The Study Of Chloroquine Interfering In Epileptogenesis Induced By Pentylenetrazole

Epilepsy, being one of the most common disorders in the central nervous system, is characterized by recurrent seizures with spontaneous episodes of synchronous neuronal hyperexcitability. Due to complex etiology for epilepsy, the mechanism of epileptogenesis remains to be fully elucidated. Therefore the strategies to planning treatment and prevention for it become a very difficult and challenging problem. Our previous studies and the literature reports have demonstrated that there are some degree disorders of neuro-immuno-endocrine network in the process of epileptogenesis and relapse. Based on the facts mentioned above, Prof. Zhu at first time in 1998 proposed the theory that epileptogenesis is closely related to the imbalance of neuro-immuno-endocrine network, and indicate that the neuronal factor plays major role, whereas the immunologic and endocrinal factors play modulator functions in the epileptogenesis. This theory provides a new route for the planning treatment and prevention of epilepsy. For this reason, how to maintain or modulate the balance status of the network is significantly important for this disease. Most previous studies elucidated the role of neuronal, immunologic, or endocrinal factors in the epileptogenesis, respectively. The facts of being considered the role of single factor only have resulted in the outcomes that the treatment and prevention of epilepsy are dissatisfied. We, therefore, believe that multiple factors, such as neural, immunologic, and endocrinal factors, and the interaction between them, should be fully thought over when strategies to planning treatment and prevention for this disease are made.Chloroquine is a traditional antimaleria drug. It has been subsequently shown to have immunomodulatory properties, suppressing the production/release of tumor necrosis factor-α(TNF-α) and interleukin 6 (IL-6), that have encouraged its application in the treatment of autoimmune diseases, such as rheumatoid arthritis, lupus erythematosus, and sarcoidosis. Given its anti-inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. Based on the effects of chloroquine on some diseases related to the immune properties, we raise the hypothesis that this drug might be of some use for the management of epilepsy. Up to now, there are no related reports in the literatures. In order to test this hypothesis, chloroquine is used as an interfering agent to be observed the effects on the seizures induced by pentylenetetrazole (PTZ) in rats. The aim of the present study is attempt to explore the effects of chloroquine on: (1) the behaviors and electroencephrogram (EEG) recording in the rat with seizures induced by PTZ; (2) the inhibition of glial fibrillary acidic protein (GFAP), proliferation cell nucleus antigen (PCNA) and Cyclin D1 in the brain of rat with seizures induced by PTZ; (3) the expression of glutamate (Glu) and N-methyl-D-asperate receptor (NMDAR1, NR1) in the brain of rat with seizures induced by PTZ; (4) suppressing the production and release of interleukin-1β(IL-1β) and TNF-αin the brain of rat with seizures induced by PTZ; (5) the modulation of estrogen receptor (ER) and progesterone receptor (PR) in the brain of rat with seizures induced by PTZ; (6) the changes of calcium/calmodulin-dependent protein kinaseⅡ, CaMKⅡ)in the expression in the brain of rat with seizures induced by PTZ.The present dissertation is classified into six parts. The design and the results of part 1-5 are as follows. Forty-eight male adult Sprague-Dawley (SD) rats (weighting 180-230g), were randomly divided into the control group (n=12, injected with 0.9% of 6μl isotonic saling solution i.c.v.), the PTZ group (n=18, 60mg/kg, injected with PTZ i.p.) and the chloroquine interfering group (n=18, PTZ was injected i.p. 2h after 0.61mg/kg of chloroquine had been injected i.c.v.). Each group was further classified into 1h, 2h, 4h, 8h, 12h, and 24h subgroups. The behaviors and EEG of rats were observed and recorded within 2h after injection. The intensity of seizure behaviors was evaluated according to Smialowski's method. The expressions of GFAP, PCNA, Glu, NR1, IL-1β, TNF-α, ER, PR, and CaMKⅡwere inspected with immunostaining. The content of Cyclin D1, NR1, TNF-α, ER, and CaMKⅡwas examined with Western blotting. The sections of immunostaining and the bands of Western blotting were semiquantitatively analyzed by digital image analysis system.The results of part 1-5 in the present study were as follows. (1) There was no seizure activity in the control group. Severe seizure activity was observed in the PTZ group (Ⅳ-Ⅴclass), whereas slight seizure activity (Ⅰ-Ⅲclass) appeared in the chloroquine interfering group (P<0.05). EEG recordings showed no epileptic spikes in the control group, but there were high amplitudes with higher rates in the PTZ group, low-amplitude and slow oscillatious in the chloroquine interfering group. (2) The expressions of GFAP and PCNA was up-regulated in the PTZ group, especially in hippocampal area, compared with chloroquine interfering and control groups (P<0.05). The content of Cyclin D1 in the PTZ group had significantly higher in hippocampus and cerebral cortex than those of the control and chloroquine interfering groups (P< 0.05). (3) Glu and NR1 were markedly increased in the PTZ group when compared with the control group, especially in the hippocampal area (P<0.05). There were no differences in the expressions of Glu and NR1 between the chloroquine interfering group and the control group. (4) Both IL-1βand TNF-αin the PTZ group were increased in the expression compared with the control group (P<0.05). There were no differences observed between the chloroquine interfering group and the control group (P>0.05). (5) The expression of ER in hippocampus and cerebral cortex in the PTZ group was increased (P<0.05), whereas there were no significant differences observed between the chloroquine interfering group and the control group (P>0.05). The expression of PR in the brains was decreased, while there were no significant differences observed between the chloroquine interfering group and the control group (P>0.05). (6) The expression of CaMKⅡwith immunohistochemistry and Western blotting in hippocampus and cerebral cortex was no significant differences observed among PTZ, chloroquine interfering and control groups (P>0.05).According to the results of our experiment mentioned above, the conclusions are made as follows. (1) Chloroquine has the abilities to restrain the seizure activities and seizure electrical discharge. (2) It can inhibit the functions and proliferation of glial cells in the hippocampus and cerebral cortex of rats with seizures induced by PTZ; (3) It can interfere in the signal transduction pathway of Glu and NR1 in the brain of rat with seizures induced by PTZ; (4) Both IL-1βand TNF-αin the brain of rat with seizures induced by PTZ can be suppressed in production and release by chloroquine; (5) It, by attenuating the expression of ER and enhancing the expression of PR, may have effects on brain excitability mediated by these hormones; (6) It may play essential role in the brain excitability by increasing the activities of CaMKⅡand not enhancing the expression of CaMKⅡ.The mechanism of chloroquine anticonvulsant may include several aspects. (1) Chloroquine has ability to combine with the secondary structure of DNA, which blocks its replication. (2) It increases the pH of lysosomal and trans-Golgi network vesicles, disrupts several enzymes including acid hydrolases and inhibits the post-translational modification of newly synthesized proteins; (3) It modulates iron metabolism within human cells by impairing the endososomal release of iron from ferreted transferring, thus decreasing the intracellular concentration of iron. This decrease in turn affects the function of several enzymes involved in pathways leading to replication of cellular DNA and expression of different genes. (4) It can combine with CaM by dehydration or static electricity, which affects the activities of CaM and its related enzymes, resulting in the decrease of intracellular Ca2+ influx and the excitation of the tissue. (5) The most important function of chloroquine affecting the epileptogenesis may be its immunomodulatory properties, suppressing the production and release of cytokines, such as TNF-α, IL-1β, and IL-6.In conclusion, chloroquine can affect the epileptogenesis and its development by several mechanisms. These effects may be significantly important in the value of theory and clinical practice for the planning treatment and prevention and elucidating the mechanism of epilepsy.Part Six In order to explore the molecular mechanism of signal transduction between astrocytes (Ast) and neurons the effects of coriaria lactone (CL)-activated Ast conditioned medium (ACM) on the expression of Glu and GluR2 in the brain of rat were observed. The cultured Asts were divided into the control group (no added any stimulating substances) and the CL group (2.5×10-5mol/L). Then the ACM was injected intracerebraventrically (i. c. v.). Forty male adult SD rats were randomly divided into the control group (n=8, 10μl ACM injected i. c. v., no added stimulate substance,) and the CL group (n=32, 10μl CL-activated ACM injected i. c. v.). The behaviors of rats were observed and the expression of Glu and GluR2 in the cerebral cortex and hippocampus of rats was demonstrated with immunohistochemistry and immunofluorescence. The content of GluR2 was tested with Western blotting. The rats injected with CL-activated ACM showed seizure activities, whereas the rats of the control group showed no seizure activities. The expression of Glu in cerebral cortex and hippocampus in the brains injected with CL-activated ACM was increased (P <0.05), but the expression of GluR2 in the cerebral cortex and hippocampus was attenuated when compared with the control group (P <0.05). The results of GluR2 inspected with Western blotting in the brains were the same as the results of GluR2 tested with immunohistochemistry and immunofluorescence (P <0.05). We, therefore, believe that CL-activated ACM can enhance the expression of Glu and attenuate the expression of GluR2 in the brain of rat. These results provide some essential data to elucidate the molecular mechanism of signal transduction between the Asts and the neurons.