepsy and in the antiepileptic drug screening researchs.4-aminopyridine (4AP) induced epileptiform events in hippocampal neurons sharing electrographic similarities with the seizure discharges seen in patients with temporal lobe epilepsy is a classic in vitro epilepsy model for screening of antiepileptic drug which could objectively reflect the antiepileptic drug effect. Epilepsy is known as "Xian Bing" in Traditional Chinese Medicine (TCM). Ancient doctors take "Stagnation of Gan, Abnormal Dispersion of Gan" as one of the basic pathogenesis of epilepsy and "Treatment from Gan" as a treatment method for epilepsy. Under the guidance of this TCM theory, the prescription of "Chaihu Shugan Tang" displayed objective curative effects in the clinical prevention and treatment of epilepsy. Further studies found that "Chaihu Shugan Tang" could decrease the level and the latency of seizure in PTZ-kindling rats, reduce the level of seizure in Li-pilocarpine incuded rat refractory epilepsy model. Based On these results, we have gradually focused on the effect of saikosaponin a (SSa), a active ingredient from Bupleurum, the monarch drug in the prescription. We found that SSa could inhibit epileptic seizure in the MES model, the acute PTZ model and the Li-pilocarpine model. The results.suggest that SSa has good anticonvulsant effects on experimental epilepsy models.In order to further investigate the antiepileptic mechanism of SSa, using the patch clamp technique we observed the inhibition effects of SSa on Low-Mg2+induced epileptiform discharges in cultured hippocampal neurons and4AP induced epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices. Furthermore, we observed the moderating effects of SSa on the NMDAR current, the sodium currents and the potassium currents in rat hippocampal neurons. To clarify the antiepileptic mechanism of SSa and provide experimental bases for the TCM theory of "Treatment form Gan".Methods1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry Technique Newborn SD rats (<24h) were sacrificed and the hippocampus was separated in sterile conditions. Cells were plated at a density of2.5×104cells/cm2onto a glial support layer that was previously plated onto poly-L-lysine-coated (0.05mg/ml) glass coverslips and cultured with Neurobasal-A medium supplemented with2%B-27. On the10th day, cultures were utilized for identification with Ⅲ β-Tubulin, GFAP and DAPI by Fluorescent Immunocytochemistry Technique. The percentage of green fluorescence positive cells in blue fluorescent cells was determined.2. Low-Mg2+induced injury of cultured hippocampal neurons and the protective effects of SSaCultures in the10th day were randomly divided into control group, Low-Mg2+group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM).The protective effects of SSa on Low-Mg2+induced cultured hippocampal neurons were evaluated with III β-Tubulin and DAPI by Fluorescent Immunocytochemistry Technique.3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neuronsCultures in the10th day were randomly divided into control group, phenytoin group (phenytoin,50μM), and SSa group (SSa,1μM). Using whole-cell patch-clamp recording, changes of the Vm before and after different treatments in cultured hippocampal neurons were measured. And the excitability of cultured hippocampal neurons was measured as the number of action potentials (AP) induced by a depolarizing current (150pA,600ms) injection. The change rates of Vm and evoked AP were determined to evaluate the effects of SSa on the excitability of cultured hippocampal neurons. 4. Induction of SREDs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SREDsCultures in the10-14th day were utilized for experimentation. A24h maintenance culturing following3h exposure of low-Mg2+solution to cultured hippocampal neurons was employed to induce SREDs. Using whole-cell patch-clamp recording, cultures displayed SREDs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SREDs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SREDs in cultured hippocampal neurons.5. Induction of SEs by Low-Mg2+treatment of cultured hippocampal neurons and the inhibition effects of SSa on SEsCultures in the10-14th day were utilized for experimentation. SEs was induced by exposing cultured hippocampal neurons to low-Mg2+solution for more than10minutes. Using whole-cell patch-clamp recording, cultures displayed typical SEs were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of SEs frequency before and after applying SSa was determined to evaluated inhibition effects of SSa on SEs in cultured hippocampal neurons.6. Induction of epileptiform discharges in CA1pyramidal neurons in rat entorhinal cortex-hippocampus slices induced by4AP solution treatment and the inhibition effects of SSaSprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400 μm in thickness) that included the entorhinal cortex and hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). To induce epileptiform events, slices were superfused with ACSF containing100μM4AP for20-40min. Using whole-cell patch-clamp recording, CA1pyramidal neurons displayed typical epileptiform discharges were randomly divided into normal saline group, valproic acid group (VPA,1mM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). The percentage inhibition of epileptiform discharges frequency and duration before and after applying SSa were quantified to evaluated inhibition effects of SSa on epileptiform discharges induced by4AP treatment entorhinal cortex-hippocampus slices.7. Effects of SSa on NMDA-evoked current in cultured hippocampal neuronsThe10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, NMDAR current was evoked by rapid local application of100μM NMDA to the cultured hippocampal neurons through a’Y-tube’ perfusion system. The percentage inhibition of peak amplitude of NMDAR current was determined to evaluate the moderating effects of SSa.8. Effects of SSa on Na+current in cultured hippocampal neuronsThe10-14d cultured hippocampal neurons were randomly divided into normal saline group, phenytoin group (phenytoin,50μM), and SSa groups (SSa,0.1μM,0.3μM,0.5μM,1μM,2μM and4μM). Using whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV, and a30 ms depolarization voltage step from-70mV to0mV was used to evoke INat. The percentage inhibition of peak amplitude of INap and INat were determined to evaluate the moderating effects of SSa.9. Effects of SSa on K+current in CA1pyramidal neurons in rat hippocampus slicesSprague-Dawley rats (25-50days old) were anesthetized with isoflurane and decapitated. The brains were rapidly removed and transverse brain slices (300-400μm in thickness) that hippocampus were cut with a Vibratome. The slices were left undisturbed in an incubation chamber for1h for stabilization at37℃in artificial cerebrospinal fluid (ACSF). Using whole-cell patch-clamp recording, to elicit ITotal, a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s, to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.he Steady-state deactivation currents of IA were elicited with an80ms test pulse to+50mV proceded by120ms prepulses to potentials between-90mV and+10mV. The effects of SSa on activation and deactivation properties of ITotal, Ik and IA were evaluated.Outward K+currents were generated by a depolarizing pulse+10mV delivered from a holding potential of-80mV4AP-sensitive and TEA-sensitive K+current were obtained by application of20mM TEA or4mM4AP, respectively. Effects of SSa on4AP-sensitive and TEA-sensitive K+currents were evaluated.10. Statistical analysisStatistical analysis was performed using Statistical Product and Service Solutions13.0(SPSS13.0) software. All results were expressed as the mean±SEM. The effects of SSa on Vm, AP, SREDs, SEs,4AP induced epilpetiform discharges, NMDAR current, INaP and Inat were evalulated by one-way ANOVA with LSD or Dunnett’s T3, and the effects on K+currents were by paired t-test. Values of P<0.05were considered to be significant.Results1. Primary cultured rat hippocampal neurons and identification of hippocampal neurons with Fluorescent Immunocytochemistry TechniqueMicroscopically, cultures in the10th d scattered in the growth, cell body full, refraction strong, axons form a dense network of nerve cells. Most of the cells were dyed by green fluorescence and III P-Tubulin positive green fluorescence accounted for96.132%±2.164%of the total cells.2. Protective effects of SSa on Low-Mg2+induced injury of cultured hippocampal neuronsMicroscopically, after incubated with Low-Mg2+solution the hippocampal neurons’bodies were shrinkage, even dissolve, axon fracture. After application of SSa the cells’bodies full and synaptics were complete. The results suggested SSa has protective effects on Low-Mg2+induced injury of cultured hippocampal neurons.3. Effects of SSa on resting membrane potential (Vm) and excitability of cultured hippocampal neuronsThere was no significant difference between groups in Vm of cultured hippocampal neurons (F=1.997, P=0.155). Compared with normal saline group, SSa has no significant effects on Vm (P=0.056). There was significant difference between groups in the inhibition rates of AP of cultured hippocampal neurons (F=108.178, P<0.001). Compared with normal saline group, SSa significantly decreased the number of AP in cultured hippocampal neurons (P<0.001). These results suggested that SSa could inhibit the excitability of cultured hippocampal neurons with no effects on the membrane properties of the neurons.4. Inhibition effects of SSa on SREDs induced by Low-Mg2+treatment of cultured hippocampal neuronsUsing whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. While,24h following exposure to low-Mg2+solution, whole-cell current-clamp recordings in pBRS showed a permanent plasticity change evidenced by the presence of SREDs.After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SREDs reduced gradually or extinguished evenly. The inhibition of SSa on SREDs was reversible after being washed out with pBRS. These results suggested the inhibition effects of SSa on SREDs were reversible. Different dose of SSa had different effects on SREDs. Compared with normal saline group SSa significantly increased the percentage inhibition of SREDs at any does and the percentage inhibitions were19.167±4.182%,66.433±7.95%,95.160±6.317%,98.000±4.216%and98.571±4.518%(P<0.001) except0.1μM (P=0.170). SSa inhibited SREDs in a concentration-dependent manner with an IC50=0.42μM.5. Inhibition effects of SSa on SEs induced by Low-Mg2+treatment of cultured hippocampal neuronsUsing whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in the hippocampal culture preparations. Removal of MgCl2(low-Mg2+) from the recording solution for more than10minutes resulted in SEs cultured hippocampal neurons.After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of SEs reduced gradually or extinguished evenly. The inhibition of SSa on SEs was reversible after being washed out with low-Mg2+solution. These results suggested the inhibition effects of SSa on SEs were reversible. Compared with normal saline group SSa significantly increased the percentage inhibition of SEs at any does (P=0.005and P<0.001), and the percentage inhibitions were0.362±0.314%,12.071±5.794%,43.418±4.441%,73.626±3.384%,93.538±4.474%and97.798±2.323%. SSa inhibited SEs in a concentration-dependent manner and the IC50was0.62μM.6. Inhibition effects of SSa on epileptiform discharges induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slicesUsing whole-cell patch-clamp recording, control neurons displayed intermittent spontaneous action potentials that were consistently observed during basal activity in CA1pyramidal neurons in entorhinal cortex-hippocampus slices. After superfusion of ACSF containing100μM4AP on entorhinal cortex-hippocampus slices for20-40min a typical epileptiform discharges in CA1pyramidal neurons was caused.After application of different does of SSa (0.1μM,0.3μM,0.5μM,1μM,2μM or4μM), the frequency and duration of epileptiform discharges reduced gradually or extinguished evenly. The inhibition of SSa on epileptiform discharges was reversible after being washed out with ACSF containing100μM4MAP.These results suggested the inhibition effects of SSa on epileptiform discharges induced by4AP were reversible. There was significant difference between groups in the inhibition rates of the frequency and duration of epileptiform discharges in CA1pyramidal neurons (F=457.286, P<0.001). Compared with normal saline group SSa significantly decreased the duration of epileptiform discharges at any does and the percentage inhibitions were26.448±6.788%,40.059±3.264%,57.212±3.231%,67.928±1.908and74.876±2.380%(P<0.001) except at0.1μM (P=0.145). And SSa significantly decreased the frequency of epileptiform discharges at any does and the percentage inhibitions were7.750±2.053%,25.875±2.475%,43.250±1.982%,57.250±3.105%,68.625±3.114%and74.750±2.493%(P<0.001). there was no significant difference between VPA group and control group in the frequency of epileptiform discharges (P=0.170). SSa inhibited epileptiform activities induced by4AP in CA1pyramidal neurons in entorhinal cortex-hippocampus slices in a concentration-dependent manner and that the IC50was0.70μM.7. Inhibition effects of SSa on NMDAR current in cultured hippocampal neuronsUsing whole-cell patch-clamp recording, after local puff of NMDA (100μM) through the "Y-tube" perfusion system, a robust inward current was evoked which could be completely inhibited by APV, a NMDAR blocker.There was significant difference between groups in the inhibition rates of NMDAR current of cultured hippocampal neurons (F=662.526, P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of NMDAR current at any does and the percentage inhibitions were1.070±0.136%,6.108±0.245%,15.015±0.219%,29.853±1.679%,37.726±1.956% and39.224±2.433%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.62μM.8. Inhibition effects of SSa on INaP and INat in cultured hippocampal neuronsUsing whole-cell patch-clamp recording, INaP was evoked by slow depolarizing voltage-ramps from-80mV to+20mV and INat was evoked by a30ms depolarization voltage step from-70mV to0mV. The two currents could be abolished by1μM TTX.There was significant difference between groups in the inhibition rates of INaP of cultured hippocampal neurons (F=4090.738,P<0.001). Compared with normal saline group, SSa significantly increased the percentage inhibition of INap at any does and the percentage inhibitions were3.483±0.097%,6.722±0.186%,21.284±2.012%,58.104±1.546%,70.546±0.991%and72.504±0.991%(P<0.001) and the inhibition effects were in a concentration-dependent manner with an IC50=0.84μM. There was significant difference between groups in the inhibition rates of INat of cultured hippocampal neurons (F=3342.278, P<0.001), while there was no significant difference between normal saline group and any does of SSa group (P<0.001). These results suggested that SSa display a selective inhibition effects on INaP.9. Effects of SSa on ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slicesUsing whole-cell patch-clamp recording, to elicit ITotal, the holding potential was-50mV and a300ms hyperpolarizing prepulse to-120mV was followed by a series of400ms steps from-60to+60mV in10mV increments, delivered every10s and to elicit Ik, a similar protocol was used, but a100ms interval at-40mV was inserted after the prepulse. IA was calculated by point-by-point subtracting Ik from ITotal.After application of1μM SSa, the current amplitudes of ITotal and IA were significant increased (P<0.001), while there was no significant changes in current amplitudes of IK (P=0.157).10. Regulation effects of SSa on the activation properties of ITotal, Ik and IA in CA1pyramidal neurons in hippocampus slicesThe Ⅰ-Ⅴ relationships for ITotal, Ik and IA were determined by Clampfit10.0soft. The increasing effects of SSa on were voltage dependent. As the membrane potential was stepped to more depolarizing values, the amplitudes of ITotal, and IA were significantly increased after application of1μM SSa, while there was no significant change in Ik.1μM SSa caused a significant change in voltage for half-maximal-activation (V1/2) for IA (28.583±0.588vs.22.317±0.662mV, t=36.729, P<0.001) with slope factor (k)(21.933±1.472vs.23.617±1.592, t=-2.139, P=0.085). The steady-state activation curves showed that SSa significantly negative shifted the voltage-dependence of the activation of IA with no change in k. There was no significant change in V1/2of ITotal (32.467±0.432vs.31.700±1.014mV,t=2.43, P=0.059), nor did the k change (33.483±1.057vs.32.867±1.648, t=2.116, P=0.088). Similarly, no significant change was observed in V1/2for Ik (28.350±1.058vs.28.450±1.810mV, t=-0.249, P=0.813) as well as k (30.233±0.942vs.31.233±0.977, t=-2.421,P=0.06).11. Regulation effects of SSa on the steady-state inactivation properties of IA inCA1pyramidal neurons in hippocampus slices Furthermore the moderate effects of SSa on the steady-state inactivationproperties of IA was observed. The the inactivation current was evoked by the protocol as follow. Neurons were held at-50mV and currents were elicited with an 80ms test pulse to+50mV preceded by120ms prepulses to potentials between-90mV and+10mV. Application of1μM SSa caused a significant depolarizing shift of the voltage-dependent steady-state inactivation of IA, and a significant changes of V1/2(t=-82.750, P<0.001), while there was no significant changes in k (t=-2.436, P=0.059).12. Regulation effects of SSa on4AP-sensitive and TEA-sensitive K+currents in CA1pyramidal neurons in hippocampus slices20mM TEA or4mM4AP was applied to separate outward K+currents pharmacologically.1μM SSa significantly enhanced a transient,4AP-sensitive outward K+current in the presence of20mM TEA (t=-20.457, P<0.001), while it did not induce a significant modification in the amplitude of a late, TEA-sensitive K+current after application of4mM4AP (t=-2.329, P=0.053).Conclusion1. SSa acted as an anticonvulsant against low-Mg2+or4AP solution induced epileptiform discharges in hippicampal neurons in a concentration-dependent manner, and the inhibition effects were reversible.2. SSa inhibited the current amplitudes of NMDA-evoked current and Na+current, at the same time increased the current amplitude of K+current in hippocampal neurons, which suggests that SSa acts as an anticonvulsant by moderating on multiple mechanisms sites, not only on excitatory ligand gating ion channels but also on voltage-gated ion channels.3. SSa could selectively decrease INap while had no effects on INat, at the same SSa selective increase IA that was4AP-sensitive K+current, hyperpolarizing shifted the activation and depolarizing shift the steady-state inactivation of IA, while had no effects on IK. These results suggest that the antiepileptic mechanisms of SSa may be lie in selective regulating different channel subunits of Na+and K+channel. |
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