eIF2α磷酸化对长波紫外线诱导的小鼠角质形成细胞氧化损伤中Nrf2-HO-1通路的影响
发布时间：2019-06-16 17:15
【摘要】：背景:紫外线辐射是导致急、慢性皮肤损伤,皮肤光老化甚至皮肤肿瘤的的一个主要的环境因素。它主要是通过激活相关信号通路诱导基因的表达和一些蛋白的重新编程,从而参与皮肤疾病发生发展的过程。真核翻译起始因子eIF2α是参与这一过程的主要的调控因子,eIF2α磷酸化能够抑制细胞内大量蛋白的合成,选择性的激活某些蛋白的合成,从而减轻内质网负荷。但是过量的内质网应激会诱导eIF2α磷酸化激活Chop和Caspase家族蛋白的表达最终诱导细胞凋亡。eIF2α磷酸化是生物适应环境变化的一种有效的方式,在应对外界刺激过程中起到了重要的作用。UVA能够诱导质网应激,激活PERK磷酸化从而诱导其下游eIF2α发生磷酸化起到保护的作用。另外UVA辐射做为一种氧化应激能够诱导ROS的产生,引起氧化损伤激活一系列的抗氧化通路的激活,其中重要的通路之一是诱导Nrf2-HO-1抗氧化通路。UVA能够激活Nrf2并且促进其转核,结合抗氧化反应原件(ARE)促进下游的抗氧化酶(HO-1、SOD、NQO1等)Ⅱ期解毒酶的合成。其中血红素氧合酶HO-1是催化血红素的起始酶和限速酶,它能够催化血红素,产生具有抗氧、抗炎作用的铁蛋白,一氧化碳(CO)以及胆红素。已有的研究表明UVA能够诱导eIF2α磷酸化以及激活Nrf2-HO-1通路的表达两种保护机制,然而目前关于对于内质网应激和氧化应激之间的关系研究较少,且对于两条信号通路之间的作用机制也不十分清楚。因此研究eIF2α磷酸化在UVA诱导的氧化损伤中Nrf2-HO-1表达中的作用以及其对UVA诱导的氧化损伤中的保护作用具有重要意义。目的:采用不同剂量的UVA(50、100、150、200、250 kJ/m2)辐射小鼠角质形成细胞(JB6),检测UVA的氧化损伤作用;验证UVA能够诱导eIF2α磷酸化,验证UVA能够激活Nrf2-HO-1通路;验证的Salubrinal,GSK2606414对eIF2α磷酸化的作用并筛选出最佳的药物浓度;探索3μM的Salubrinal,0.5μM的GSK2606414分别预处理对150kJ/m2UVA辐射细胞对Nrf2,Bach1,H0-1 mRNA的影响;探索Salubrinal,GSK2606414分别预处理对150 kJ/m2UVA辐射细胞后Grp78,Nrf2,Bach1,HO-1,P-eIF2α,eIF2α蛋白水平的影响;探索eIF2α磷酸化的改变对细胞形态,细胞活力,细胞内ROS的水平以及细胞周期的影响,探讨在UVA造成的氧化损伤中eIF2α磷酸化的保护作用。研究方法:采用:mts检测uva对jb6细胞活力的影响;fitc-dapi免疫荧光染色法检测不同剂量的uva对jb6细胞形态的影响;dcfh-da荧光探针检测eif2α磷酸化改变对细胞内ros的影响;实时定量pcr检测nrf2、bach1、ho-1的表达的影响;westernblotting检测grp78、nrf2、bach1、ho-1、p-eif2α、eif2α蛋白表达的改变;流式细胞术检测eif2α磷酸化改变对细胞周期的影响。结果:1.长波紫外线uva辐射能够诱导jb6细胞氧化损伤,且损伤程度呈剂量依赖性:与未辐射组相比,细胞形态,细胞活力在小剂量50kj/m2uva辐射时无明显的影响(p0.05)。100、150、200、250kj/m2uva辐射之后随剂量的增加细胞碎片增多,细胞活力显著性降低且具有统计学差异(p0.05)。2.uva辐射诱导eif2α磷酸化呈剂量和时间依赖性:与未辐射组相比,不同剂量的uva(50、100、150、200、250kj/m2)能够诱导eif2α磷酸化,磷酸化程度随着uva剂量的增加而增高,在250kj/m2的剂量辐射较200kj/m2的磷酸化程度稍微有所降低。因此我们选择中等生理剂量的150kj/m2uva辐射jb6细胞检测不同时间点eif2α的磷酸化情况,与未辐射组相比,uva辐射之后eif2α磷酸化即刻增加且程度最高,随着时间的延长磷酸化程度下降。3.uva辐射能够激活nrf2-are通路且呈时间和剂量依赖性:与未辐射组相比,随着uva剂量的增加,nrf2蛋白水平显著增加,但250kj/m2uva辐射有所降低。而ho-1蛋白水平随着uva剂量的增加而显著增加。选择150kj/m2uva辐射jb6细胞后检测不同时间点nrf2,ho-1基因和蛋白水平的改变,nrf2,ho-1的转录水平随着时间点的增加而增加nrf2在4h达到高峰,ho-1在12h达到最高峰。nrf2,ho-1的蛋白水平先增加后降低,ho-1的蛋白水平在12h达到高峰。4.salubrinal,gsk2606414分别预处理对150kj/m2uva辐射jb6细胞eif2α磷酸化的影响:与对照组相比,salubrinal预处理对细胞活力未发现显著性影响(p0.05)。与对照组相比,不同浓度的gsk2606414预处理细胞活力未发现显著性影响(p0.05)。不同浓度salubrinal预处理对150kj/m2uva辐射jb6细胞eif2α的磷酸化具有增强的作用呈现出剂量的依赖性,但在5μm的浓度下较3μm组eif2α的磷酸化稍微有所降低。不同浓度gsk2606414预处理对150kj/m2uva辐射jb6细胞eif2α的磷酸化具有抑制作用,在0.5μm时磷酸化程度最低。5.eif2α磷酸化的改变对nrf2、bach1、ho-1等rna和蛋白的影响:3μM Salubrinal预处理联合150 k J/m2UVA辐射与单独150 kJ/m2UVA组相比eIF2α磷酸化增加,0.5μM GSK2606414预处理联合150 kJ/m2UVA辐射与单独150kJ/m2UVA组相比eIF2α磷酸化降低。Salubrinal联合UVA组较单独UVA辐射组对Bach1,Nrf2,HO-1基因水平影响不大,可以促进Grp78蛋白表达,、诱导Nrf2、HO-1蛋白表达延迟。GSK2606414联合UVA组较单独UVA组延迟Grp78表达,诱导Nrf2,HO-1蛋白表达提前。6.eIF2α磷酸化的改变对细胞生物学的影响:与未辐射组相比,3μM Salubrinal预处理,0.5μM GSK2606414预处理对细胞活力无显著性影响。与150 kJ/m2UVA辐射组相比,3μM Salubrinal预处理联合150kJ/m2UVA辐射细胞的活力增加,0.5μM GSK2606414预处理联合150 kJ/m2UVA辐射细胞的活力降低。与未辐射组相比,3μM Salubrinal预处理,0.5μM GSK2606414预处理对细胞周期无显著性影响,与150 kJ/m2UVA辐射组相比,3μM Salubrinal预处理联合150 kJ/m2UVA辐射细胞周期S期增加7.28±1.03(P0.05),0.5μM GSK2606414预处理联合150 kJ/m2UVA辐射细胞的周期S期降低差异不显著。结论:UVA辐射能够激活内质网应激,诱导eIF2α磷酸化且eIF2α磷酸化呈现出剂量和时间的依赖性。UVA辐射作为一种氧化应激也能够诱导Nrf2-ARE通路的激活且也呈现出剂量和时间的依赖性。使用Sal预处理能够维持eIF2α磷酸化,使用GSK2606414能够抑制eIF2α磷酸化。eIF2α磷酸化参与Nrf2-HO-1通路的调控。维持eIF2α磷酸化的增加能够减轻UVA诱导的氧化损伤,促进细胞的增殖。而抑制eIF2α磷酸化会加重UVA诱导的氧化损伤抑制细胞的增殖。
[Abstract]:BACKGROUND: UV radiation is a major environmental factor leading to acute, chronic skin lesions, skin photoaging, and even skin tumors. It is mainly the process of participating in the development of the skin disease by activating the expression of the related signal path and the reprogramming of some proteins. EIF2 is the main control factor involved in this process, and the phosphorylation of eIF2 can inhibit the synthesis of a large amount of protein in the cell, selectively activate the synthesis of certain proteins, thereby reducing the load of the endoplasmic reticulum. However, excessive endoplasmic reticulum stress induces the expression of eIF2 and the expression of Caspase family proteins to induce apoptosis. The phosphorylation of eIF2 is an effective way to adapt to the change of the environment, and plays an important role in the process of external stimulation. UVA can induce stress, activate the phosphorylation of PERK, and induce the phosphorylation of eIF2 in its downstream. In addition, UVA radiation is an oxidative stress that can induce ROS production, which causes oxidative damage to activate a series of activation of the antioxidant pathway, one of which is the induction of the Nrf2-HO-1 antioxidant pathway. UVA can activate Nrf2 and promote its transformation, and combine the anti-oxidation reaction original (ARE) to promote the synthesis of the downstream anti-oxidation enzyme (HO-1, SOD, NQO1, etc.) phase II detoxification enzyme. Wherein the heme oxygenase HO-1 is a starting enzyme and a speed-limiting enzyme for catalyzing the heme, which can catalyze the heme to generate ferritin, carbon monoxide (CO) and bilirubin with anti-oxygen and anti-inflammatory effects. Studies have shown that UVA can induce the phosphorylation of eIF2 and the activation of two mechanisms of protection of the Nrf2-HO-1 pathway, however, there are few studies on the relationship between endoplasmic reticulum stress and oxidative stress, and the mechanism of action between the two signal pathways is not clear. Therefore, it is of great significance to study the role of eIF2 in the expression of Nrf2-HO-1 in the oxidative damage induced by UVA and its protective effect on the oxidative damage induced by UVA. Objective: To study the effects of UVA (50,100,150,200,250 kJ/ m2) on the oxidative damage of UVA, and to verify that UVA can induce the phosphorylation of eIF2 and to verify that UVA can activate Nrf2-HO-1 pathway. The effects of pre-treatment on Nrf2, Bach1, H0-1 mRNA of 150 kJ/ m2UVA radiation cells and the effect of the pretreatment on the level of Grp78, Nrf2, Bach1, HO-1, P-eIF2 and eIF2 in the radiation cells of 150 kJ/ m2UVA were respectively pre-treated with 3. m u.M of Salubrinal and 0.5. m u.M of GSK2606414. The changes of the phosphorylation of eIF2 to the cell morphology were explored. The cell activity, the level of ROS in the cell and the effect of cell cycle were discussed, and the protective effect of eIF2 on the phosphorylation of eIF2 in the oxidative damage caused by UVA was discussed. Methods: the effect of uva on the activity of jb6 cells was detected by the method of mts; the effect of uva on the morphology of jb6 cells was detected by the fitc-dapi immunofluorescent staining method; the effect of the changes of the ethec-da fluorescent probe on the ros in the cells was detected by the dcfh-da fluorescent probe; the effect of nrf2, bachi1, ho-1 was detected in real time by the real-time quantitative pcr. The changes of the expression of grp78, nrf2, bachi1, ho-1, p-eif2, and eif2 were detected by the western blotting, and the effect of the alteration of eisf2 on the cell cycle was detected by flow cytometry. Results:1. long-wave ultraviolet uva radiation can induce the oxidative damage of jb6 cells, and the degree of damage is dose-dependent: compared with the non-radiation group, the cell morphology and the cell viability do not have a significant effect on the dose of 50 kj/ m2uva radiation (p0.05).100,150,200,250 kj/ m2uva radiation, The significant decrease in cell viability and statistically significant (p0.05) .2.uva radiation induced the dose and time-dependent phosphorylation of eif2: the different doses of uva (50,100,150,200,250 kj/ m2) were able to induce esif2 phosphorylation, and the degree of phosphorylation increased with the increase of the uva dose, The level of phosphorylation at 250 kj/ m2 was slightly lower than the level of phosphorylation of 200 kj/ m2. Therefore, we selected a moderate physiological dose of 150 kj/ m2uva radiation jb6 cells to detect the phosphorylation of eif2 antigen at different time points, and the phosphorylation of eif2 was increased immediately after uva irradiation compared to the non-irradiated group, The decrease of .3.uva radiation over time allows for the activation of the nrf2-are pathway and in time and dose-dependent: as the uva dose increases, the nrf2 protein level increases significantly as the uva dose increases, but the 250 kj/ m2uva radiation is reduced. While the level of ho-1 protein increased significantly with the increase of the uva dose. The changes of nrf2, ho-1 and protein levels of nrf2, ho-1 at different time points were detected after the selection of 150 kj/ m2uva radiation jb6 cells, and the transcription level of nrf2, ho-1 reached the peak at 4 h with the increase of the time point, and ho-1 reached its peak at 12 h. The protein level of nrf2 and ho-1 decreased firstly, and the protein level of ho-1 reached a peak at 12 h. Compared with the control group, no significant effect was found on the activity of the pre-treated cells of gsk2606414 in different concentrations (p0.05). Phosphorylation of the eif2 cells of the 150kj/ m2uva radiation jb6 cells with different concentrations of salubrinal pretreatment showed a dose-dependent manner, but the phosphorylation of eif2 cells at a concentration of 5. m u.m was slightly reduced. the pre-treatment of gsk2606414 at different concentrations had an inhibitory effect on the phosphorylation of eif2 cells of the 150kj/ m2uva radiation jb6 cells, the lowest phosphorylation of 5. m The effect of ho-1, such as rna and protein, was that the phosphorylation of eIF2 was increased with the addition of 150 kJ/ m2UVA radiation compared with the 150 kJ/ m2UVA group alone, and the phosphorylation of eIF2 was reduced by 0.5. m The effect of UVA radiation on Bach1, Nrf2 and HO-1 gene level in the combined UVA group was not significant, and the expression of Grp78 protein could be promoted, and the expression of Nrf2 and HO-1 protein could be induced. GSK2606414 delayed the expression of Grp78 in the UVA group and induced Nrf2 and HO-1 protein expression in advance. Compared with the 150 kJ/ m2UVA radiation group, the activity of the 3. m M Salubrinal pretreatment combined with 150 kJ/ m2UVA radiation cells increased, and the activity of the 0.5. mu.M GSK2606414 pre-treatment combined with 150 kJ/ m2UVA radiation cells was reduced. in contrast to that non-radiation group, the pre-treatment of 3. mu. M Salubrinal pretreatment, 0.5. mu.M GSK2606414 pre-treatment had no significant effect on the cell cycle, Compared with the 150 kJ/ m2UVA radiation group, the cell cycle S phase of the combined 150kJ/ m2UVA radiation cell increased by 7.28-1.03 (P0.05), and the difference between the pre-treatment of 0.5 & mu; M GSK2606414 and the cycle S phase of the 150 kJ/ m2UVA radiation cell was not significant. Conclusion: UVA radiation can activate endoplasmic reticulum stress, induce eIF2-induced phosphorylation, and eIF2-induced phosphorylation presents a dose-and time-dependent dependence. UVA radiation as an oxidative stress can also induce activation of the Nrf2-ARE pathway and also exhibit a dose and time-dependent dependence. The phosphorylation of eIF2 was maintained by using the Sal pretreatment, and the phosphorylation of eIF2 was inhibited using GSK2606414. The phosphorylation of eIF2 is involved in the regulation of Nrf2-HO-1 pathway. The increased phosphorylation of eIF2 can reduce the oxidative damage induced by UVA and promote cell proliferation. While inhibiting the phosphorylation of eif2 will increase the proliferation of uva-induced oxidative damage-inhibiting cells.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R751
本文编号：2500686
[Abstract]:BACKGROUND: UV radiation is a major environmental factor leading to acute, chronic skin lesions, skin photoaging, and even skin tumors. It is mainly the process of participating in the development of the skin disease by activating the expression of the related signal path and the reprogramming of some proteins. EIF2 is the main control factor involved in this process, and the phosphorylation of eIF2 can inhibit the synthesis of a large amount of protein in the cell, selectively activate the synthesis of certain proteins, thereby reducing the load of the endoplasmic reticulum. However, excessive endoplasmic reticulum stress induces the expression of eIF2 and the expression of Caspase family proteins to induce apoptosis. The phosphorylation of eIF2 is an effective way to adapt to the change of the environment, and plays an important role in the process of external stimulation. UVA can induce stress, activate the phosphorylation of PERK, and induce the phosphorylation of eIF2 in its downstream. In addition, UVA radiation is an oxidative stress that can induce ROS production, which causes oxidative damage to activate a series of activation of the antioxidant pathway, one of which is the induction of the Nrf2-HO-1 antioxidant pathway. UVA can activate Nrf2 and promote its transformation, and combine the anti-oxidation reaction original (ARE) to promote the synthesis of the downstream anti-oxidation enzyme (HO-1, SOD, NQO1, etc.) phase II detoxification enzyme. Wherein the heme oxygenase HO-1 is a starting enzyme and a speed-limiting enzyme for catalyzing the heme, which can catalyze the heme to generate ferritin, carbon monoxide (CO) and bilirubin with anti-oxygen and anti-inflammatory effects. Studies have shown that UVA can induce the phosphorylation of eIF2 and the activation of two mechanisms of protection of the Nrf2-HO-1 pathway, however, there are few studies on the relationship between endoplasmic reticulum stress and oxidative stress, and the mechanism of action between the two signal pathways is not clear. Therefore, it is of great significance to study the role of eIF2 in the expression of Nrf2-HO-1 in the oxidative damage induced by UVA and its protective effect on the oxidative damage induced by UVA. Objective: To study the effects of UVA (50,100,150,200,250 kJ/ m2) on the oxidative damage of UVA, and to verify that UVA can induce the phosphorylation of eIF2 and to verify that UVA can activate Nrf2-HO-1 pathway. The effects of pre-treatment on Nrf2, Bach1, H0-1 mRNA of 150 kJ/ m2UVA radiation cells and the effect of the pretreatment on the level of Grp78, Nrf2, Bach1, HO-1, P-eIF2 and eIF2 in the radiation cells of 150 kJ/ m2UVA were respectively pre-treated with 3. m u.M of Salubrinal and 0.5. m u.M of GSK2606414. The changes of the phosphorylation of eIF2 to the cell morphology were explored. The cell activity, the level of ROS in the cell and the effect of cell cycle were discussed, and the protective effect of eIF2 on the phosphorylation of eIF2 in the oxidative damage caused by UVA was discussed. Methods: the effect of uva on the activity of jb6 cells was detected by the method of mts; the effect of uva on the morphology of jb6 cells was detected by the fitc-dapi immunofluorescent staining method; the effect of the changes of the ethec-da fluorescent probe on the ros in the cells was detected by the dcfh-da fluorescent probe; the effect of nrf2, bachi1, ho-1 was detected in real time by the real-time quantitative pcr. The changes of the expression of grp78, nrf2, bachi1, ho-1, p-eif2, and eif2 were detected by the western blotting, and the effect of the alteration of eisf2 on the cell cycle was detected by flow cytometry. Results:1. long-wave ultraviolet uva radiation can induce the oxidative damage of jb6 cells, and the degree of damage is dose-dependent: compared with the non-radiation group, the cell morphology and the cell viability do not have a significant effect on the dose of 50 kj/ m2uva radiation (p0.05).100,150,200,250 kj/ m2uva radiation, The significant decrease in cell viability and statistically significant (p0.05) .2.uva radiation induced the dose and time-dependent phosphorylation of eif2: the different doses of uva (50,100,150,200,250 kj/ m2) were able to induce esif2 phosphorylation, and the degree of phosphorylation increased with the increase of the uva dose, The level of phosphorylation at 250 kj/ m2 was slightly lower than the level of phosphorylation of 200 kj/ m2. Therefore, we selected a moderate physiological dose of 150 kj/ m2uva radiation jb6 cells to detect the phosphorylation of eif2 antigen at different time points, and the phosphorylation of eif2 was increased immediately after uva irradiation compared to the non-irradiated group, The decrease of .3.uva radiation over time allows for the activation of the nrf2-are pathway and in time and dose-dependent: as the uva dose increases, the nrf2 protein level increases significantly as the uva dose increases, but the 250 kj/ m2uva radiation is reduced. While the level of ho-1 protein increased significantly with the increase of the uva dose. The changes of nrf2, ho-1 and protein levels of nrf2, ho-1 at different time points were detected after the selection of 150 kj/ m2uva radiation jb6 cells, and the transcription level of nrf2, ho-1 reached the peak at 4 h with the increase of the time point, and ho-1 reached its peak at 12 h. The protein level of nrf2 and ho-1 decreased firstly, and the protein level of ho-1 reached a peak at 12 h. Compared with the control group, no significant effect was found on the activity of the pre-treated cells of gsk2606414 in different concentrations (p0.05). Phosphorylation of the eif2 cells of the 150kj/ m2uva radiation jb6 cells with different concentrations of salubrinal pretreatment showed a dose-dependent manner, but the phosphorylation of eif2 cells at a concentration of 5. m u.m was slightly reduced. the pre-treatment of gsk2606414 at different concentrations had an inhibitory effect on the phosphorylation of eif2 cells of the 150kj/ m2uva radiation jb6 cells, the lowest phosphorylation of 5. m The effect of ho-1, such as rna and protein, was that the phosphorylation of eIF2 was increased with the addition of 150 kJ/ m2UVA radiation compared with the 150 kJ/ m2UVA group alone, and the phosphorylation of eIF2 was reduced by 0.5. m The effect of UVA radiation on Bach1, Nrf2 and HO-1 gene level in the combined UVA group was not significant, and the expression of Grp78 protein could be promoted, and the expression of Nrf2 and HO-1 protein could be induced. GSK2606414 delayed the expression of Grp78 in the UVA group and induced Nrf2 and HO-1 protein expression in advance. Compared with the 150 kJ/ m2UVA radiation group, the activity of the 3. m M Salubrinal pretreatment combined with 150 kJ/ m2UVA radiation cells increased, and the activity of the 0.5. mu.M GSK2606414 pre-treatment combined with 150 kJ/ m2UVA radiation cells was reduced. in contrast to that non-radiation group, the pre-treatment of 3. mu. M Salubrinal pretreatment, 0.5. mu.M GSK2606414 pre-treatment had no significant effect on the cell cycle, Compared with the 150 kJ/ m2UVA radiation group, the cell cycle S phase of the combined 150kJ/ m2UVA radiation cell increased by 7.28-1.03 (P0.05), and the difference between the pre-treatment of 0.5 & mu; M GSK2606414 and the cycle S phase of the 150 kJ/ m2UVA radiation cell was not significant. Conclusion: UVA radiation can activate endoplasmic reticulum stress, induce eIF2-induced phosphorylation, and eIF2-induced phosphorylation presents a dose-and time-dependent dependence. UVA radiation as an oxidative stress can also induce activation of the Nrf2-ARE pathway and also exhibit a dose and time-dependent dependence. The phosphorylation of eIF2 was maintained by using the Sal pretreatment, and the phosphorylation of eIF2 was inhibited using GSK2606414. The phosphorylation of eIF2 is involved in the regulation of Nrf2-HO-1 pathway. The increased phosphorylation of eIF2 can reduce the oxidative damage induced by UVA and promote cell proliferation. While inhibiting the phosphorylation of eif2 will increase the proliferation of uva-induced oxidative damage-inhibiting cells.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R751
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1 薛方方;eIF2α磷酸化对长波紫外线诱导的小鼠角质形成细胞氧化损伤中Nrf2-HO-1通路的影响[D];重庆大学;2016年
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