癫痫大鼠海马CA3区Nav1.2、Nav1.6的动态表达变化

Dynamic expressions of Nav1.2 and Nav1.6 in hippocampal CA3 region of epileptic rats

目的 动态观察Nav1.2、Nav1.6在氯化锂-匹罗卡品癫痫模型大鼠海马CA3区的表达变化,探讨两者在癫痫发病机制中的可能作用.方法 采用随机数字表法,将90只健康雄性SD大鼠随机分为实验组（n=45）和对照组（n=45）,实验组予以腹腔注射氯化锂-匹罗卡品致痫,对照组予生理盐水假处理.根据时间点又分为3个亚组：24 h（n=15）组（n=15）、7d组（n=15）和60 d组（n=15）,采用随机数字表法将每亚组再随机分为3个小组（n=5）分别进行免疫组化、Western印迹及RT-PCR方法检测Nav1.2、Nav1.6在大鼠海马CA3区的动态表达.结果 （1）免疫组化检测示实验24 h组海马CA3区Nav1.2蛋白表达相对对照组差异无统计学意义（P=0.492）,而实验组Nav1.6蛋白表达（0.398±0.019）较对照组（0.313 ±0.017）显著升高,差异具有统计学意义（P =0.034）;实验7d组海马CA3区Nav1.2和Nav1.6蛋白相对对照组差异无统计学意义（P=0.157,0.109）;实验60 d组Nav1.2蛋白表达（0.117±0.009）较对照组（0.155±0.010）显著降低,差异具有统计学意义（P =0.002）,而实验组Nav1.6蛋白表达（0.400±0.009）较对照组（0.318±0.010）显著升高,差异具有统计学意义（P=0.018）.（2） Western印迹检测示实验24 h组海马CA3区Nav1.2蛋白表达较对照组差异无统计学意义（P=0.472）,实验组Nav1.6蛋白表达（0.419±0.027）较对照组（0.290±0.007）显著升高,差异具有统计学意义（P=0.001）;7 d组Nav1.2和Nav1.6蛋白表达较对照组差异无统计学意义（P=0.517,0.514）;60 d组实验组Nav1.2蛋白表达（0.209 ±0.077）较对照组（0.339±0.080）明显降低,差异具有统计学意义（P=0.024）,实验组Nav1.6蛋白表达（0.772±0.029）较对照组（0.489±0.014）显著升高,差异具有统计学意义（P=0.00I）.（3）RT-PCR检测示实验24 h组海马CA3区Nav1.2 mRNA表达较对照组差异无统计学意义（P =0.453）,实验组Nav1.6mRNA表达（2.250±0.117）较对照组（0.998±0.139）显著升高,差异具有统计学意义（P=0.001）;7d组Nav1.2和Nav1.6 mRNA表达较对照组差异无统计学意义（P=0.493,0.624）;60 d组实验组Nav1.2 mRNA表达（0.718±0.056）较对照组（1.000±0.026）明显降低,差异具有统计学意义（P=0.027）,实验组Nav1.6 mRNA表达（2.445±0.167）较对照组（1.003±0.060）显著升高,差异具有统计学意义（P =0.001）.结论 Nav1.2和Nav1.6均参与氯化锂-匹罗卡品癫痫大鼠慢性自发性癫痫形成,且Nav1.6在大鼠急性期痫性发作中发挥了作用.

Objective To observe the expressions of Nav1.2 and Nav1.6 in the hippocampal CA3 region of lithium chloride-pilocarpine epileptic rats to explore their potential roles in epileptogenesis.Methods A total of 90 healthy male SD rats were randomly divided into normal control group （physiological saline） and epilepstic group （lithium chloride-pilocarpine）.According to different timepoints,the control and epilepstic groups were randomly divided into 3 subgroups of 24-hour,7-day and 60-day.Then immunohistochemistry,Western blot and reverse transcription-polymerase chain reaction （RT-PCR） were employed to detect the expressions of Nav1.2 and Nav1.6 in hippocampal CA3 region of rats.Results Immunohistochemisty showed that,at 24 hours,the expression of Nav1.2 had no significant difference （P =0.492） while Nav1.6 significantly increased as compared with controls（0.398 ±0.019 vs 0.313 ±0.017,P =0.034）.At Day 7,both Nav1.2 and Nav1.6 had no significant change （P =0.157,0.109）.Nav1.2 significantly decreased at Day 60 （0.117 ±0.009 vs 0.155 ±0.010,P =0.002）.But Nav1.6 significantly increased（0.400 ±0.009 vs 0.318 ±0.010,P =0.018）.Western blot showed that Nav1.2 protein had no significant difference at 24 hours （P =0.472） while Nav1.6 significantly increased （0.419 ± 0.027 vs 0.290 ±0.007,P =0.001）.At Day 7,Nav1.2 and Nav1.6 proteins had no significant change （P =0.517,0.514）.At Day 60,Nav1.2 protein significantly decreased （0.209 ±0.077 vs 0.339 ±0.080,P=0.024） while Nav1.6 significantly increased （0.772 ± 0.029 vs 0.489 ± 0.014,P =0.001）.RT-PCR showed the same results as Western blot and immunohistochemisty.Nav1.2 mRNA had no significantly difference at 24 hours（P =0.453） while Nav1.6 mRNA significantly increased （2.250 ± 0.117 vs 0.998 ± 0.139,P =0.001） ; at Day 7,both Nav1.2 mRNA and Nav1.6 mRNA had no significant change （P =0.493,0.624）.Nav 1.2 mRNA significantly decreased at Day 60 （0.718 ± 0.056 vs 1.000 ± 0.026,P =0.027）.But Nav1.6 mRNA significantly increased （2.445 ± 0.167 vs 1.003 ± 0.060,P =0.001）.Conclusion Both Nav1.2 and Nav1.6 are involved in the formation of chronic spontaneous recurrent seizures.And Nav1.6 also plays an important role in acute phase of seizures.