神经药理学报

神经药理学报 ›› 2018, Vol. 8 ›› Issue (4): 23-25.

• Session 1A: CNS Diseases: Unique Roles of Traditional Chinese • 上一篇    下一篇

Research of Quercetin’s Estrogen-Like Action on Central Nervous System and Its Mechanisms

SHEN li-xia1，LIU Liang-liang1，ZHANG Ming1，LIU Yang1，ZHANG Dan-shen 2*   

  1. 1. Hebei North University，Zhangjiakou，075000，China
    2. Hebei University of Science and Technology，Shijiazhuang，050018，China
  • 出版日期:2018-08-26 发布日期:2018-11-16
  • 通讯作者: 张丹参，女，教授，博士研究生导师，研究方向：神经药理学，E-mail：zhangds2011@126.com
  • 作者简介:沈丽霞，女，教授，硕士研究生导师，研究方向：神经药理学

Research of Quercetin’s Estrogen-Like Action on Central Nervous System and Its Mechanisms

SHEN li-xia1，LIU Liang-liang1，ZHANG Ming1，LIU Yang1，ZHANG Dan-shen 2*   

  1. 1. Hebei North University，Zhangjiakou，075000，China
    2. Hebei University of Science and Technology，Shijiazhuang，050018，China
  • Online:2018-08-26 Published:2018-11-16
  • Contact: 张丹参，女，教授，博士研究生导师，研究方向：神经药理学，E-mail：zhangds2011@126.com
  • About author:沈丽霞，女，教授，硕士研究生导师，研究方向：神经药理学

摘要： 目的：大量临床和流行病学数据表明，女性更年期雌激素水平下降可能诱发阿尔茨海默病（Alzheimer's disease，AD），雌激素有提高神经元活力和减少Aβ聚集的作用，可通过多种方式对神经元发挥保护作用。但绝经后妇女的临床试验数据显示，雌激素疗法存在不良反应，使其临床应用受限。植物雌激素（Phytoestrogen）与雌激素化学结构类似，被称为选择性雌激素受体调节剂，具有雌激素样神经保护作用，同时减轻因雌激素替代疗法产生的副作用。研究表明，槲皮素（quercetin，Que）作为植物雌激素，对大脑缺血再灌注、β-淀粉样蛋白和重金属导致的神经元损伤都具有一定的保护作用。本课题组的前期研究也证实槲皮素可以通过结合雌激素α受体表现出雌激素样作用，促进人乳腺癌MCF-7细胞系的增殖。此研究延续前期研究，继续探讨槲皮素雌激素样作用对脑神经元的保护作用及其作用机制。方法：利用原代培养的大鼠海马神经元细胞、皮层神经元细胞，以及老化的Aβ25-35处理的PC12细胞，分别观察使用不同浓度的槲皮素进行药物干预24小时的作用。通过MTT实验检测槲皮素对神经元、PC12细胞活性的影响；通过免疫荧光染色比较槲皮素对海马、皮层神经元突触形态的影响；通过蛋白免疫印迹法检测PC12细胞模型中相关受体和蛋白的表达变化。并通过使用雌激素受体拮抗剂ICI182，780、雌激素受体α拮抗剂MPP和雌激素受体β拮抗剂PHTPP，深入探讨槲皮素发挥雌激素样神经保护作用的机制。结果：MTT实验结果显示，槲皮素可以提高大鼠海马神经元的活性（P<0.05），且槲皮素对海马神经元的保护作用呈剂量依赖性增加。在低密度海马神经元中25μmol•L-1槲皮素对海马神经元有保护作用（P<0.05）。当雌激素受体拮抗后，50μmol•L-1槲皮素仍能提高海马神经元细胞的活性（P<0.05），延长作用时间后三组不同浓度的槲皮素都可以提高海马神经元的活性。在PC12细胞的研究中，随着Aβ25-35作用浓度的增加，细胞活力所受抑制程度逐渐加强，与对照组相比Aβ25-3510μmol•L-1作用细胞24h后能够显著降低细胞存活率（P<0.01）。与模型组相比，三个不同浓度的槲皮素组的细胞存活率均显著增强（P<0.05）。免疫荧光染色结果显示，槲皮素可以提高海马神经元的突触数目和长度（P<0.05），促进突触发生。槲皮素（50,100μmol•L-1）组明显促进皮层神经元突触形成，大部分神经元胞体饱满，神经元突触明显增粗，增长并连接成致密的神经网络。雌激素受体完全拮抗后，槲皮素仍可提高海马神经元的突触数目和长度（P<0.05）。槲皮素可以提高雌激素受体β拮抗后海马神经元的突触数目和长度，高于雌激素受体α拮抗（P<0.05）。Western blot结果显示，槲皮素可以提高雌激素受体α蛋白的表达。与模型组相比，槲皮素提高ERα和p-ERK1/2蛋白表达量（P<0.05），对于ERβ蛋白表达量虽有增加但无显著性差异（P>0.05）。并且在PC12细胞实验中，Western blot结果显示，实验组凋亡相关蛋白Bcl-2/Bax表达比值增加，Caspase-3蛋白表达下降（P<0.05）。当槲皮素雌激素受体受到ICI182，780抑制后，p-ERK1/2蛋白表达下降（P<0.05），Bcl-2/Bax比值下降及Caspase-3蛋白表达增加（P<0.05）。当U0126作用于槲皮素后，Bcl-2/Bax比值下降及Caspase-3蛋白表达增加（P<0.05）。结论：槲皮素可上调雌激素受体α蛋白的表达，增加神经元突触的数目及长度，促进突触的生长及发生，从而改善神经元细胞的生长率和存活率，对大鼠海马和皮层的神经元发挥保护作用；槲皮素对Aβ25-35诱导的PC12细胞损伤的AD模型也具有保护作用，主要提高雌激素受体α蛋白的表达，通过经典的雌激素受体通路发挥其雌激素样神经保护作用，并可通过ERα介导激活MAPK信号通路，减轻凋亡。因此，槲皮素雌激素样作用对脑神经元和拟AD损伤具有保护作用，主要作用机制与调控雌激素受体α有关，并可影响ERα介导的MAPK信号通路。

关键词: 雌激素, 槲皮素, 神经元, 阿尔茨海默病, 雌激素受体

Abstract: Objective: A large number of clinical and epidemiological data showed that decreased estrogen levels in women during menopause may induce Alzheimer's disease (AD). Estrogen can Protect neurons by increasing neuronal activity and reducing Aβ aggregation in a variety of ways. However, clinical trial data of postmenopausal women showed that estrogen therapy has many adverse reactions, which limits its clinical application. Phytoestrogens, similar to the chemical structure of estrogens, are known as selective estrogen receptor modulators. And they have estrogen-like neuroprotective effects while reducing the adverse reactions of estrogen replacement therapy. Studies have shown that Quercetin (Que), as a phytoestrogens, has a protective effect on cerebral ischemia-reperfusion, and neuronal damage induced by β-amyloid and heavy metal. Our previous study also confirmed that Que can exhibit estrogen-like effects by binding to estrogen alpha receptors and promote the proliferation of human breast cancer MCF-7 cell line. This study continues the previous works and deeply explore the protective effects of Que estrogen-like effects on brain neurons and its mechanisms. Methods: Primary cultured rat hippocampal neurons, cortical neurons, and PC12 cells treated by Aβ25-35 were used to observe the effects of different concentrations of Que for 24 and 48 hours. The effects of Que on the activity of neurons and PC12 cells were detected by MTT assay. The effects of Que on synaptic morphology of hippocampus and cortical neurons were compared by immunofluorescence staining. It was examined by Western blotting that the changes in the expression of related receptors and proteins in the PC12 cell model. And we studied in-depth on the mechanisms of estrogen-like neuroprotection of Que by using the estrogen receptor antagonist, such as ICI182,780, and the estrogen receptor alpha antagonist MPP, and the estrogen receptor beta antagonist PHTPP. Results: The results of MTT assay showed that Que could increase the activity of rat hippocampal neurons (P<0.05), and the protective effect of Que on hippocampal neurons increased in a dose-dependent manner. In low-density hippocampal neurons, 25μmol•L-1 Que had protective effects on hippocampal neurons (P<0.05). After estrogen receptor antagonism, 50μmol•L-1 Que could still increase the activity of hippocampal neurons (P<0.05). After prolonging the action time, three different concentrations of Que could increase the activity of hippocampal neurons. In the test of PC12 cells, with the increase of Aβ25-35 concentration, the inhibition of cell viability was gradually enhanced. Compared with the control group, Aβ25-3510μmol•L-1 could significantly reduce cell viability after 24h treatment (P<0.01). Compared with the model group, the cell viability of the three different concentrations of Que groups was significantly increased (P<0.05). The results of immunofluorescence staining test showed that Que could increase the number and length of synapses in hippocampal neurons (P<0.05) and promote synapse development. The Que (50, 100 μmol•L-1) groups significantly promoted synapse formation in cortical neurons. Most of the neurons were full of cells, and the synapses of the neurons were significantly thickened, which grew and connected into a dense neural network. After the estrogen receptor was completely antagonized, Que could still increase the number and length of synapses in hippocampal neurons (P<0.05). Que could increase the number and length of synapses in hippocampal neurons after estrogen receptor β antagonism, which was higher than that of estrogen receptor α (P<0.05). Western blot results showed that Que could increase the expression of estrogen receptor α protein. Compared with the model group, Que increased the expression of ERα and p-ERK1/2 protein (P<0.05), but there was no significant difference in the expression of ERβ protein (P>0.05). In the PC12 cell experiment, the results of Western blot showed that the expression ratio of apoptosis-related protein Bcl-2/Bax was increased and the expression of Caspase-3 protein was decreased (P<0.05). When Que estrogen receptor was inhibited by ICI182,780, p-ERK1/2 protein expression decreased (P<0.05), Bcl-2/Bax ratio decreased and Caspase-3 protein expression increased (P<0.05). When U0126 was applied to Que, the ratio of Bcl-2/Bax decreased and the expression of Caspase-3 protein increased (P<0.05). Conclusion: It was further confirmed that Que coulld up-regulate the expression of estrogen receptor α protein, increase the number and length of neuronal synapses, promote the growth and occurrence of synapses, and thus improve the growth rate and survival rate of neuronal cells. Then Que played a protective role of neurons in the hippocampus and cortex in rats. The Que also protected the AD model of PC12 cell injury induced by Aβ25-35, mainly enhancing the expression of estrogen receptor α protein. It exerted its estrogen- like neuroprotective effect through the classical estrogen receptor pathway. And it can attenuate apoptosis through ERα-mediated activation of the MAPK signaling pathway. In a word, Estrogen-like effect of Que has protective effects on brain neurons and AD-like injury. The main mechanism is related to the regulation of estrogen receptor α and the ERα-mediated MAPK signaling pathway.

Key words: estrogen, quercetin, neuron, Alzheimer's disease, estrogen receptor