Nrf2及其激動劑在星形膠質(zhì)細胞活化中的作用與機制研究
發(fā)布時間：2018-07-29 08:19
【摘要】：星形膠質(zhì)細胞(Astrocyte)是中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)中數(shù)量最多的細胞,越來越多的證據(jù)表明星形膠質(zhì)細胞不只是單純的支持細胞,而在中樞神經(jīng)系統(tǒng)中發(fā)揮著重要的生理作用,包括構(gòu)成血腦屏障、參與突觸活動、介導炎癥反應等。在病理狀態(tài)下星形膠質(zhì)細胞可因內(nèi)環(huán)境改變而發(fā)生活化(Astrogliosis),主要表現(xiàn)為細胞增殖、肥大以及中間絲、細胞外基質(zhì)、炎癥因子等的高表達。星形膠質(zhì)細胞活化是眾多CNS疾病的共同病理表現(xiàn),目前認為輕度、早期的星形膠質(zhì)細胞活化有助于限制炎癥擴散,然而過度的星形膠質(zhì)細胞活化在后期可形成膠質(zhì)瘢痕并伴隨大量細胞外基質(zhì)成分CSPG等的沉積,成為多種CNS疾病尤其是CNS創(chuàng)傷后軸突再生的主要障礙。故如何減輕星形膠質(zhì)細胞的過度活化是神經(jīng)再生研究的熱點方向之一。大量研究表明,過度的氧化應激和炎癥反應是導致星形膠質(zhì)細胞活化的主要因素之一,抑制過度的氧化應激和炎癥反應是減輕星形膠質(zhì)細胞過度活化的主要策略。Nrf2是機體調(diào)控氧化應激水平的重要轉(zhuǎn)錄因子,研究表明其在多種中樞神經(jīng)系統(tǒng)疾病中發(fā)揮神經(jīng)保護作用。但既往在中樞神經(jīng)系統(tǒng)疾病中對Nrf2的研究多集中于對神經(jīng)元的直接保護作用,而較少關(guān)注其對神經(jīng)再生和膠質(zhì)瘢痕的作用。萊菔硫烷(Sulforaphane,SFN)是提取自天然植物中的活性成分,可有效激活Nrf2發(fā)揮抗炎、抗氧化等作用,并在多項研究中作為Nrf2的特異性激動劑而用于Nrf2及相關(guān)信號通路的研究中,并且有研究證實其在抑制腫瘤細胞增殖中亦可發(fā)揮積極作用,此外SFN可通過血腦屏障、毒性低、可由食物中獲得等特點使其在中樞神經(jīng)疾病中具有較大的潛在應用價值。綜上,我們猜想通過調(diào)控Nrf2能否有效抑制星形膠質(zhì)細胞活化?Nrf2的激動藥物能否應用于中樞神經(jīng)系統(tǒng)以控制星形膠質(zhì)細胞的過度增殖而減少膠質(zhì)瘢痕形成?基于上述猜想我們采用離體研究的方式進行了以下2個部分研究:一、Nrf2在小鼠星形膠質(zhì)細胞活化中的作用與機制目的在體外使用LPS誘導星形膠質(zhì)細胞活化,并對Nrf2進行調(diào)控,探討Nrf2在LPS誘導的星形膠質(zhì)細胞活化中的作用與可能涉及的分子機制。方法體外培養(yǎng)小鼠星形膠質(zhì)細胞,以不同劑量的LPS(0、0.2、0.4、0.8、1.0ug/ml)刺激星形膠質(zhì)細胞,通過免疫熒光和WB檢測細胞核和細胞漿Nrf2,觀察LPS對Nrf2核轉(zhuǎn)位的影響,并通過WB檢測Nrf2下游NQO1、HO-1兩個抗氧化酶的表達間接反映Nrf2的活化情況;使用SFN激活Nrf2并通過免疫熒光和WB檢測GFAP、Neurocan兩個星膠活化的標志蛋白的表達以探討上調(diào)Nrf2對LPS刺激下的星形膠質(zhì)細胞活化的影響;使用敲除Nrf2的細胞以失活Nrf2,通過免疫熒光和WB檢測以上活化指標探討Nrf2失活對LPS刺激下的星形膠質(zhì)細胞活化的影響;使用DCFH-DA法檢測ROS,WB檢測P38的磷酸化對可能涉及的細胞活化相關(guān)信號分子進行檢測以揭示可能的機制。結(jié)果1、LPS可以促進體外星形膠質(zhì)細胞Nrf2核轉(zhuǎn)位,并促進NQO1和HO-1的表達,且這種現(xiàn)象呈濃度依賴性;2、Nrf2的激動劑SFN可以減少LPS刺激下星形膠質(zhì)細胞GFAP、Neurocan的表達;3、Nrf2敲除的星形膠質(zhì)細胞在LPS刺激后較野生型細胞表達更多的GFAP和Neurocan;4、LPS干預24h后星形膠質(zhì)細胞內(nèi)ROS產(chǎn)生增加,SFN可以抑制這種效應,而Nrf2敲除的星形膠質(zhì)細胞較野生型細胞產(chǎn)生更多ROS;SFN可以抑制LPS導致的P38MAPK的磷酸化,而Nrf2敲除星形膠質(zhì)細胞P38MAPK磷酸化水平較野生型細胞更高。結(jié)論LPS可以促進星形膠質(zhì)細胞的內(nèi)源性Nrf2激活,增加抗氧化酶的表達;Nrf2可以影響LPS刺激的星形膠質(zhì)細胞活化水平,并能調(diào)控ROS的產(chǎn)生及P38MAPK的磷酸化,這可能是Nrf2影響星形膠質(zhì)細胞活化可能的機制。二、萊菔硫烷對LPS誘導的小鼠星形膠質(zhì)細胞增殖的影響目的探討萊菔硫烷對體外LPS誘導的小鼠星形膠質(zhì)細胞增殖的作用及可能的機制。方法體外培養(yǎng)原代小鼠星形膠質(zhì)細胞,首先使用CCK8法檢測不同濃度(0、10、15、20u M)SFN對LPS誘導的星形膠質(zhì)細胞細胞增值率的影響;選取20u M SFN作為后續(xù)實驗干預劑量通過Ki67免疫熒光染色檢測Ki67陽性細胞數(shù);流式細胞儀檢測各組細胞細胞周期;Western blot實驗檢測PCNA、Cyclin D1及P27kip1表達情況。結(jié)果1、CCK8實驗結(jié)果提示SFN可以抑制LPS誘導的星形膠質(zhì)細胞的增殖率,其中20u M抑制效果更明顯;Ki67免疫熒光雙標結(jié)果也顯示20u M SFN可以降低LPS誘導的星形膠質(zhì)細胞Ki67陽性細胞數(shù);2、流式細胞周期檢測顯示20u M SFN可以降低LPS誘導的星形膠質(zhì)細胞處于S期及S+G2/M期細胞的比例;3、Western blot結(jié)果顯示20u M SFN可以降低PCNA、Cyclin D1的表達,提高P27kip1的表達。結(jié)論20u M的SFN可以抑制體外LPS誘導的星形膠質(zhì)細胞的增殖,其機制可能與調(diào)控P27kip1、Cyclin D1等周期相關(guān)蛋白的表達,抑制細胞周期有關(guān)。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R741
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R741
【參考文獻】
相關(guān)期刊論文 前1條
1 廖小俊;袁繼超;朱海濤;劉偉;陳亞星;胡勝利;李蘭;林江凱;;萊菔硫烷對小鼠脊髓損傷后后肢功能的作用[J];第三軍醫(yī)大學學報;2016年10期
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