激動素對谷氨酸誘導(dǎo)的HT22細(xì)胞氧化損傷干預(yù)作用及相關(guān)機(jī)制研究
發(fā)布時間：2019-05-15 23:51
【摘要】：背景:阿爾茨海默病(Alzheimer’s disease,AD)是一種嚴(yán)重危害老年人身心健康和生活質(zhì)量的神經(jīng)退行性疾病。越來越多的證據(jù)表明,氧化應(yīng)激很可能在AD發(fā)病和發(fā)展過程中發(fā)揮著極其重要的作用。激動素是腺嘌呤的甲基呋喃衍生物,研究表明其能夠在動物個體、組織和細(xì)胞水平上發(fā)揮顯著的抗氧化應(yīng)激和抗衰老作用。HT22細(xì)胞是來源于小鼠海馬神經(jīng)元的永生化細(xì)胞系,谷氨酸能誘導(dǎo)其發(fā)生氧化應(yīng)激和細(xì)胞死亡,因此它是研究神經(jīng)元氧化損傷的良好細(xì)胞模型。本研究探索了激動素對谷氨酸誘導(dǎo)的HT22細(xì)胞氧化損傷的保護(hù)作用,對抗AD藥物的篩選具有重要意義。目的:研究激動素對谷氨酸誘導(dǎo)的HT22細(xì)胞氧化損傷的保護(hù)作用及其相關(guān)機(jī)制。方法:首先,通過DPPH清除試驗(yàn)、三價鐵還原試驗(yàn)、羥自由基抑制試驗(yàn)、超氧陰離子抑制試驗(yàn)和二價鐵離子螯合試驗(yàn),對激動素體外抗氧化能力予以評估,同時通過CCK8細(xì)胞活力試驗(yàn)對激動素的安全濃度范圍和最佳處理時間進(jìn)行了篩選。然后,通過CCK8細(xì)胞活力試驗(yàn)和乳酸脫氫酶(Lactate dehydrogenase,LDH)細(xì)胞毒性試驗(yàn)研究了激動素對谷氨酸處理的HT22細(xì)胞活力下降及LDH泄露的影響;通過流式細(xì)胞術(shù)研究了激動素對細(xì)胞內(nèi)活性氧(Reactive oxygen species,ROS)和Ca~(2+)水平的影響;通過相關(guān)試劑盒研究了激動素對細(xì)胞內(nèi)谷胱甘肽(Glutathione,GSH)水平、抗氧化酶活性和細(xì)胞抗氧化能力的影響;通過流式細(xì)胞術(shù)評估了激動素對細(xì)胞凋亡的影響;通過ATP試劑盒檢測細(xì)胞內(nèi)ATP含量,流式細(xì)胞術(shù)檢測細(xì)胞線粒體膜電位(Mitochondrial membrane potential,MMP)以及Western Blot(WB)方法檢測凋亡誘導(dǎo)因子(Apoptosis inducing factor,AIF)核轉(zhuǎn)移,評估了激動素對線粒體功能的影響;通過WB方法研究了激動素對谷氨酸處理的HT22細(xì)胞中凋亡信號調(diào)節(jié)激-1(Apoptosis signal-regulating kinase 1,ASK-1)、c-jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和絲裂原激活蛋白激酶p38(Mitogen activated protein kinase p38,p38)活化的影響。最后,通過WB、實(shí)時定量PCR(Quantitative real-time polymerase chain reaction,qRT-PCR)研究了激動素對Nrf2核轉(zhuǎn)移和血紅素加氧酶-1(Heme oxygenase-1,HO-1)表達(dá)影響,通過siRNA沉默和CCK8細(xì)胞活力試驗(yàn)研究了Nrf2和HO-1對激動素細(xì)胞保護(hù)作用的影響。結(jié)果:(1)和陽性對照相比,激動素自身幾乎沒有抗氧化能力;(2)激動素的安全濃度范圍在5mg/L之下,最佳預(yù)處理時間為8h;(3)激動素能夠抑制谷氨酸誘導(dǎo)的HT22細(xì)胞活力下降和LDH泄露增加;(4)激動素能夠抑制細(xì)胞內(nèi)ROS和Ca~(2+)水平上升,提高細(xì)胞內(nèi)抗氧化酶的活性和GSH水平,增強(qiáng)細(xì)胞抗氧化能力;(5)相對于谷氨酸處理組,激動素能部分恢復(fù)細(xì)胞內(nèi)ATP水平,抑制MMP的去極化和AIF的核轉(zhuǎn)移,同時能夠抑制谷氨酸誘導(dǎo)的HT22細(xì)胞凋亡;(6)激動素能夠抑制谷氨酸誘導(dǎo)的ASK-1、JNK和p38的磷酸化;(7)激動素能夠顯著促進(jìn)Nrf2的核轉(zhuǎn)移,且具有時間依賴性;能夠顯著促進(jìn)Nrf2下游基因HO-1在轉(zhuǎn)錄和翻譯水平上的表達(dá),且呈現(xiàn)出時間和劑量依賴性。此外,使用Nrf2 siRNA靶向沉默Nrf2幾乎完全消除了激動素的細(xì)胞保護(hù)作用,而使用HO-1siRNA靶向沉默HO-1只能部分消除激動素的細(xì)胞保護(hù)作用。結(jié)論:激動素自身基本不具備抗氧化能力,但其能夠抑制谷氨酸誘導(dǎo)的HT22細(xì)胞毒性,抑制細(xì)胞內(nèi)ROS聚集和Ca~(2+)水平升高,抑制細(xì)胞內(nèi)抗氧化酶活性的下降,且激動素自身對細(xì)胞抗氧化酶系統(tǒng)具有活化作用。同時,激動素能夠抑制線粒體功能障礙和細(xì)胞凋亡,抑制谷氨酸誘導(dǎo)的HT22細(xì)胞中ASK-1、JNK和p38的活化。此外,激動素能夠激活Nrf2信號通路,并能增強(qiáng)其下游抗氧化基因HO-1的表達(dá),而且激動素細(xì)胞保護(hù)作用的發(fā)揮依賴Nrf2的活化和HO-1的表達(dá)。這些結(jié)果表明激動素具有神經(jīng)保護(hù)作用,可能在預(yù)防和治療AD的研究和實(shí)踐中具有應(yīng)用潛力。
[Abstract]:BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the physical and mental health and quality of life of the elderly. An increasing number of evidence suggests that oxidative stress is likely to play a very important role in the pathogenesis and development of AD. Akinetin is a methorphan derivative of the adenoid, which shows that it can play a significant anti-oxidative stress and anti-aging effect on the individual, tissue and cellular level of the animal. HT22 cells are immortalized cell lines derived from mouse hippocampal neurons, and glutamate can induce oxidative stress and cell death, so it is a good cell model for the study of neuronal oxidative damage. This study explored the protective effect of the agonist on the oxidative damage of the HT22 cell induced by glutamate, which is of great significance to the screening of AD drugs. Objective: To study the protective effect and related mechanism of agonist on glutamate-induced oxidative damage of HT22 cells. The method comprises the following steps of: firstly, performing a DPPH clearance test, a trivalent iron reduction test, a hydroxyl radical inhibition test, a super-oxygen anion inhibition test and a divalent iron ion agglutination test, and evaluating the in vitro antioxidant capacity of the agonist, At the same time, the safe concentration range and the optimal treatment time of the agonist were selected through the cell viability test of the CCK8 cell. Then, the effects of the activation of the agonist on the activity of the HT22 cells and the leakage of the LDH were studied by the cell viability test of the CCK8 and the cell toxicity of the lactate dehydrogenase (LDH), and the reactive oxygen species (reactive oxygen species) in the cells were studied by flow cytometry. The effects of kinetin on the level of Glutathione (GSH), the activity of anti-oxidation enzyme and the anti-oxidation ability of the cells were studied by the related reagent kit, and the effect of the agonist on the apoptosis of the cells was assessed by flow cytometry. The intracellular ATP content, the mitochondrial membrane potential (MMP) and the Western Blot (WB) method were used to detect the nuclear transfer of the apoptosis-inducing factor (AIF). The effects of agonist-1 (ASK-1), c-jun N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38) on glutamate-treated HT22 cells were studied by WB method. Finally, by WB, real-time quantitative PCR (qRT-PCR), the effect of agonist on the nuclear transfer of Nrf2 and the expression of heme oxygenase-1 (HO-1) was studied. The effect of Nrf2 and HO-1 on the cytoprotective effect of Nrf2 and HO-1 was studied by siRNA silencing and cell viability test of CCK8. Results: (1) In contrast to the positive control, the agonist itself had little anti-oxidation ability; (2) the safe concentration of the agonist was below 5 mg/ L, the optimal pretreatment time was 8 h; (3) the agonist was able to inhibit the decrease of the activity of the HT22 cells induced by glutamate and the increase of LDH leakage; (4) The agonist can inhibit the level of ROS and Ca ~ (2 +) in the cells, increase the activity and GSH level of the antioxidant enzymes in the cells, enhance the anti-oxidation ability of the cells, and (5) restore the intracellular ATP level with respect to the glutamate treatment group. (6) AIF can inhibit the phosphorylation of TASK-1, JNK and p38 induced by glutamate, and (7) the agonist can significantly promote the nuclear transfer of Nrf2 and has time-dependence; The expression of the downstream gene HO-1 in the transcription and translation level of the Nrf2 downstream gene can be significantly promoted and time and dose-dependent. In addition, the use of the Nrf2 siRNA targeting silencing Nrf2 substantially completely eliminates the cytoprotective effect of the agonist, whereas the use of the HO-1 siRNA targeting silent HO-1 can only partially eliminate the cytoprotective effect of the agonist. Conclusion: The agonist itself does not possess the ability of anti-oxidation, but it can inhibit the toxicity of glutamate-induced HT22, inhibit the increase of ROS in the cells and increase the level of Ca ~ (2 +), and inhibit the decrease of the activity of antioxidant enzymes in the cells. And the agonist itself has an activating effect on the cell anti-oxidation enzyme system. In addition, the activation of ASK-1, JNK, and p38 in the HT22 cells induced by glutamate was inhibited by the inhibition of mitochondrial dysfunction and apoptosis. In addition, the agonist can activate the Nrf2 signal path and enhance the expression of the downstream anti-oxidation gene HO-1, and the activation of the agonist cell protection depends on the activation of Nrf2 and the expression of HO-1. These results suggest that the agonist has a neuroprotective effect and may have an application potential in the study and practice of prevention and treatment of AD.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R749.16
本文編號：2477858
[Abstract]:BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the physical and mental health and quality of life of the elderly. An increasing number of evidence suggests that oxidative stress is likely to play a very important role in the pathogenesis and development of AD. Akinetin is a methorphan derivative of the adenoid, which shows that it can play a significant anti-oxidative stress and anti-aging effect on the individual, tissue and cellular level of the animal. HT22 cells are immortalized cell lines derived from mouse hippocampal neurons, and glutamate can induce oxidative stress and cell death, so it is a good cell model for the study of neuronal oxidative damage. This study explored the protective effect of the agonist on the oxidative damage of the HT22 cell induced by glutamate, which is of great significance to the screening of AD drugs. Objective: To study the protective effect and related mechanism of agonist on glutamate-induced oxidative damage of HT22 cells. The method comprises the following steps of: firstly, performing a DPPH clearance test, a trivalent iron reduction test, a hydroxyl radical inhibition test, a super-oxygen anion inhibition test and a divalent iron ion agglutination test, and evaluating the in vitro antioxidant capacity of the agonist, At the same time, the safe concentration range and the optimal treatment time of the agonist were selected through the cell viability test of the CCK8 cell. Then, the effects of the activation of the agonist on the activity of the HT22 cells and the leakage of the LDH were studied by the cell viability test of the CCK8 and the cell toxicity of the lactate dehydrogenase (LDH), and the reactive oxygen species (reactive oxygen species) in the cells were studied by flow cytometry. The effects of kinetin on the level of Glutathione (GSH), the activity of anti-oxidation enzyme and the anti-oxidation ability of the cells were studied by the related reagent kit, and the effect of the agonist on the apoptosis of the cells was assessed by flow cytometry. The intracellular ATP content, the mitochondrial membrane potential (MMP) and the Western Blot (WB) method were used to detect the nuclear transfer of the apoptosis-inducing factor (AIF). The effects of agonist-1 (ASK-1), c-jun N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38) on glutamate-treated HT22 cells were studied by WB method. Finally, by WB, real-time quantitative PCR (qRT-PCR), the effect of agonist on the nuclear transfer of Nrf2 and the expression of heme oxygenase-1 (HO-1) was studied. The effect of Nrf2 and HO-1 on the cytoprotective effect of Nrf2 and HO-1 was studied by siRNA silencing and cell viability test of CCK8. Results: (1) In contrast to the positive control, the agonist itself had little anti-oxidation ability; (2) the safe concentration of the agonist was below 5 mg/ L, the optimal pretreatment time was 8 h; (3) the agonist was able to inhibit the decrease of the activity of the HT22 cells induced by glutamate and the increase of LDH leakage; (4) The agonist can inhibit the level of ROS and Ca ~ (2 +) in the cells, increase the activity and GSH level of the antioxidant enzymes in the cells, enhance the anti-oxidation ability of the cells, and (5) restore the intracellular ATP level with respect to the glutamate treatment group. (6) AIF can inhibit the phosphorylation of TASK-1, JNK and p38 induced by glutamate, and (7) the agonist can significantly promote the nuclear transfer of Nrf2 and has time-dependence; The expression of the downstream gene HO-1 in the transcription and translation level of the Nrf2 downstream gene can be significantly promoted and time and dose-dependent. In addition, the use of the Nrf2 siRNA targeting silencing Nrf2 substantially completely eliminates the cytoprotective effect of the agonist, whereas the use of the HO-1 siRNA targeting silent HO-1 can only partially eliminate the cytoprotective effect of the agonist. Conclusion: The agonist itself does not possess the ability of anti-oxidation, but it can inhibit the toxicity of glutamate-induced HT22, inhibit the increase of ROS in the cells and increase the level of Ca ~ (2 +), and inhibit the decrease of the activity of antioxidant enzymes in the cells. And the agonist itself has an activating effect on the cell anti-oxidation enzyme system. In addition, the activation of ASK-1, JNK, and p38 in the HT22 cells induced by glutamate was inhibited by the inhibition of mitochondrial dysfunction and apoptosis. In addition, the agonist can activate the Nrf2 signal path and enhance the expression of the downstream anti-oxidation gene HO-1, and the activation of the agonist cell protection depends on the activation of Nrf2 and the expression of HO-1. These results suggest that the agonist has a neuroprotective effect and may have an application potential in the study and practice of prevention and treatment of AD.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R749.16
【相似文獻(xiàn)】
相關(guān)期刊論文 前3條
1 孫江宏;劉玉梅;曹統(tǒng);歐陽五慶;;激動素對D-半乳糖致衰雌性小鼠卵巢和子宮的影響[J];生理學(xué)報;2013年04期
2 俞旭平;方堅(jiān);徐秀瑛;孫昌高;鄭俊波;;激動素處理對人工老化條件下龍膽種子活力的影響[J];現(xiàn)代應(yīng)用藥學(xué);1991年03期
3 TrishaGura,李大衛(wèi),何方淑;化學(xué)激動素登上了炎癥的中心舞臺[J];世界科學(xué);1997年10期
相關(guān)博士學(xué)位論文 前3條
1 魏云鵬;激動素對谷氨酸誘導(dǎo)的HT22細(xì)胞氧化損傷干預(yù)作用及相關(guān)機(jī)制研究[D];西北農(nóng)林科技大學(xué);2017年
2 唐立剛;激動素對大鼠乳腺的作用及相關(guān)機(jī)制研究[D];西北農(nóng)林科技大學(xué);2012年
3 李夢云;激動素抗大鼠免疫衰老的作用研究[D];西北農(nóng)林科技大學(xué);2014年
相關(guān)碩士學(xué)位論文 前6條
1 劉丹;激動素對AlCl_3和D-半乳糖聯(lián)合誘導(dǎo)的小鼠AD模型正向干預(yù)作用的研究[D];西北農(nóng)林科技大學(xué);2017年
2 張亞瓊;丙烯酰胺誘導(dǎo)HT22小鼠海馬神經(jīng)元損傷作用的研究[D];廣東藥學(xué)院;2015年
3 韓麗蓉;SD-BMSC-Exosome對氧化損傷的神經(jīng)細(xì)胞HT22的保護(hù)修復(fù)[D];蘭州大學(xué);2017年
4 黎小艷;共振瑞利散射法和熒光法分析測定天然植物性激素及其分析應(yīng)用研究[D];西南大學(xué);2012年
5 李亞蒙;Exendin-4改善Aβ31-35所致小鼠海馬HT22神經(jīng)細(xì)胞Per2異常表達(dá)及機(jī)制研究[D];山西醫(yī)科大學(xué);2017年
6 趙學(xué)玲;Exendin-4通過拮抗鈣超載改善Aβ31-35誘導(dǎo)的HT22海馬神經(jīng)細(xì)胞Per1異常表達(dá)[D];山西醫(yī)科大學(xué);2017年
，本文編號：2477858
本文鏈接：http://sikaile.net/yixuelunwen/jsb/2477858.html
最近更新
教材專著
