Nrf2及其激动剂在星形胶质细胞活化中的作用与机制研究
发布时间：2018-07-29 08:19
【摘要】：星形胶质细胞(Astrocyte)是中枢神经系统(Central nervous system,CNS)中数量最多的细胞,越来越多的证据表明星形胶质细胞不只是单纯的支持细胞,而在中枢神经系统中发挥着重要的生理作用,包括构成血脑屏障、参与突触活动、介导炎症反应等。在病理状态下星形胶质细胞可因内环境改变而发生活化(Astrogliosis),主要表现为细胞增殖、肥大以及中间丝、细胞外基质、炎症因子等的高表达。星形胶质细胞活化是众多CNS疾病的共同病理表现,目前认为轻度、早期的星形胶质细胞活化有助于限制炎症扩散,然而过度的星形胶质细胞活化在后期可形成胶质瘢痕并伴随大量细胞外基质成分CSPG等的沉积,成为多种CNS疾病尤其是CNS创伤后轴突再生的主要障碍。故如何减轻星形胶质细胞的过度活化是神经再生研究的热点方向之一。大量研究表明,过度的氧化应激和炎症反应是导致星形胶质细胞活化的主要因素之一,抑制过度的氧化应激和炎症反应是减轻星形胶质细胞过度活化的主要策略。Nrf2是机体调控氧化应激水平的重要转录因子,研究表明其在多种中枢神经系统疾病中发挥神经保护作用。但既往在中枢神经系统疾病中对Nrf2的研究多集中于对神经元的直接保护作用,而较少关注其对神经再生和胶质瘢痕的作用。莱菔硫烷(Sulforaphane,SFN)是提取自天然植物中的活性成分,可有效激活Nrf2发挥抗炎、抗氧化等作用,并在多项研究中作为Nrf2的特异性激动剂而用于Nrf2及相关信号通路的研究中,并且有研究证实其在抑制肿瘤细胞增殖中亦可发挥积极作用,此外SFN可通过血脑屏障、毒性低、可由食物中获得等特点使其在中枢神经疾病中具有较大的潜在应用价值。综上,我们猜想通过调控Nrf2能否有效抑制星形胶质细胞活化?Nrf2的激动药物能否应用于中枢神经系统以控制星形胶质细胞的过度增殖而减少胶质瘢痕形成?基于上述猜想我们采用离体研究的方式进行了以下2个部分研究:一、Nrf2在小鼠星形胶质细胞活化中的作用与机制目的在体外使用LPS诱导星形胶质细胞活化,并对Nrf2进行调控,探讨Nrf2在LPS诱导的星形胶质细胞活化中的作用与可能涉及的分子机制。方法体外培养小鼠星形胶质细胞,以不同剂量的LPS(0、0.2、0.4、0.8、1.0ug/ml)刺激星形胶质细胞,通过免疫荧光和WB检测细胞核和细胞浆Nrf2,观察LPS对Nrf2核转位的影响,并通过WB检测Nrf2下游NQO1、HO-1两个抗氧化酶的表达间接反映Nrf2的活化情况;使用SFN激活Nrf2并通过免疫荧光和WB检测GFAP、Neurocan两个星胶活化的标志蛋白的表达以探讨上调Nrf2对LPS刺激下的星形胶质细胞活化的影响;使用敲除Nrf2的细胞以失活Nrf2,通过免疫荧光和WB检测以上活化指标探讨Nrf2失活对LPS刺激下的星形胶质细胞活化的影响;使用DCFH-DA法检测ROS,WB检测P38的磷酸化对可能涉及的细胞活化相关信号分子进行检测以揭示可能的机制。结果1、LPS可以促进体外星形胶质细胞Nrf2核转位,并促进NQO1和HO-1的表达,且这种现象呈浓度依赖性;2、Nrf2的激动剂SFN可以减少LPS刺激下星形胶质细胞GFAP、Neurocan的表达;3、Nrf2敲除的星形胶质细胞在LPS刺激后较野生型细胞表达更多的GFAP和Neurocan;4、LPS干预24h后星形胶质细胞内ROS产生增加,SFN可以抑制这种效应,而Nrf2敲除的星形胶质细胞较野生型细胞产生更多ROS;SFN可以抑制LPS导致的P38MAPK的磷酸化,而Nrf2敲除星形胶质细胞P38MAPK磷酸化水平较野生型细胞更高。结论LPS可以促进星形胶质细胞的内源性Nrf2激活,增加抗氧化酶的表达;Nrf2可以影响LPS刺激的星形胶质细胞活化水平,并能调控ROS的产生及P38MAPK的磷酸化,这可能是Nrf2影响星形胶质细胞活化可能的机制。二、莱菔硫烷对LPS诱导的小鼠星形胶质细胞增殖的影响目的探讨莱菔硫烷对体外LPS诱导的小鼠星形胶质细胞增殖的作用及可能的机制。方法体外培养原代小鼠星形胶质细胞,首先使用CCK8法检测不同浓度(0、10、15、20u M)SFN对LPS诱导的星形胶质细胞细胞增值率的影响;选取20u M SFN作为后续实验干预剂量通过Ki67免疫荧光染色检测Ki67阳性细胞数;流式细胞仪检测各组细胞细胞周期;Western blot实验检测PCNA、Cyclin D1及P27kip1表达情况。结果1、CCK8实验结果提示SFN可以抑制LPS诱导的星形胶质细胞的增殖率,其中20u M抑制效果更明显;Ki67免疫荧光双标结果也显示20u M SFN可以降低LPS诱导的星形胶质细胞Ki67阳性细胞数;2、流式细胞周期检测显示20u M SFN可以降低LPS诱导的星形胶质细胞处于S期及S+G2/M期细胞的比例;3、Western blot结果显示20u M SFN可以降低PCNA、Cyclin D1的表达,提高P27kip1的表达。结论20u M的SFN可以抑制体外LPS诱导的星形胶质细胞的增殖,其机制可能与调控P27kip1、Cyclin D1等周期相关蛋白的表达,抑制细胞周期有关。
[Abstract]:Astrocytes (Astrocyte) are the most abundant cells in the Central nervous system (CNS). More and more evidence shows that star shaped glial cells are not only simple support cells, but play an important physiological role in the central nervous system, including the blood brain barrier, synapse activities, and inflammatory reactions. Astrocytes can live (Astrogliosis) in the pathological condition, including cell proliferation, hypertrophy, and high expression of the intermediate filament, extracellular matrix, and inflammatory factors. Astrocyte activation is a common pathological manifestation of many CNS diseases. At present, it is considered mild and early astrocytes. It is helpful to restrict the spread of inflammation, but excessive astrocyte activation can form glial scar in the later period and with the deposition of a large number of extracellular matrix components CSPG, which is the main obstacle to the regeneration of axon after multiple CNS diseases, especially CNS. A large number of studies have shown that excessive oxidative stress and inflammatory response are one of the major factors leading to astrocyte activation. Inhibition of excessive oxidative stress and inflammatory response is the main strategy to reduce the overactivation of astrocytes..Nrf2 is an important transcription factor for the regulation of oxidative stress. The neuroprotective effect is played in various central nervous system diseases. But the previous study of Nrf2 in central nervous system disease focused on the direct protection of neurons, but less attention to its effect on nerve regeneration and glial scar. Sulforaphane (SFN) is the active ingredient in the extraction of natural plants. Activation of Nrf2 plays a role in anti-inflammatory and antioxidant activities, and is used in a number of studies as a specific agonist for Nrf2 in the study of Nrf2 and related signaling pathways, and studies have demonstrated that it also plays an active role in inhibiting the proliferation of tumor cells. In addition, SFN can be used as a blood brain barrier with low toxicity and can be obtained from food. It is of great potential application in central nervous disease. To sum up, we suspect that whether or not Nrf2 can effectively inhibit astrocytes activation by Nrf2 can be used in the central nervous system to control the proliferation of astrocytes to reduce the formation of glial marks? Based on the above hypothesis we use in vitro The following 2 parts are studied: first, the role and mechanism of Nrf2 in the activation of astrocytes in mice and the purpose of using LPS to induce astrocyte activation in vitro, and to regulate and control Nrf2, and to explore the possible molecular mechanism of Nrf2 in the activation of astrocytes induced by LPS. In mice astrocytes, astrocytes were stimulated with different doses of LPS (0,0.2,0.4,0.8,1.0ug/ml). By immunofluorescence and WB detection of nucleus and cytoplasm Nrf2, the effects of LPS on the transposition of Nrf2 nuclei were observed, and NQO1 in the lower reaches of Nrf2 was detected by WB, and the expression of the two antioxidant enzymes of HO-1 indirectly reflected Nrf2 activation. The effect of up regulation of Nrf2 on the activation of astrocytes under LPS stimulated by Nrf2 was detected by immunofluorescence and WB to investigate the effect of up regulation of Nrf2 on the activation of astrocytes under LPS stimulation. The activation of Nrf2 by knockout Nrf2 cells was used to detect the activity of Nrf2 inactivation on astrocytes stimulated by LPS. The effects of ROS and WB detection of the phosphorylation of P38 by WB to detect possible cellular activation related signal molecules to reveal possible mechanisms. Results 1, LPS can promote the Nrf2 nuclear transposition of astrocytes in vitro, and promote the expression of NQO1 and HO-1, and this phenomenon is concentration dependent; 2, Nrf2 agonist SFN can The expression of GFAP and Neurocan in astrocytes was reduced by LPS stimulation; 3, Nrf2 knockout astrocytes expressed more GFAP and Neurocan than wild type cells after LPS stimulation; 4, LPS increased ROS production in astrocytes after 24h, and SFN could inhibit this effect, while Nrf2 knockout astrocytes produced more than wild type cells. Multiple ROS; SFN can inhibit the phosphorylation of P38MAPK caused by LPS, and Nrf2 knockout astrocytes P38MAPK phosphorylation level is higher than that of wild type cells. Conclusion LPS can promote endogenous Nrf2 activation in astrocytes and increase the expression of antioxidant enzymes; Nrf2 can affect the activation level of astrocytes stimulated by LPS, and can regulate ROS. Production and phosphorylation of P38MAPK, which may be the possible mechanism of Nrf2 affecting the activation of astrocytes. Two, the effect of sulforaphane on the proliferation of mouse astrocytes induced by LPS objective to explore the effect of sulforaphane on the proliferation of mouse astrocytes induced by LPS in vitro. In glial cells, CCK8 method was used to detect the effect of different concentrations (0,10,15,20u M) SFN on the proliferation of astrocytes induced by LPS; 20u M SFN was selected as a follow-up experimental intervention dose to detect the number of Ki67 positive cells by Ki67 immunofluorescence staining, and the cell cycle of each group was detected by flow cytometry; Western blot experiment was used to detect the cell cell cycle. CNA, Cyclin D1 and P27kip1 expression. Results 1, CCK8 experimental results suggest that SFN can inhibit the proliferation rate of astrocytes induced by LPS, and 20u M inhibition effect is more obvious; Ki67 immunofluorescence double labeling results also show that 20u M can reduce the number of astrocyte positive cells induced by astrocytes; 2, flow cytometric detection shows 20 U M SFN can reduce the proportion of astrocytes in S and S+G2/M cells induced by LPS. 3, Western blot results show that 20u M SFN can reduce the expression of PCNA, and improve the expression of astrocytes. The expression of equal cycle related proteins inhibits cell cycle.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741
本文编号：2152070
[Abstract]:Astrocytes (Astrocyte) are the most abundant cells in the Central nervous system (CNS). More and more evidence shows that star shaped glial cells are not only simple support cells, but play an important physiological role in the central nervous system, including the blood brain barrier, synapse activities, and inflammatory reactions. Astrocytes can live (Astrogliosis) in the pathological condition, including cell proliferation, hypertrophy, and high expression of the intermediate filament, extracellular matrix, and inflammatory factors. Astrocyte activation is a common pathological manifestation of many CNS diseases. At present, it is considered mild and early astrocytes. It is helpful to restrict the spread of inflammation, but excessive astrocyte activation can form glial scar in the later period and with the deposition of a large number of extracellular matrix components CSPG, which is the main obstacle to the regeneration of axon after multiple CNS diseases, especially CNS. A large number of studies have shown that excessive oxidative stress and inflammatory response are one of the major factors leading to astrocyte activation. Inhibition of excessive oxidative stress and inflammatory response is the main strategy to reduce the overactivation of astrocytes..Nrf2 is an important transcription factor for the regulation of oxidative stress. The neuroprotective effect is played in various central nervous system diseases. But the previous study of Nrf2 in central nervous system disease focused on the direct protection of neurons, but less attention to its effect on nerve regeneration and glial scar. Sulforaphane (SFN) is the active ingredient in the extraction of natural plants. Activation of Nrf2 plays a role in anti-inflammatory and antioxidant activities, and is used in a number of studies as a specific agonist for Nrf2 in the study of Nrf2 and related signaling pathways, and studies have demonstrated that it also plays an active role in inhibiting the proliferation of tumor cells. In addition, SFN can be used as a blood brain barrier with low toxicity and can be obtained from food. It is of great potential application in central nervous disease. To sum up, we suspect that whether or not Nrf2 can effectively inhibit astrocytes activation by Nrf2 can be used in the central nervous system to control the proliferation of astrocytes to reduce the formation of glial marks? Based on the above hypothesis we use in vitro The following 2 parts are studied: first, the role and mechanism of Nrf2 in the activation of astrocytes in mice and the purpose of using LPS to induce astrocyte activation in vitro, and to regulate and control Nrf2, and to explore the possible molecular mechanism of Nrf2 in the activation of astrocytes induced by LPS. In mice astrocytes, astrocytes were stimulated with different doses of LPS (0,0.2,0.4,0.8,1.0ug/ml). By immunofluorescence and WB detection of nucleus and cytoplasm Nrf2, the effects of LPS on the transposition of Nrf2 nuclei were observed, and NQO1 in the lower reaches of Nrf2 was detected by WB, and the expression of the two antioxidant enzymes of HO-1 indirectly reflected Nrf2 activation. The effect of up regulation of Nrf2 on the activation of astrocytes under LPS stimulated by Nrf2 was detected by immunofluorescence and WB to investigate the effect of up regulation of Nrf2 on the activation of astrocytes under LPS stimulation. The activation of Nrf2 by knockout Nrf2 cells was used to detect the activity of Nrf2 inactivation on astrocytes stimulated by LPS. The effects of ROS and WB detection of the phosphorylation of P38 by WB to detect possible cellular activation related signal molecules to reveal possible mechanisms. Results 1, LPS can promote the Nrf2 nuclear transposition of astrocytes in vitro, and promote the expression of NQO1 and HO-1, and this phenomenon is concentration dependent; 2, Nrf2 agonist SFN can The expression of GFAP and Neurocan in astrocytes was reduced by LPS stimulation; 3, Nrf2 knockout astrocytes expressed more GFAP and Neurocan than wild type cells after LPS stimulation; 4, LPS increased ROS production in astrocytes after 24h, and SFN could inhibit this effect, while Nrf2 knockout astrocytes produced more than wild type cells. Multiple ROS; SFN can inhibit the phosphorylation of P38MAPK caused by LPS, and Nrf2 knockout astrocytes P38MAPK phosphorylation level is higher than that of wild type cells. Conclusion LPS can promote endogenous Nrf2 activation in astrocytes and increase the expression of antioxidant enzymes; Nrf2 can affect the activation level of astrocytes stimulated by LPS, and can regulate ROS. Production and phosphorylation of P38MAPK, which may be the possible mechanism of Nrf2 affecting the activation of astrocytes. Two, the effect of sulforaphane on the proliferation of mouse astrocytes induced by LPS objective to explore the effect of sulforaphane on the proliferation of mouse astrocytes induced by LPS in vitro. In glial cells, CCK8 method was used to detect the effect of different concentrations (0,10,15,20u M) SFN on the proliferation of astrocytes induced by LPS; 20u M SFN was selected as a follow-up experimental intervention dose to detect the number of Ki67 positive cells by Ki67 immunofluorescence staining, and the cell cycle of each group was detected by flow cytometry; Western blot experiment was used to detect the cell cell cycle. CNA, Cyclin D1 and P27kip1 expression. Results 1, CCK8 experimental results suggest that SFN can inhibit the proliferation rate of astrocytes induced by LPS, and 20u M inhibition effect is more obvious; Ki67 immunofluorescence double labeling results also show that 20u M can reduce the number of astrocyte positive cells induced by astrocytes; 2, flow cytometric detection shows 20 U M SFN can reduce the proportion of astrocytes in S and S+G2/M cells induced by LPS. 3, Western blot results show that 20u M SFN can reduce the expression of PCNA, and improve the expression of astrocytes. The expression of equal cycle related proteins inhibits cell cycle.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741
【参考文献】
相关期刊论文 前1条
1 廖小俊;袁继超;朱海涛;刘伟;陈亚星;胡胜利;李兰;林江凯;;莱菔硫烷对小鼠脊髓损伤后后肢功能的作用[J];第三军医大学学报;2016年10期
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