The Research Of Relationship Among IL-1β, IL-6 And Epileptogenesis

Epilepsy refers to a central nervous system clinical syndrome characterized by the recurrence of sudden, excessive and synchronous ultra-discharge of neurons. It is harmful to the people's health,decrease severely the patient's quality of life,and bring about heavy burden to society and family. Its etiopathogenisis is complicate and mechanisms of its onset are not yet completely clear. Thus, it has important meaning to explore its mechanism. Prof. Zhu Changgeng introduce first the theory of"imbalance of immune-neuro-endocrine network", which suggests that nervous factors are predominant in epileptogenesis, while immune and endocrine factors co-participate. Cytokine is a group of polypeptides or glycoproteins, which classically mediate and regulate immune response, stimulate haematogenesis and participate in tissue repair. However, the recent researches indicate that immune factors play an important role in nervous system. Neurons and glial cells express cytokine receptors, and they may synthesis and excrete many kinds of cytokines. Interleukin-1β(IL-1β) and Interleukin-6(IL-6) are two important cytokines . Their concentrations in serum of patients increase, which are conjected to relate with epileptic pathogenesis. Glutaminic acid (Glu) andγ-aminobutyric acid ( GABA ) are supreme excitatory and inhibitory neurotransmitter respectively which maintain the balance between excitation and inhibition of cerebral neurons, their imbalance is believed as one of initiate reasons of epileptic onset. Nitrogen monoxidum(NO) is a intracellular and intercellular message in many physiological activity, relate to many nervous system disease, and is considered as a neuromodulator. NOS content increase obviously in many encephalic regions of epilepstic animal induced by sound, electricity and some epilepsy-inducing agents, which indicate that NO relates to epileptic mechanism. Protein kinase A (PKA) is an important signal transduction molecule. cAMP is a second messenger within the cells, which can activate PKA and the latter can phosphorylate many intra-cellular proteins, then play biologic role. There are high-level of cAMP and its metabolic regulatory enzymes which play an important regulatory role in central nervous system. Researches suggested that cAMP-PKA signal transduction system participate in epileptic onset. Besides, glial cells play an important role in epileptic recurrence. Therefore, immunocytochemistry, electrophysiology, western blot, cell culture, flow cytometry and so on were utilized in this study to explore IL-1βand IL-6's influence on the intra-cellular signal transduction of neurons, neurotransmitter and glial cell cycle in epileptogenesis, in order to explore the relation between IL-1β, IL-6 and epileptic mechanism.PartⅠ: The effects on GABA and glutamate immunereaction in brain of rats with epilepsy induced by IL-1βor IL-6. Glutamate ( Glu ) and GABA are respectively the most aboundent excitatory and inhibitive amino acid neurotransmitters in the mammalian central nervous system ( CNS ) and relate directly with the pathogenesis of epilepsy. Interleukin-1 (IL-1) and Interleukin-6 (IL-6) are two important proinflammatory cytokines. It has two subtypes as IL-1αand IL-1β. In the brain IL-1βis the main subtype. Researches discovered that the concentration of IL-1βincreased obviously in the brain and cerebrospinal fluid of epileptic patients and animal models. In order to explore the relation between IL-1βor IL-6 and neurotransmitter, experimental rats were randomly divided into 6 groups : saline group , IL-1βgroup, IL-6 group, cardiazol group, IL-1ra (IL-1 receptor antagonist ) + cardiazol group and dexamethasone+ cardiazol group. Behaviour changes were observed, Glu and GABA were examined by means of immunohistochemistry in the cerebral cortex and hippocampus of rats. Results: Seizure in middle degree was observed in IL-1βgroup or IL-6 group. Seizure in severe degree was observed in cardiazol group. Compared with cardiazol group, the degree of epileptic onset was decreased in IL-1ra + cardiazol group. While no obvious seizure happened in dexamethasone+ cardiazol group. Compared with saline group, the expression of Glu was significantly increased, while the expression of GABA was obviously decreased in cerebral cortex and hippocampus in IL-1βgroup, IL-6 group and cardiazol group, the differences were significant. Compared with cardiazol group, the expression of Glu was decreased, and the expression of GABA was increased, their differences were significant in IL-1ra+cardiazol group and dexamethasone+cardiazol group. The above results indicated that the Glu content increased while GABA decreased in epileptic rats induced by IL-1βor IL-6, then neuronal excitability was elevated. It broke the balance between Glu and GABA, thus promoted epileptic onset and maintain. Immunosuppressive agent could inhibit seizure induced by cardiazol which is classic epilepsy-inducing agent, and influence the content of Glu and GABA, thus it might be effective to treat epilepsy.PartⅡ: Effect on electroencephalogram and the expression of NOS in cerebral cortex of epileptic rat induced by of IL-1βor IL-6. NO is an intra-cellular and intercellular message in many physiological activities and relate with many pathogenesis of nervous system diseases. NOS is synthetase of NO and reflect indirectly the content of local NO. Researches indicated that the content of NOS increased obviously in every region of animal brain after seizure induced by sound, electricity and some epilepsy-inducing agents. To explore the effect of IL-1βor IL-6 on the signal transduction passway, experimental rats were randomly divided into 6 groups : saline group , IL-1βgroup, IL-6 group, Dex +IL-1βgroup, Dex +IL-6 group and Dex group. The changes of behaviour, electroencephalogram and expression of NOS in cerebral cortex were observed by means of behaviour observation, electroencephalogram tracting and Western blot assay respectively. Behaviour observation showed that the extent of epileptic attack is middle in IL-1βand IL-6 group. There is no obvious epileptic attack in Dex +IL-1βgroup, Dex +IL-6 group and Dex group. Electroencephalogram of Cortex and hippocampus in IL-1βand IL-6 group showed vertex sharp transient wave ,sharp wave and composite wave and so on. There were no obvious epileptic waves in saline group, Dex+IL-1βgroup, Dex+IL-6 group. The frequency and amplitude of electroencephalogram in Dex group were decreased compared with the saline group. Western blot result indicated that the expression of NOS in cerebral cortex was significantly increased in IL-1βgroup and IL-6 group ( P<0.05) compared with the saline group, while there was no significantly difference in Dex +IL-1βgroup, Dex +IL-6 group and Dex group (P>0.05)compared with the saline group. The above results indicated that there were obvious epileptic wave in cerebral cortex and hippocampus in seizure rat induced by IL-1βor IL-6. The pretreatment of immunosuppressive agent might effectually inhibit the formation of epileptic wave. The signal transduction of epilepsy induced by IL-1βor IL-6 might relate to NO signal transduction passway.PartⅢ: Effect on the expression of PKAcβin the brain of epileptic rats induced by IL-1βor IL-6. cAMP-PKA pathway is an important intra-cellular signal transduction pathway. Excitatory Gs protein activate adenyl cyclase(AC), heighten cAMP which catalysis the phosphorylation of substrate proteins through activating protein kinase PKA, thus switch on a series of cascade reactions and produce biological effect. Therefore, PKA is an important link of intra-cellular signal transduction. To explore the effect on the expression of PKAcβin the brain of epileptic rat induced by IL-1βor IL-6, the expression of PKAβcatalysis subunit (PKAcβ)in cerebral cortex and hippocampus was observed by immunohistochemistry method in epileptic rat induced by IL-1βor IL-6. Rats were divided randomly into 4 groups: control group, IL-1βgroup, IL-6 group and dexamethason group. Results: Epilepsy was markedly attacked and immunereaction of PKAcβwas obviously increased in hippocampus of rats in IL-1βgroup and IL-6 group compared with control group, their differences were significant (P<0.05). Epilepsy was inhibited and immunereaction of PKAcβwas decreased in brain in dexamethason group compared with control group, their differences were significant (P<0.05). The above results indicated that IL-1β, IL-6 participated in epilepsy induction, which related to signal transduction mediated by PKA in the neurons. Immunosuppressive agent might suppress the signal transduction passway and then inhibit epileptic pathogenisis.PartⅣ: Effects of IL-1βor IL-6 on the cell cycle of astrocytes in cerebral cortex of neonatal rats. The CNS is a complicated network of neurons and glia. Astrocytes closely related to neurons. They not only have supporting, nutrient and protecting roles for neurons as described in traditional theory, but also take parts in the genesis, development and pathology of the CNS deseases. Thus, pathological changes of nervous system inevitably influence astrocytes. The new progress of epileptic genesis has shown that glial cell play an important role in epilepsy recurrence. To explore IL-1βor IL-6 whether or not promoted astrocyte's proliferation and function and thus participate in epilepsy induction, cell culture in vitro and flow cytometry were applied to detect the effects of IL-1βor IL-6 on astrocyte cell cycle in this study. Pure cultured cerebral cortex astrocytes were obtained and divided into two groups in which IL-1βor IL-6 were respectively added to the culture medium. The effects of IL-1βor IL-6 on astrocyte cell cycle were detected by means of flow cytometry after 6h,12h and 24h after the treatment. Results: IL-1βor IL-6 might cause obvious changes of cell cycle of astrocyte. The cell population of G1 phase decreased, while S and G2/M phases increased, the changes were significant at 12h. The above results indicated that IL-1βor IL-6 promoted astrocyte proliferation, which might participate in epilepsy induced by IL-1βor IL-6 with neurons.Summary: IL-1βor IL-6 could induce epilepsy onset. IL-1ra and GC inhibited this effect, which were confirmed by electroencephalogram detection. In seizure onset, on one hand, IL-1βor IL-6 activated AC-cAMP-PKA signal transduction passway of brain cells, produced a series of cascade reactions, and then made intra-cellular protein phosphorylation and biological effects through their specific receptors respectively. They might also increase NOS content and activation, which augmented Ca2+ concentration in brain or regulated genetic transcription. Seizure might be induced ubder the coaction of this above two signal transduction passway. On the other hand, IL-1βor IL-6 might enhance cerebral neuronal excitability by increasing excitatory neurotransmitter Glu and decreasing inhibitory neurotransmitter GABA through breaking the metabolic balance between Glu and GABA or regulating their genetic transcription levels. NMDA receptor could also be activated by increased Glu and NO, which could heighten the intra-cellular Ca2+ of neurons and the latter could produce excessive depolarization and abnormal discharge. IL-1 or IL-6 receptor antagonist and immunodepressant agent glucocorticoid could counteract the effects of IL-1 or IL-6 on PKA, NO,Glu and GABA. In addition, IL-1βor IL-6 in vitro could promote the proliferation of purified cultured astrocytes which might affect the normal physiological function of brain through interacting with neurons or forming glial scar. So, they could also facilitate the onset of seizures. To summary, the mechanism of IL-1 or IL-6 in inducing epilepsy is an extremely complicated process. They could mediate the epilepsy inducing signal through activating different signal transduction passway. For example IL-1 or IL-6 could change the content of Glu and GABA which related closely to seizure onset and then heighten neuronal excitability. They might also promote the progress of AC cell cycle and make Ast proliferate. The roles of neuron together with the roles of glial cells promote epilepsy formation and maintenance.