Abstract: Objective To investigate the protective effect and mechanism of tanshinone Ⅱ A on hippocampal neurons in epileptic mice.Methods 100 adult male ICR mice were randomly divided into sham group,kainic acid group,Tanshinone IIA low dose group,Tanshinone IIA high dose group and LY294002 group.Intracerebroventricular injection of kainic acid (1 μg/μL,1 μL) induced epilepsy model in mice.Sham group was injected with 1 μL PBS solution.Among them,Tanshinone IIA low dose group,Tanshinone IIA high dose group and LY294002 group were intraperitoneally injected with tanshinone IIA (50 mg/kg,100 mg/kg) and LY294002(10 mg/kg) 30 minutes before modeling.Sham group and the kainic acid group were injected with PBS solution.The mice were killed 24 hours after modeling.HE staining,Nissl staining and TUNEL staining were used to detect the damage and apoptosis of hippocampal neurons in mice;Immunofluorescence staining was used to detect the expression of Beclin1 and p62 in hippocampus;Transmission electron microscopy was used to detect the autophagosome number in hippocampus;Western blot was used to detect the expression of Cleaved-Caspase-3,B cell lymphoma-2 (Bcl-2),Bcl-2 related X(Bax),Microtubule associated protein light chain 3 (LC3I/II),Beclin1,p62,protein kinase B (AKT),p-AKT,mammalian target of rapamycin (mTOR),p-mTOR.Results In sham group,neurons in hippocampal CA1 area were closely arranged and Nissl bodies were abundant.Compared with sham group,the kainic acid group hippocampal neurons disordered,Nissl bodies decreased,the number of TUNEL positive neurons and autophagosomes increased,the expression of Bcl-2 and p62 proteins decreased,and the expression of Bax,Cleaved-Caspase-3,LC3II/I,Beclin1,p-AKT/AKT,p-mTOR/mTOR proteins increased.Compared with the kainic acid group,the neuronal damage in the low dose group of tanshinone IIA,the high dose group of tanshinone IIA,and the LY294002 group were reduced,the number of TUNEL positive neurons and autophagosomes were decreased,the expression of Bcl-2 and p62 proteins were increased,and the expression of Bax,Cleaved-Caspase-3,LC3II/I,Beclin1,p-AKT/AKT,p-mTOR/mTOR proteins were decreased.Conclusion Tanshinone ⅡA can protect hippocampal neurons in epileptic mice by inhibiting AKT/mTOR signal pathway,neuronal apoptosis and autophagy.

Key words: Epilepsy, Tanshinone IIA, Apoptosis, Autophagy

摘要： 目的 探讨丹参酮ⅡA对癫痫小鼠海马神经元损伤的保护作用及机制。方法 将100只成年雄性ICR小鼠随机分为假手术组、红藻氨酸组、丹参酮ⅡA低剂量组、丹参酮ⅡA高剂量组、LY294002组。采用侧脑室注射红藻氨酸(1 μg/μL,1 μL)诱导小鼠癫痫模型,假手术组注射1 μL PBS溶液。其中丹参酮ⅡA低剂量组、丹参酮ⅡA高剂量组、LY294002组分别于造模前30 min腹腔注射丹参酮ⅡA(50 mg/kg、100 mg/kg)、LY294002(10 mg/kg)。假手术组、红藻氨酸组注射PBS溶液。造模24 h后处死小鼠。HE染色、尼氏染色、TUNEL染色检测小鼠海马神经元损伤和凋亡;免疫荧光染色检测海马组织Beclin1和p62表达;透射电镜检测海马组织自噬小体数目;Western blot检测海马组织剪切型半胱氨酸天冬氨酸蛋白酶-3(Cleaved-Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、微管相关蛋白轻链3(LC3I/II)、Beclin1、p62、蛋白激酶B(AKT)、p-AKT、雷帕霉素靶蛋白(mTOR)、p-mTOR蛋白表达。结果 假手术组海马CA1区神经元排列紧密,尼氏体丰富。与假手术组比较,红藻氨酸组海马神经元排列紊乱,尼氏体减少,TUNEL阳性神经元和自噬小体数量增加,Bcl-2、p62蛋白表达减少,Bax、Cleaved-Caspase-3、LC3II/I、Beclin1、p-AKT/AKT、p-mTOR/mTOR蛋白表达增加。与红藻氨酸组比较,丹参酮ⅡA低剂量组、丹参酮ⅡA高剂量组、LY294002组神经元损伤减轻,TUNEL阳性神经元和自噬小体数量减少,Bcl-2、p62蛋白表达增加,Bax、Cleaved-Caspase-3、LC3II/I、Beclin1、p-AKT/AKT、p-mTOR/mTOR蛋白表达减少。结论 丹参酮ⅡA通过抑制AKT/mTOR信号通路,抑制神经元凋亡和自噬,对癫痫小鼠海马神经元具有保护作用。

关键词: 癫痫, 丹参酮ⅡA, 凋亡, 自噬, 磷脂酰肌醇3激酶, 蛋白激酶B, 雷帕霉素靶蛋白通路