The Effects Of Chloroquine On Astrocytes And Expressions Of GluRr2、nNOS In The Rat With Epilepsy

Objective:1Observation the effectes of different intervention dose of chloroquine on glial fibrillary acidic protein(GFAP), amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA-GluR2) receptor and nitric oxide synthase(nNOS) expressions on Pentylenetetrazole(PTZ)-activated hippocampal astrocytes of rats. State of the effects of chliroquine on the proliferation index and neural index, to explore the role of chloroquine in the development of epileptogenesis.2Observation the effectes of different intervention dose of chloroquine on GluR2and nNOS expressions on PTZ-activated chronic epilepsy rats, observation of the effects of chloroquine on the index of astrocyte proliferation.Methods:Experiment1Epilepsy model in vitro and choric epilepsy model of rats1The cultivation of astrocytes in vitroNew born SD rats within24hours, male and female unlimited, after executed by broken neck, soaking in75%ethanol disinfection5min. Separation hippocampal, stripping capsule by asana microscope, sheer ground and centrifugal, press106/cm2inoculation in25cm2cell culture flask, cultured cell in37℃,5%CO2incubator, subcultring until cell with bottle90%, appraisal the astrocytes and divided into immune control group, PTZ group, PTZ and different dosage of chloroquine groups (25,50,75mg/L).2Preparation of chronic epilepsy model of ratsFifty healthy male SD rats randomly divided into control group(10rats) and chronic epilepsy model groups(40rats). Control group injected with saline according to40mg/kg, models of chronic epilepsy injected with PTZ according to40mg/kg, injected PTZ every day till successed of chronic epilepsy model. According to Racine marking where a row of at least5times made by two or more are considered modeling success. After the success of modeling, pentylenetetrazol injection by half. After the forty rats modeled successful, The forty rats were randomly divided into PTZ-induced epilepsy group and chloroquine intervention groups(20,40,60mg/kg,10rats). After PTZ injection and chloroquine treatment2hours, observe and record the behavior of5groups and the EEG changes. Chloroquine injected two weeks.Experiment2MTT detect the effects of chloroquine on hippocampal astrocytes of rats in vitroCultivation of astrocytes in vitro, methods in alexandrine. Appraisal the astrocytes and subcultring, according to8×104/cm2density inoculation96-well plates, cultured24hours by medium which including10%tire bovine serum, continue to cultivate24hours after joining100μl dosing medium each hole, each group of six complex hole. Abandon medium, washed2times with D-Hanks, using MTT colorimetric detect the activation of astrocytes in each group. In microplate measure on OD value, determination wavelength of490nm, reflect the activation of astrocytes by OD value.Experiment3Immune fluorescence detected the expressions of GFAP Cultured hippocampal astrocytes of rats, methods in alexandrine. Appraisal the astrocytes and subcultring, climbing slice, cultured cell in37℃,5%CO2incubator for24hours, compound drug with5%tire bovine serum medium, grouping, using immuno fluorescence test the expression of GFAP. Observation and photographic with fluorescence inverted microscope, analysis the IOD values by pathological picture analytical system, reflect the situation of each group by IOD.Experiment4Western Blot detect the expressions of GluR2and nNOS Cultured hippocampal astrocytes of rats in vitro and preparation of chronic epilepsy model of rats, grouping, methods in alexandrine. Extracted proteins on the ice. Using western blot detect the expressions of GluR2and nNOS of each group. Scan images, analyzes the experimental grayscale value. Comparing the different experimental protein’s expression quantity by grayscale value of purpose protein.Experiment5Immunohistochemical detect the expressions of GluR2and nNOS Preparation of chronic epilepsy model of rats, grouping, methods in alexandrine. Extraction experimental animals cerebral cortex and hippocampal organization on the ice, fixed by4%polyphosphate formaldehyde, routine organization processing, producer. Using immunohistochemistry detected the expressions of GluR2and nNOS in different tissue. Analysis images by HPLAS-1000high-definition color pathological graphic analysis system, each section in10by40times horizon random take10visions, counting the number of positive cells, taking its average;detecting the OD value of positive cells simultaneous, taking its average OD value.Results(One) Results of cell culture1Former generation training hippocampal astrocytes of rats, the astrocytes growth well, stick wall well, cell protruding from the different length of swelled, clear oval nuclei visible in the cytoplasm, and rich cytoplasmic, which visible in the fluorescence inverted microscopically. The former generation training astrocytes by immune fluorescence appraisal, GFAP immune response rate to90%.2The results of MTT:PTZ could activated the hippocampal astrocytes in vitro significantly, the chloroquine could significant inhibited the activation of astrocytes which activitied by PTZ, with the increasing of chloroquine dose, the inhibition gradually strengthened, among them with75mg/L concentration of chloroquine have the best inhibit effection.3The results of immune fluorescence:compared with control group, PTZ could activated the hippocampal astrocytes in vitro significantly, the expression of GFAP was increased (P<0.05), the different dose of chloroquine could significant inhibited the activation of astrocytes which activitied by PTZ, make GFAP express reduced, among them with75mg/L concentration of chloroquine have the best inhibit effection, the IOD value can be maintained in the normal range (P<0.05).4The results of western blot:compared with control group, the content of GluR2reduced in PTZ-activated group, with significant difference (P<0.05), compared with PTZ-activated group, with the increasing of chloroquine dose, the content of GluR2was increased, among them with75mg/L concentration of chloroquine have the best effection, with significant difference (P<0.05). Compared with control group, the content of nNOS was highest in PTZ group, with significant difference (P<0.05), with the increasing of chloroquine dose, the content of nNOS was reduced (P<0.05).(Two) The results of animal experiments1Behavior observation: most of the experiment animals first appeared Ⅱ class above attacks after intraperitoneal injection drug about7days, all of the animals appeared Ilclass above attacks after injection14days. The control group without epileptic seizures occur more commonly, compared with PTZ group, chloroquine intervention20mg/Kg group with no significant difference (P>0.05); chloroquine intervention40,60mg/Kg group with significant difference compared with PTZ group (P<0.05)2Electroencephalogram(EEG) recording:the control group without epileptiform discharges, appeared gently, match waveform; epilepsy group showed frequent high-amplitude sharp waves, spikes and slow wave or spike-slow wave complex, the intervention group showed slow-wave and small spines wave, with the intervention dose increase, epilepsy waves weakened.3The results of western blot:compared with control group, the content of GluR2reduced in PTZ-activated group, with significant difference (P<0.05), compared with PTZ-activated group, with the increasing of chloroquine dose, the content of GluR2was increased, among them with40,60mg/Kg concentration of chloroquine have the significant effection, with significant difference (P<0.05). Compared with control group, the content of nNOS was highest in PTZ group, with significant difference (P<0.05), with the increasing of chloroquine dose, the content of nNOS was reduced (P<0.05).4The results of immunohistochemical:the positive results of GluR2and nNOS are in the cytoplasm is dyed brown, positive cells for neurons. Compared with control group, the expression of GluR2lowest in PTZ group, with significant difference (P<0.05), compared with PTZ-activated group, the expression of GluR2was increased, with40,60mg/Kg concentration of chloroquine have the significant effection, with significant difference (P<0.05). Compared with control group, the expression of nNOS was strong in the hippocampus CA1and DG areas, with significant difference (P<0.05). Compared with PTZ-activated group, the expression of nNOS was reduced in hippocampus CA1and DG areas, with significant difference (P<0.05), compared with control group, with40,60mg/Kg concentration of chloroquine with no significant difference (P>0.05). Compared with CA1area, the expression of nNOS was weakened in DG area (P>0.05).Conclusion (One) Cell culture110mmol/L PTZ can activated the hippocampal astrocytes significantly.2Chloroquine could inhibited the activation of astrocytes which activitied by PTZ, with the increasing of chloroquine dose, the inhibition gradually strengthened, among them with75mg/L concentration of chloroquine have the best inhibit effection.3Chloroquine could inhibited the expressions of GFAP, GluR2and nNOS, thus inhibit stellate cell proliferation.(Two) Animal experiment1Using0.5%PTZ solution can be successfully copied chronic epilepsy animal models, the over proliferation of astrocytes was closely related to epileptic state.2Chloroquine could inhibited the rats chronic epilepsy seizure state which activated by PTZ, and with the increasing of chloroquine dose, the inhibited effects presents the enhanced tends to be a stable state.3Chloroquine could inhibited the rats chronic epilepsy seizure state by adjusting the expression of GluR2and inhibiting the expression of nNOS, then play antiepileptic effect.