丹参酮ⅡA注射液对缺血缺氧性脑损伤新生大鼠皮质神经元自噬及Akt-mTOR通路的影响
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摘要
目的观察丹参酮ⅡA注射液对缺血缺氧性脑损伤新生大鼠皮质神经元自噬及Akt-mTOR通路影响并探讨其机制。方法 7日龄SD新生大鼠80只,随机平均分为5组,分别为假手术组、模型组及经丹参酮ⅡA处理的高剂量组(0.1 mg/g)、中剂量组(0.05 mg/g)、低剂量组(0.02 mg/g)。Rice法造模,透射电镜观察新生大鼠大脑皮质神经元变化情况,TTC染色检测脑梗死情况,蛋白免疫印迹检测脑组织LC3-Ⅰ、LC3-Ⅱ、Beclin-1、p-Akt、p-mTOR、p-S6蛋白表达情况。结果假手术组神经元结构正常,模型组中观察到皮质神经元中存在大量的自噬体和自噬溶酶体,其中泡状自噬体中包裹有可见的细胞质结构。相较于模型组,高剂量组、中剂量组、低剂量组中自噬体数量明显减少,且呈一定的剂量依赖性(P <0.05)。与假手术组相比,模型组中幼鼠脑梗死面积显著增加(P <0.05)。与模型组相比,高剂量组、中剂量组、低剂量组中脑梗死面积显著减小,且呈一定的剂量依赖性(P <0.05)。与假手术组相比,模型组中LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ)的比值与Beclin-1蛋白表达水平显著增高(P <0.05)。与模型组相比,高剂量组、中剂量组、低剂量组中LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ)的比值与Beclin-1蛋白表达水平显著降低,且呈剂量依赖性(P <0.05)。与假手术组相比,模型组中pAkt、p-mTOR、p-S6蛋白表达水平均显著降低(P <0.05)。与模型组相比,高剂量组、中剂量组、低剂量组中pAkt、p-mTOR、p-S6蛋白表达水平均显著增加,且呈剂量依赖性(P <0.05)。结论丹参酮ⅡA对新生大鼠缺血缺氧性脑损伤大脑皮质神经元自噬具有显著的抑制作用,推测其机制可能为通过激活Akt-mTOR信号转导通路实现的。
Objective: To investigate the effects of Tanshinone Ⅱ A Injection on autophagy and Akt-mTOR pathway in cortical neurons of neonatal rats with hypoxic-ischemic brain damage. Methods: 80 7-day old SD rats were randomly divided into 5 groups: sham operation group(Sham),the model group(HIBD) and Tanshinone A high dose group(0.1 mg/g,HD),middle dose group(0.05 mg/g,MD) and low dose group(0.02 mg/g,LD). The HIBD model was constructed by Rice method. Changes of neurons in cerebral cortex of neonatal rats were observed by transmission electron microscopy;TTC staining was used to detect the cerebral infarction,and the expression of LC3-I,LC3-Ⅱ,Beclin-1,p-Akt,p-mTOR and p-S6 protein was detected in brain tissue with Western blot. Results: Neurons in sham-operated group had normal structure. In HIBD group,a large number of autophages and autophagic lysosomes were observed in cortical neurons and the vesicular autophages contained visible cytoplasmic structure;the infarct area of young rats increased significantly(P < 0.05). The ratio of LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ) and the expression of Beclin-1 protein increased significantly(P < 0.05). The expression levels of p-Akt,p-mTOR and p-S6 decreased significantly(P < 0.05). Compared with HIBD group,the number of autophages and the area of cerebral infarction and the ratio of LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ) and the expression level of Beclin-1 protein decreased significantly,and the expression levels of p-Akt,p-mTOR and p-S6 protein increased significantly in a dose-dependent manner in other groups(P < 0.05). Conclusion: TanshinoneⅡA can significantly inhibit the autophagy of cortical neurons in neonatal rats with hypoxic-ischemic brain injury,which may be mediated by activating Akt-mTOR signal transduction pathway.
Objective: To investigate the effects of Tanshinone Ⅱ A Injection on autophagy and Akt-mTOR pathway in cortical neurons of neonatal rats with hypoxic-ischemic brain damage. Methods: 80 7-day old SD rats were randomly divided into 5 groups: sham operation group(Sham),the model group(HIBD) and Tanshinone A high dose group(0.1 mg/g,HD),middle dose group(0.05 mg/g,MD) and low dose group(0.02 mg/g,LD). The HIBD model was constructed by Rice method. Changes of neurons in cerebral cortex of neonatal rats were observed by transmission electron microscopy;TTC staining was used to detect the cerebral infarction,and the expression of LC3-I,LC3-Ⅱ,Beclin-1,p-Akt,p-mTOR and p-S6 protein was detected in brain tissue with Western blot. Results: Neurons in sham-operated group had normal structure. In HIBD group,a large number of autophages and autophagic lysosomes were observed in cortical neurons and the vesicular autophages contained visible cytoplasmic structure;the infarct area of young rats increased significantly(P < 0.05). The ratio of LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ) and the expression of Beclin-1 protein increased significantly(P < 0.05). The expression levels of p-Akt,p-mTOR and p-S6 decreased significantly(P < 0.05). Compared with HIBD group,the number of autophages and the area of cerebral infarction and the ratio of LC3-Ⅰ/(LC3-Ⅰ+LC3-Ⅱ) and the expression level of Beclin-1 protein decreased significantly,and the expression levels of p-Akt,p-mTOR and p-S6 protein increased significantly in a dose-dependent manner in other groups(P < 0.05). Conclusion: TanshinoneⅡA can significantly inhibit the autophagy of cortical neurons in neonatal rats with hypoxic-ischemic brain injury,which may be mediated by activating Akt-mTOR signal transduction pathway.
引文
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[17] Xue Z,Guo Y,Zhang S,et al. Beta-asarone attenuates amyloid beta-induced autophagy via Akt/mTOR pathway in PC12cells[J]. European Journal of Pharmacology,2014,741(1):195-204.
[18] Makhov P,Golovine K,Teper E,et al. Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death[J]. Br J Cancer,2014,110(4):899-907.
[2]陈长春.锂对缺氧缺血性脑损伤神经修复机制研究进展[J].中国儿童保健杂志,2016,24(2):156-159.
[3] Martin S,Harper CB,May LM,et al. Inhibition of PIKfyve by YM-201636 dysregulates autophagy and leads to apoptosisindependent neuronal cell death[J]. Plos One,2013,8(3):e60152.
[4] Xu S,Liu P. TanshinoneⅡ-A:new perspectives for old remedies[J]. Expert Opinion on Therapeutic Patents,2013,23(2):149-153.
[5] Hei M,Luo Y,Zhang X,et al. Tanshinone IIa alleviates the biochemical changes associated with hypoxic ischemic brain damage in a rat model[J]. Phytotherapy Research,2011,25(12):1865.
[6] Wang YJ,Liu YH,Liao RX. Protective effect of tanshinoneⅡA on neurocyte apoptosis in rats with hypoxia ischemic brain damage and its mechanism[J]. Chinese Pharmacological Bulletin,2015,31(3):443-444.
[7]任陈,杜莎莎.丹参酮ⅡA在体外对海马神经元细胞放射性损伤的保护作用[J].热带医学杂志,2017,17(5):588-591.
[8] Im SH,Yu JH,Park ES,et al. Induction of striatal neurogenesis enhances functional recovery in an adult animal model of neonatal hypoxic-ischemic brain injury[J]. Neuroscience,2010,169(1):259-268.
[9] Seo Y,Kim GT,Choi JW. Early detection of neonatal hypoxic-ischemic white matter injury:an MR diffusion tensor imaging study[J]. Neuroreport,2017,28(13):845-855.
[10] Koike M,Shibata M,Tadakoshi M,et al. Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury[J]. American Journal of Pathology,2008,172(2):454-469.
[11] Qian M,Fang X,Wang X. Autophagy and inflam mation[J].Clinical&Translational Medicine,2017,6(1):24.
[12] Conza D,Mirra P,CalìG,et al. The SGK1 inhibitor SI113 induces autophagy, apoptosis, and endoplasmic reticulum stress in endometrial cancer cells[J]. Journal of Cellular Physiology,2017,232(12):3735-3743.
[13] Lu Q,Harris VA,Kumar S,et al. Autophagy in neonatal hypoxia ischemic brain is associated with oxidative stress[J].Redox Biology,2015,6(1):516-523.
[14] Sharma M,Bhattacharyya S,Nain M,et al. Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I-and EDEM1-containing membranes[J]. Autophagy,2014,10(9):1637-1651.
[15] Spencer B,Potkar R,Trejo M,et al. Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology inα-synuclein models of parkinson′s and lewy body disease[J]. Journal of Neuro science,2009,29(43):13578-13588.
[16] Bertacchini J,Heidari N,Mediani L,et al. Targeting PI3K/AKT/mTOR network for treatment of leukemia.[J]. Cellular&Molecular Life Sciences Cmls,2015,72(12):2337-2347.
[17] Xue Z,Guo Y,Zhang S,et al. Beta-asarone attenuates amyloid beta-induced autophagy via Akt/mTOR pathway in PC12cells[J]. European Journal of Pharmacology,2014,741(1):195-204.
[18] Makhov P,Golovine K,Teper E,et al. Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death[J]. Br J Cancer,2014,110(4):899-907.