丹参酮ⅡA对癫痫小鼠海马神经元损伤的保护作用及机制

doi:10.19854/j.cnki.1008-2425.2023.04.0003

摘要/Abstract

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

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

中图分类号:

R742.1

曹慧, 周敏, 舒芸芸, 王仕丽. 丹参酮ⅡA对癫痫小鼠海马神经元损伤的保护作用及机制[J]. 立体定向和功能性神经外科杂志, 2023, 36(4): 205-212.

Cao Hui, Zhou Min, Shu Yunyun, et al. Protective effect and mechanism of Tanshinone ⅡA on hippocampal neuron injury in epileptic mice[J]. Chinese Journal of Stereotactic and Functional Neurosurgery, 2023, 36(4): 205-212.

参考文献

[1] Wu Q,Yi XW.Down-regulation of long noncoding RNA MALAT1 protects hippocampal neurons against excessive autophagy and apoptosis via the PI3K/Akt signaling pathway in rats with epilepsy[J].J Mol Neurosci,2018,65(2):234~245.
[2] 刘冲冲,董笑克,李中浩,等.柴贝止痫汤添加治疗对耐药性癫痫患者血清炎症因子的影响[J].时珍国医国药,2021,32(10):2451~2455.
[3] 王淑湘,吴可佳,石瑞平,等.红藻氨酸致痫大鼠海马小窝蛋白(Cav-1)和核因子(NF-κB)的变化及灵芝多糖的干预[J].广东化工,2021,48(2):50~52.
[4] Hosseinzadeh M,Nikseresht S,Khodagholi F,et al.Cannabidiol post-treatment alleviates rat epileptic-related behaviors and activates hippocampal cell autophagy pathway along with antioxidant defense in chronic phase of pilocarpine-induced seizure[J].J Mol Neurosci,2016,58(4):432~440.
[5] Ren B,Zhang YX,Zhou HX,et al.Tanshinone IIA prevents the loss of nigrostrial dopaminergic neurons by inhibiting NADPH oxidase and iNOS in the MPTP model of Parkinson's disease[J].J Neurol Sci,2015,348(1-2):142~152.
[6] Tang LM,Wang LX,Wang ZY,et al.Tanshinone IIA ameliorates lead(Pb)-induced cognitive deficits and oxidative stress in a rat pup model[J].Bratisl Lek Listy,2017,118(4):196~201.
[7] 许凯,成薇婷,胡作为,等.丹参酮ⅡA抑制奥沙利铂诱发周围神经病变大鼠神经生长因子的表达[J].肿瘤防治研究,2015,42(7):648~651.
[8] Buenafe OE,Orellana-Paucar A,Maes J,et al.Tanshinone IIA exhibits anticonvulsant activity in zebra and mouse seizure models[J].ACS Chem Neurosci,2013,4(11):1479~1487.
[9] Tan XQ,Cheng XL,Yang Y,et al.Tanshinone II-A sodium sulfonate(DS-201)enhances human BKCa channel channel activity by selectively argeting the pore-formingαsubunit[J].Acta Pharmacol Sin,2014,35(11):1351~1363.
[10] Zhu YC,Tang QQ,Wang GP,et al.Tanshinone IIA Protects Hippocampal Neuronal Cells from Reactive Oxygen Species Through Changes in Autophagy and Activation of Phosphatidylinositol 3-Kinase,Protein Kinas B,and Mechanistic Target of Rapamycin Pathways[J].Curr Neurovasc Res,2017,14(2):132~140.
[11] Xu ZR,Han X,Ou DM,et al.Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy[J].Appl Microbiol Biotechnol,2020,104(2):575~587.
[12] Yang N,Guan QW,Chen FH,et al.Antioxidants Targeting Mitochondrial Oxidative Stress:Promising Neuroprotectants for Epilepsy[J].Oxid Med Cell Longev,2020,2020:6687185.
[13] Li XG,Lou XF,Xu SZ,et al.Knockdown of miR-372 Inhibits Nerve Cell Apoptosis Induced by Spinal Cord Ischemia/Reperfusion Injury via Enhancing Autophagy by Up-regulating Beclin-1[J].J Mol Neurosci,2018,66(3):437~444.
[14] Calderó J,Brunet N,Tarabal O,et al.Lithium prevents excitotoxic cell death of motoneurons in organotypic slice cultures of spinal cord[J].Neuroscience,2010,165(4):1353~1369.
[15] Fang CZ,Xie LL,Liu CM,et al.Tanshinone IIA improves hypoxic ischemic encephalopathy through TLR4 mediated NFkappaB signal pathway[J].Mol Med Rep,2018,18(2):1899~1908.
[16] 姜珊,陆西萍,高华,等.丹参酮ⅡA对癫痫大鼠认知功能障碍的治疗作用[J].神经解剖学杂志,2014,30(4):452~456.
[17] Jia C,Zhang R,Wei LM,et al.Investigation of the mechanism of tanshinone IIA to improve cognitive function via synaptic plasticity in epileptic rats[J].Pharm Biol,2023,61(1): 100~110.
[18] Li M,Wang JY,Ding LX,et al.Tanshinone IIA attenuates nerve transection injury associated with nerve regeneration promotion in rats[J].Neurosci Lett,2017,659:18~25.
[19] Toscano ECDB,Vieira ÉLM,Portela ACDC,et al.Bcl-2/Bax ratio increase does not prevent apoptosis of glia and granular neurons in patients with temporal lobe epilepsy[J].Neuropathology,2019,39(5):348~357.
[20] Mahdavi S,Khodarahmi P,Roodbari NH,et al.Effects of cadmium on Bcl-2/ Bax expression ratio in rat cortex brain and hippocampus[J].Hum Exp Toxicol,2018,37(3):321~328.
[21] 刘杰.奥卡西平在戊四氮诱导癫痫小鼠模型治疗中对Caspase-3蛋白表达和细胞凋亡的影响[J].解剖学研究,2018,40(2):118~121+136.
[22] Wang J,Li Y,Huang WH,et al.The protective effect of aucubin from eucommia ulmoides against sTSIIAtus epilepticus by inducing autophagy and inhibiting necroptosis[J].Am J Chin Med,2017,45(3):557~573.
[23] Zhang HY,Tao JY,Zhang SX,et al.LncRNA MEG3 Reduces Hippocampal Neuron Apoptosis via the PI3K/AKT/mTOR Pathway in a Rat Model of Temporal Lobe Epilepsy[J].Neuropsychiatr Dis Treat,2020,16:2519~2528.
[24] Xing Y,Xue WW,Teng YH,et al.Raddeanin A promotes autophagy-induced apoptosis by inactivating PI3K/AKT/mTOR pathway in lung adenocarcinoma cells [J].Naunyn Schmiedebergs Arch Pharmacol,2023,396(9):1987~1997.
[25] Macias M,Blazejczyk M,Kazmierska P,et al.Spatiotemporal characterization of mTOR kinase activity following kainic acid induced status epilepticus and analysis of rat brain response to chronic rapamycin treatment[J].PLoS One,2013,8(5):e64455.
[26] 徐璇.丹参酮ⅡA对大鼠脑缺血后PTEN-PI3K/AKT/mTOR信号通路作用机制的研究[D].安徽医科大学,2020.