摘要
目的:探讨灵芝孢子粉对戊四氮(PTZ)致痫大鼠大脑皮质及海马区增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)变化的影响,进一步研究癫痫的发病机制及灵芝孢子粉的作用机制。方法:健康雄性Wistar大鼠30只,(体重200±20g购自佳木斯大学实验动物中心),随机分为对照组、癫痫模型组和灵芝孢子粉干预组,每组各10只。癫痫模型组和灵芝孢子粉干预组用亚惊厥剂量的PTZ(35mg/kg体重)腹腔注射,注射时浓度为10g/L,每日1次,持续28天,同时对照组以等容生理盐水腹腔注射;灵芝孢子粉干预组经灌胃给予灵芝孢子粉(150mg/kg体重),灌胃时浓度为30g/L;同时癫痫模型组和对照组以等容生理盐水灌胃。每天对每只大鼠在PTZ注射后进行观察并记录癫痫发作的潜伏期,发作级别及持续时间,用药28天后停药一周,再用相同剂量的PTZ测试,凡出现连续5次Ⅱ级以上痫性发作为达到点燃标准。实验动物麻醉后断头处死,在低温条件下迅速取脑,一侧甲醛固定用于免疫组化法检测皮质和海马区PCNA及CyclinD1的免疫反应性;另一侧冰上迅速分离海马用western-blot检测PCNA、CyclinD1蛋白含量的变化。结果:对照组无痫样发作,癫痫模型组有重度的痫样发作(Ⅳ-Ⅴ级),灵芝孢子粉干预组有轻度的痫样发作(Ⅰ-Ⅱ级),与癫痫模型组动物相比,灵芝孢子粉干预组大鼠潜伏期明显延长,发作级别降低,鲜有大发作。癫痫模型组PCNA、CyclinD1阳性细胞数目明显增多与对照组比较差异有显著性(P<0.01),灵芝孢子粉干预组与癫痫模型组比较阳性细胞数明显减少差异有显著性(P<0.01); PCNA、CyclinD1在癫痫模型组的大脑海马区的蛋白含量与对照组比较明显增多差异有显著性(P<0.01),灵芝孢子粉干预组与癫痫模型组比较明显减少差异有显著性(P<0.01)。结论:灵芝孢子粉可以通过调节PCNA、CyclinD1蛋白含量抑制星形胶质细胞(AS)增生,从而影响戊四氮致痫大鼠癫痫发作的发生与发展。
Purpose:To investigate the effect of ganoderma lucidum spores on the change of PCNA and CyclinD1 in cerebral cortex and hippocampus of rats with epilepsy induced by PTZ. And further the study of epilepsy mechanism and the anti-epilepsy mechanism of ganoderma lucidum spores. Methods:30 male Wistar rats(weighing 200±20g, purchased from the center of animal experiment in Jiamusi university) were randomly divided into three groups: (1) the control group(n = 10), in which rats received intraperitoneal injections and oral administration of the normal saline and, (2)PTZ group (n = 10), in which rats were injected with a subconvulsant dose PTZ (35mg/kg in 0.9% saline, 10g/L) intraperitoneally and received saline orally; (3) ganoderma lucidum spores intervening group (n = 10),in which rats were injected intraperitoneally with PTZ (35 mg/kg in 0.9% saline, 10g/L) and received oral administration of ganoderma lucidum spores (150mg/kg suspended in normal saline, 30g/L). 28days later withdrawal drugs for a week,then these rats received a final challenge dose of PTZ (35 mg/kg) to check the persistence of enhanced susceptibility to the convulsant. Control rats received injections of saline instead of the PTZ challenge dose. At the end of the experiment, all rats in each group were anesthetized with diethyl ether and decapitated, take brain rapidly at low temperature, every brain were devided two parts allow the sagittal suture. one part were fixed with neutral formalin for detection of the immunoreactivity of PCNA and CyclinD1 with immunohistochemistry; the cerebral cortex and hippocampus were segregated rapidly from the other part of the brain on the ice for detection of the contents of PCNA and CyclinD1 with western-blot. Result:The control group showed no seizure activity. Severe seizure activity was observed in PTZ group (Ⅲ-Ⅴclass).slight seizure activity (Ⅰ-Ⅱclass) appeared in ganoderma lucidum spores intervening group. the control group showed no seizure activity. Compared with epilepsy model group, the latent period of ganoderma lucidum spores intervening group had obvious difference, but they had no obvious difference at duration. The expressions of PCNA and CyclinD1 were stronger in PTZ group than those of control group (P<0.01). The significant differences were observed in PCNA and CyclinD1 expressions between ganoderma lucidum spores intervening group and PTZ group (P<0.01). The contents of PCNA and CyclinD1 in PTZ group was significantly higher in hippocampus as compared with control group (P<0.01), while the significant difference were observed between the ganoderma lucidum spores intervening group and PTZ group (P<0.01). Conclusion:Ganoderma lucidum spores can inhibit the function and proliferation of astrocyte in cerebral cortext and hippocampus by regulate the contents of PCNA and CyclinD1, can alleviate the seizure activity, which suggests that ganoderma lucidum spores may be an ideal anticonvulsant in preventing and treating epilepsy.
引文
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