加味柴胡疏肝汤调控miRNA-204对癫痫小鼠海马自噬的影响

Effect of Modified Chaihu Shugantang Regulat miRNA-204 on Hippocampus Autophagy in Epileptic Mice

目的:观察加味柴胡疏肝汤对癫痫小鼠海马微小非编码RNA 204(miRNA-204)的表达影响,探讨其发挥神经保护作用的机制。方法:将60只小鼠随机分为6组:正常组,模型组(盐酸匹罗卡品180 mg·kg-1),加味柴胡疏肝汤组(7 g·kg-1·d-1),加味柴胡疏肝汤+miRNA-204模拟物组(7 g·kg-1·d-1+2μL),加味柴胡疏肝汤+miRNA-204抑制物组(7 g·kg-1·d-1+2μL),卡马西平组(30 mg·kg-1·d-1),各灌胃2周;采用匹罗卡品使小鼠致痫,6组分别灌胃给药、抑制及过表达miRNA-204后,处死小鼠,取其海马组织,观察每组的指标情况,采用实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠海马miRNA-204的表达;蛋白免疫印迹法(Western blot)检测自噬相关蛋白微管相关蛋白轻链3(LC3),自噬相关标志性蛋白7(ATG7)的表达情况,苏木素-伊红(HE)染色观察每组小鼠海马病理学变化;透射电镜观察各组小鼠海马组织自噬情况。结果:与正常组比较,模型组小鼠miRNA-204表达显著降低(P<0.01),海马C1区病理改变最明显,ATG7,LC3Ⅱ/LC3Ⅰ表达显著升高(P<0.01),自噬小体数量最多;与模型组比较,加味柴胡疏肝组小鼠海马组织miRNA-204表达明显升高(P<0.05),海马C1区病理改变减轻,ATG7,LC3Ⅱ/LC3Ⅰ表达明显降低(P<0.05),自噬小体数量减少;加味柴胡疏肝汤+miRNA-204模拟物组小鼠海马组织miRNA-204表达显著升高(P<0.01),海马C1区病理改变最轻,ATG7,LC3Ⅱ/LC3Ⅰ表达显著降低(P<0.01),自噬小体数量最少;与加味柴胡疏肝汤比较,加味柴胡疏肝汤+miRNA-204抑制物组上述指标变化趋势相同,变化幅度明显减少(P<0.05)。结论:加味柴胡疏肝汤可以改善癫痫小鼠海马组织病理改变,其作用机制可能是通过升高癫痫小鼠海马miRNA-204的表达,抑制神经元过度自噬,减少细胞凋亡。

Objective:To observe the effect of modified Chaihu Shugantang on the expression of miRNA-204 in hippocampus of epileptic mice,and to explore its mechanism of neuroprotection.Method:The sixty mice were randomly divided into 6 groups:normal group,model group(pilocarpine 180 mg·kg-1),and modified Chaihu Shugantang group(7 g·kg-1·d-1),modified Chaihu Shugantang+miRNA-204 mimic group(7 g·kg-1·d-1+2μL),modified Chaihu Shugantang+miRNA-204 inhibitor group(7 g·kg-1·d-1+2μL),carbamazepine group(30 mg·kg-1·d-1),each was given intragastric administration for 2 weeks,using pilocarpine to cause epilepsy in mice,respectively,add flavor to Bupleurum after intragastric administration,inhibition and overexpression of miRNA-204,the mice were sacrificed and their hippocampus tissues were harvested.The indicators of each group were observed,Real-time quantitative PCR detecting system(Real-time PCR)was used to detect mouse hippocampal miRNA-204 expression,Western blot analysis of autophagy-related protein microtubule-associated protein light chain 3(LC3),autophagy-associated marker protein 7(ATG7)expression,hematoxylin pathological condition of hippocampus in each group was observed by hematoxylin-eosin(HE)staining.The autophagy of hippocampus in each group was observed by transmission electron microscopy.Result:Compared with normal group,the expression of miRNA-204 was significantly decreased in model group(P<0.01),the pathological changes in the hippocampal C1 area were the most obvious,the expression of ATG7,LC3Ⅱ/LC3Ⅰwas increased(P<0.01),and the autophagy was small.Compared with model group,the expression of miRNA-204 in the hippocampus of the modified Chaihu Shugantang group was increased(P<0.05),the pathological changes in the hippocampal C1 area were alleviated,the expression of ATG7,LC3Ⅱ/LC3Ⅰwas decreased(P<0.05),and the autophagy was small.The number of body decreased,the expression of miRNA-204 in hippocampus of modified Chaihu Shugantang+miRNA-204 mimic group was significantly increased(P<0.01),the pathological changes in hippocampal C1 area were the lightest,and the expression of ATG7,LC3Ⅱ/LC3Ⅰwas decreased(P<0.01),the number of autophagosomes was the least.Compared with modified Chaihu Shugantang group,the above-mentioned indicators of modified Chaihu Shugantang+miRNA-204 inhibitor group had the same change trend and the change range decreased(P<0.05).Conclusion:Modified Chaihu Shugantang can improve the pathological changes of hippocampus in mice with epilepsy and play a neuroprotective role.The mechanism may be to increase the expression of miRNA-204 in hippocampus of mice with epilepsy,inhibit excessive autophagy of neurons and reduce apoptosis.