| Epilepsy is a chronic brain disorder characterized by recurrent seizure cause by abnormal synchronized discharge of nerve cells. This disorder affects over 40 million people worldwide, and about 6 million people in China. Despite the availability of a wide range of antiepileptic drugs(AEDs), about one-third of individuals with epilepsy still experience seizures that do not respond to medication, and be named as patients with refractory epilepsy. Understanding the mechanisms that are involved in the generation of epilepsy should aid the development of novel drugs that modify the epileptic process.The targeting of neuronal ion channels, neurotransmitters, mitochondrial abnormalities, recombinant neural network, and signal transduction has been the primary approach to eliminate seizures. Over the past years an increasing body of clinical and experimental evidence has provided strong support to the hypothesis that inflammatory processes within the brain might constitute a common and crucial mechanism in the pathophysiology of seizures and epilepsy. Cytokines, the most basicelement of inflammation, actualize their biological effect through their combination with corresponding receptors and the downstream reactions. Inflammatory cytokines, including interleukin-1β(IL-1β) and TNFα, are released from astrocytes and microglia after seizures. These cytokines would trigger signaling pathways, such as NF-κB, COX-2, TLR, at neuron, glia cells, and blood-brain barrier. The primary issues studied, neuronal ion channels abnormalities, neurotransmitters disorders, recombinant neural network, blood-brain barrier dysfunction, may be the consequences of brain inflammation. Eventually these pathologic changes caused by inflammation will result in a rapid change in neuronal excitability and a decreased seizure threshold. To date, only few cytokines are thoroughly studied, like IL-1β, IL-6, TNFα, HMGB1. New cytokines involved in epileptogenesis means new therapeutic target.On the other hand, another important research area of refractory epilepsy is why it is resistant to medication, how does it become refractory. Inspired by the researches of oncology, multidrug transporter has been postulated to cause an enhanced extrusion of antiepileptic drugs back into the capillary lumen associated with an effective limitation of their brain penetration rates. Amongst these transporter proteins, P-glycoprotein(P-gp) is the most effective one. P-gp is highly increased in epileptogenic brain tissue of rodents and higher in AED-resistant rats than in responsive animals, and coadministration of the highly selective P-gp inhibitor, tariquidar, reverses AED resistance. Overexpression of P-gp can be found in neuron, endothelial cells, astrocyte, and correlated with AEDs resistance. Mechanism studies revealed that COX-2 may play a crucial role in synthesis of P-gp. The COX-2(PTGS2) is an enzymes catalyze the conversion of arachidonic acid to prostaglandins, and participates in almost all kinds of inflammation process. It recommends there is a close relationship between multidrug resistance, P-gp, and inflammation.Our previous clinical studies has demonstrated traditional Chinese medicine rhubarb could reduce seizure frequency. Its active ingredient Emodin may suppress EPSP of pyramidal neurons of CA1 by inhibiting excitatory neurotransmitter release and down regulate exitotoxicity conducted by glutamate and NMDA receptor. Emodin may affect lots of pathophysiological pathways such as reducing brain edema, up-regulating activity of SOD and Na+-K+ ATPase, down-regulating MDA. Rhubarb was first used as an anti-inflammatory medicine by Chinese herbalist doctors, and its anti-inflammatory mechanisms was confirmed in recent years. Lots of inflammatory cytokines, signal pathways, such as NF-κB, COX-2, can be modulated by emodin. It suggests us Emodin may play an interventional role in epilepsy via inflammatory pathways and consequently modulate multidrug resistance. To verify this hypothesis, we determined the cytokines alternation in KA induced epileptic rats and the effect of emodin. The P-gp, COX-2, NMDA receptor were also determined in KA induced epileptic rats to demonstrate the mechanisms underlined.Part 1: Effect of Emodin on Pathogenesis of Epilepsy in Ratsand its Mechanismvia Inflammatory PathwaysObjective: To determine theexpression of inflammatory cytokines in hippocampustissue of kainic acid induced epileptic rats, using rat RayBio? antibody microarray. And to determine the effect of emodin on these cytokines and the epileptic rats.Methods: Rats were divided into three groups, control(group C), model(group M), and treatment(group T). Kainic acid was intraperitoneally injected with a dose of 12mg/kg in rats of M and T. Ten minitus after KA administration, emodin was intraperitoneally injected with a dose of 200mg/kg in rats of T. Seizure was assessed according to Racine scale. Electroencephalogram was performed in all rats. Pathological changes of hippocampus were determined using hematoxylin and eosin stain and scanning electron microscope methods. The alternations of expression of inflammatory cytokines were determined by rat RayBio? antibody microarray.Results: Rats exhibited symptoms of seizure after KA injection, epileptic discharge could be observed on electroencephalogram. According to Racine scale, level of seizure insult was lower in group T. Neuronal cell loss can be detected in CA3, CA4 8 hours after KA injection, and increased until 48 hours. A great quantity of cytokines up-regulation or down-regulation could be detected after KA injection. Inflammatory cytokines IL-2, IL-3, IL-4, IL-5, IL-12,IL-13, TGF-β1, TGF-β2 were up-regulated shortly after KA injection, and returned to normal in 24 hours. Anti-inflammatory cytokines B7-1/CD80, CNTF, CNTF R, BASIC-FGF,and CSK were down-regulated after KA injection. Cytokines promote glycoprotein synthesis, like TGF-β1,TGF-β2,RAGE were up-regulated after KA injection. While cytokines inhibitedglycoprotein synthesis, FRACTALKINE were down-regulated. All these alternation of cytokines could be inhibited by emodin.Conclusion: Kainic acid model reproduces major features of temporal lobe epilepsy and is a relatively ideal experimental model. A great quantity of cytokines up-regulation or down-regulation could be detected after KA injection. Inflammatory cytokines were up-regulated while anti-inflammatory cytokines were down-regulated. These cytokines may interfere with COX-2 pathway through Jak-STAT, PI3K-Akt,and MAPK signaling pathways. Emodin suppresses intensity of seizures, epileptic discharge, up-regulation of inflammatory cytokines, and down-regulation of anti-inflammatory cytokines. Glycoprotein synthesis is also promoted and can be inhibited by emodin.Part 2: Effect of Emodin on Drug Resistance in Epileptic Ratsand its MechanismObjective: To determine the effect of Emodin on COX-2, P-glycoprotein, and NMDA receptor in epileptic rats, and analyze the underling mechanisms.Methods: Rats were divided into five groups, control(group C), model(group M), and emodin administrated(group E), Phenytoin administrated(group P), and Combined administration(group S). Brain capillaries were freshly isolated and incubated in confocal imaging chambers with the fluorescent P-gp substrate, NBD-CSA. P-gp transport activity was assessed using confocal laser scanning microscopy. P-gp, COX-2, and NMDA receptor of hippocampus were assessed using immunohistochemistry and Western blotting. Messenger RNA of P-gp, COX-2, and NMDA receptor were also assessed using RT-PCR method.Results: Fluorescence intensity of P-gp increased after seizure and reached its peak at day 9. Immunohistochemistry showed lots of COX-2 positive cells could be found in CA3 and CA4 in group M and P. There were no differences in COX-2 positive cells between groups E, S and normal control. The same pattern of positive cells could be detected in P-gp immunohistochemistry. NMDA receptor positive cells were increased in group M, E, P and S. Western blotting experiment showed P-gp and COX-2 protein were elevated in group M and P. Emodin could reverse their elevation in group E and S. NMDA receptor was up-regulated in group M, E, P and S. RT-PCR experiment showed amplification of MDR1, PTGS2 were elevated ingroup M and P.Emodin could reverse their elevation in group E and S. Amplification of NR1 was up-regulated in group M, E, P and S.Conclusion: Expression of P-gp, COX-2 protein, and NMDA receptoris highly elevated after seizure. Antiepileptic drug, Phenytoin, has no influences on P-gp, COX-2 protein, and NMDA receptor. Up-regulation of P-gp and COX-2 protein can be reversed by emodin. Emodin may modulate P-gp through COX-2 pathway, but not NMDA receptor.Conclusions1.Kainic acid model reproduces major features of temporal lobe epilepsy and is a relatively ideal experimental model. A great quantity of cytokines up-regulation or down-regulation could be detected after KA injection. Inflammatory cytokines were up-regulated while anti-inflammatory cytokines were down-regulated. These cytokines may interfere with COX-2 pathway through Jak-STAT, PI3K-Akt,and MAPK signaling pathways. Emodin suppresses the up-regulation of inflammatory cytokines, and down-regulation of anti-inflammatory cytokines.2.Expression of P-gp, COX-2 protein, and NMDA receptor is highly elevated after seizure. Antiepileptic drug, Phenytoin, has no influences on P-gp, COX-2 protein, and NMDA receptor. Up-regulation of P-gp and COX-2 protein can be reversed by emodin. Emodin may modulate P-gp through COX-2 pathway, but not NMDA receptor.3.Emodin suppresses intensity of seizures, epileptic discharge after KA injection. Emodin can enhance Antiepileptic effect of phenytoin... |
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