Bach1在紫檀芪防御UV致HaCaT细胞急性光损伤中的作用
发布时间：2019-06-22 17:36
【摘要】：[研究背景]皮肤是被覆于体表的人体最大的器官,保护人体避免遭受各种环境因素尤其是紫外线(ultraviolet,UV)照射等造成的损伤。UV根据波长可分为长波紫外线(ultraviolet A,UVA,320 - 400nm)、中波紫外线(ultraviolet B,UVB,280 - 320nm)及短波紫外线(ultraviolet C,UVC,200 - 280nm)三类,作用于人体表面的主要是UVA和UVB。长期或者大量UV辐射可以造成皮肤损伤,如急性日晒伤、慢性光化性皮炎、光老化及皮肤癌等。随着环境污染的不断加重,UV所致皮肤疾病日益严重。目前研究认为,UV导致皮肤光损伤的机制复杂,除引起DNA双链的破坏和产生嘧啶光产物等直接损伤外,更重要的是氧化应激导致过量的活性氧簇(reactive oxygen species,ROS)堆积,从而对细胞的蛋白质、DNA、脂质等造成损伤,诱发多种光线性皮肤病。如何清除过量的ROS、增强抗氧化酶活性、阐明皮肤氧化应激防御机制以及寻找科学有效的UV防护剂成为目前的研究热点。核因子 E2 相关因子 2 (Nuclear factor erythroid 2-relatedfactor 2,Nrf2 )信号通路是目前最重要的抗氧化应激通路之一。当过量的ROS产生时,Nrf2与Kelch 样 ECH 联合蛋白 1 (kelch-like-Ech-associated - protein 1,Keap1)解偶联转位进入细胞核,原本和Maf识别元件(Maf recognition elements,MAREs)结合的转录因子BTB-CNC异体同源体-1 (BTB CNC homology 1, Bach1)解偶联,胞核内Bach1转位至胞浆内,Nrf2和MAREs形成异二聚体后识别并结合抗氧化反应元件(antioxidant response element,ARE),调控下游Ⅱ相解毒酶和抗氧化蛋白的表达,发挥其抗氧化作用。Bach1之前被认为只是同Nrf2竞争ARE的结合位点,对抗氧化基因的表达起到负性调控作用。最近研究发现Bach1可直接调控氧化还原、细胞周期等相关靶基因的表达,从而在氧化应激反应以及多种疾病发生中发挥重要的调控作用,可能成为氧化应激相关疾病的新型治疗靶点。国内外研究表明,多种天然植物成分如紫檀芪、茶多酚、姜黄素、枸杞多糖、莱菔硫烷、白藜芦醇等具有抗氧化作用,其中紫檀芪在多种肿瘤细胞中可通过调节Nrf2信号通路发挥抗氧化作用。但紫檀芪对UV辐照所致皮肤急性光损失是否具有保护作用以及可能的调控机制,目前尚不清楚。因此,本研究拟探讨紫檀苗对UV致人永生化角质细胞(Human keratinocytes,HaCaT)急性光损伤中的防护作用和Bach1的调控作用,为新型光防护剂的开发提供理论依据。[目的]1.探讨紫檀芪对UVA/UVB致HaCaT细胞急性光损伤是否有防护作用;2.探讨紫檀芪对UVA/UVB致HaCaT细胞急性光损伤可能防护机制;3.探讨Bach1在紫檀芪防御UVA/UVB致HaCaT细胞急性光损伤中的机制。[方法]1.分别以 UVA (30 J/cm2)和 UVB (300 mJ/cm2)剂量照射 HaCaT 细胞,采用MTT法检测和倒置显微镜观察照射后的细胞活性及细胞形态,以制备HaCaT细胞急性光损伤模型;2. CCK - 8法和倒置显微镜观察筛选紫檀芪非细胞毒性作用浓度;3.用MTT法和彗星实验检测紫檀芪对UVA/UVB照射HaCaT细胞前后的细胞活性和DNA损伤情况;4.用WST-1法和DCFH-DA法检测紫檀芪对UVA/UVB照射HaCaT细胞前后SOD活性和ROS水平的变化;5.用WesternBlot法检测紫檀芪对Nrf2/Bach1蛋白在细胞质和细胞核中的分布;6.用Western Blot检测磷酸激酶抑制剂Genistein对Nrf2/Bach1蛋白在细胞质和细胞核中的分布;7.用CCK-8法检测磷酸激酶抑制剂Genistein对UVA/UVB照射紫檀芪孵育HaCaT细胞前后的细胞活性变化;8.构建过表达的干扰短发夹RNA (short hairpin RNA,shRNA)的慢病毒载体的克隆,将HaCaT细胞中Nrf2敲低,获得Nrf2KD细胞;9.采用qRT-PCR技术分析HaCaT细胞和Nrf2KD细胞中Bach1可能调控的相关靶基因。[结果]1.分别以30 J/cm2的UVA和300 mJ/cm2的UVB剂量照射HaCaT细胞后其细胞活性下降(P0.05),细胞形态变圆,体积增大,胞内颗粒增加。2.用CCK-8法和倒置显微镜筛选出紫檀芪的非细胞毒性作用浓度5μM。3.用MTT法检测紫檀芪对UVA/UVB照射HaCaT细胞前后的细胞活性,与对照相比较,UVA/UVB照射后细胞活性下降(P0.05);紫檀芪预处理后细胞活性上升(P 0.05)。用彗星实验检测紫檀芪对UVA/UVB照射HaCaT细胞前后DNA损伤,与对照相比较,细胞核的尾长、尾矩、尾部DNA含量、尾部DNA百分比均显著增加(P 0.05);紫檀芪预处理后细胞核的尾长、尾矩、尾部DNA含量、尾部DNA百分比均减少(P0.05)。4.用WST-1法检测紫檀芪对UVA/UVB照射HaCaT细胞前后SOD活性,与对照组比较,SOD活性上升(P 0.05),紫檀芪处理后SOD活性上升(P0.05);用DCFH-DA法检测紫檀芪孵育后HaCaT细胞ROS水平,与对照相比较,ROS水平上升(P0.05),紫檀芪预处理后ROS水平下降(P0.05)。5.用Western Blot法检测紫檀芪对Nrf2/Bach1蛋白在细胞质和细胞核中的分布,胞核内Nrf2增加,Bach1减少。6.用Western Blot法检测磷酸酶抑制剂Genistein处理紫檀芪孵育HaCaT细胞,胞核内Bach1增加,Nrf2无变化。7.用CCK-8法检测磷酸激酶抑制剂Genistein对UVA/UVB照射紫檀芪孵育HaCaT细胞活性的变化,与对照组比较,磷酸酶抑制剂Genistein对HaCaT细胞的活性无影响(P0.05),磷酸酶抑制剂Genistein对UVA/UVB照射紫檀芪孵育HaCaT细胞的活性下降( 0.05)。8.采用慢病毒将Nrf2-shRNA转染至HaCaT细胞中,稳转细胞中Nrf2蛋白抑制率80%。9.采用qRT-PCR技术分析HaCaT细胞,与未加磷酸酶抑制剂Genistein对照相比较,bach1、bcl2l1 1、ftl、hmga2、hmox1、mafg、mapt、mmp13、mmp9、sqstm1、tfe3、vrna1-1和vrna1-2基因的表达上升(P0.05);采用qRT-PCR技术分析Nrf2KD细胞,与未加磷酸酶抑制剂Genistein对照相比较,gclc、gclm、hmga2、hmox1、itpr2、me1和vrnal- 基因的表达下降(P0.05),mafg、mapt和vegf基因的表达上升(P0.05)。[结论]1.紫檀芪对UVA/UVB致HaCaT细胞的急性光损伤具有防护作用;2.紫檀芪对UVA/UVB致HaCaT细胞急性光损的防护作用机制可能是激活抗氧化信号通路、提高细胞活性、降低DNA损伤;3. Bach1在紫檀芪防御UVA/UVB致HaCaT细胞急性光损伤中起调控作用,其可能是通过与Nrf2协同上调gclc、gclm、hmga2、hmox1、itpr2、me1和vrna1-2基因的表达,同时单独上调mafg、mapt和vegf基因的表达。
[Abstract]:[Study Background] The skin is the largest organ of the human body, which is covered on the body surface, and protects the human body from damage caused by various environmental factors, in particular ultraviolet (UV) irradiation, and the like. The UV wavelength can be divided into three types: long-wave ultraviolet (UVB,320-400 nm), medium-wave ultraviolet (UVB,280-320 nm) and short-wave ultraviolet (ultraviolet C, UVC,200-280 nm). Long-term or large amounts of UV radiation may cause skin lesions, such as acute sunburn, chronic actinic dermatitis, photoaging, and skin cancer. With the increasing of environmental pollution, the skin diseases caused by UV are becoming more and more serious. In the present study, the mechanism of UV-induced skin-light damage is complex, and in addition to the direct injury of the DNA double-chain and the generation of photogenic products, it is more important that oxidative stress causes an excess of reactive oxygen species (ROS) to accumulate, so that the proteins, DNA, The lipid and the like can cause damage and induce a plurality of light-sensitive skin diseases. How to remove excess ROS, enhance the activity of antioxidant enzymes, elucidate the defense mechanism of oxidative stress of the skin, and find a scientific and effective UV protective agent become the current research hotspot. The nuclear factor E2-related factor 2 (Nrf2) signal pathway is one of the most important anti-oxidative stress pathways. When an excess of ROS is produced, Nrf2 is coupled with the Kelch-like-Ech-associated-protein 1, Keap1, to the cell nucleus, and the transcription factor BTB-CNC homologue-1 (BTB CNC homology 1, Bach1), which is bound to the Maf recognition element (MAREs), is de-coupled. Bach1 in the nucleus of the cell is indexed to the cytoplasm, and the Nrf2 and MAREs form heterodimers and then recognize and bind to the anti-oxidation reaction element (ARE) to control the expression of the downstream II-phase detoxification enzyme and the anti-oxidation protein to play an anti-oxidation effect. Bach1 was previously thought to be a binding site to the Nrf2 competition, and a negative regulatory effect on the expression of the anti-oxidation gene. Recent studies have found that Bach1 can directly regulate the expression of related target genes such as redox and cell cycle, so as to play an important regulatory role in oxidative stress reaction and various diseases, and may be a new therapeutic target for oxidative stress-related diseases. The anti-oxidation effect of various natural plant components, such as rosewood, tea polyphenol, curcumin, lycium barbarum polysaccharide, leanthionane, and aloe, is shown at home and abroad, and the anti-oxidation effect can be exerted by adjusting the Nrf2 signal pathway in a variety of tumor cells. However, it is not clear whether the red sandalwood has a protective effect on the acute light loss caused by UV irradiation and the possible regulatory mechanism. Therefore, this study is to explore the protective effect of red sandalwood on the acute light damage of human immortalized keratinocytes (HaCaT) and the regulation of Bach1, and provide a theoretical basis for the development of novel photoprotective agent. [Objective] 1. To investigate the protective effect of red sandalwood on the acute light injury of HaCaT cells induced by UVA/ UVB. Objective To study the possible protective mechanism of red sandalwood on the acute light damage of HaCaT cells induced by UVA/ UVB. To study the mechanism of Bach1 in the treatment of acute light damage to HaCaT cells induced by UVB and UVA/ UVB. [Method] 1. HaCaT cells were irradiated with UVA (30 J/ cm2) and UVB (300 mJ/ cm2) doses, and the cell activity and cell morphology after irradiation were examined by MTT method and inverted microscope to prepare the acute light damage model of HaCaT cells. The non-cytotoxic effect of red sandalwood was observed by CCK-8 and inverted microscope. The cell activity and DNA damage before and after the irradiation of HaCaT cells with UVA/ UVB were detected by MTT and comet assay. The changes of SOD activity and ROS level in red sandalwood were detected by WST-1 method and DCFH-DA method before and after the irradiation of HaCaT cells with UVA/ UVB. The distribution of Nrf2/ Bach1 protein in cytoplasm and nucleus was detected by Western Blot method. The distribution of the Nrf2/ Bach1 protein in the cytoplasm and the nucleus of the phosphokinase inhibitor Genistein was detected by Western Blot. The changes of cell activity before and after the incubation of HaCaT cells with a phosphokinase inhibitor Genistein for UVA/ UVB were detected by the CCK-8 method. The expression of the lentiviral vector with short hairpin RNA (shRNA) was constructed, and the Nrf2 in the HaCaT cell was knocked down to obtain the Nrf2KD cell;9. The related target genes of Bach1 in HaCaT cells and Nrf2KD cells were analyzed by qRT-PCR. [Results] 1. After irradiation of HaCaT cells with UVB dose of 30 J/ cm2 and UVB dose of 300 mJ/ cm2, the cell activity decreased (P0.05). The non-cytotoxic effect of red sandalwood was 5. mu.M.3 by CCK-8 method and inverted microscope. The cell activity before and after the irradiation of HaCaT cells with UVA/ UVB was detected by MTT method, and the cell activity decreased after the irradiation with UVA/ UVB (P0.05); and the cell activity increased after the pre-treatment of the red sandalwood (P 0.05). The DNA damage before and after the irradiation of HaCaT cells with UVA/ UVB was detected by the comet assay, and the tail length, the tail moment, the tail DNA content and the tail DNA percentage of the nucleus were significantly increased (P 0.05), and the tail length, the tail moment and the tail DNA content of the nucleus after the pretreatment of the red sandalwood were significantly increased (P 0.05). The percentage of tail DNA was decreased (P0.05). The activity of SOD in red sandalwood was detected by WST-1 method before and after the irradiation of HaCaT cells with UVA/ UVB. Compared with the control group, the activity of SOD increased (P 0.05), and the activity of SOD in the treatment of the red sandalwood was higher than that of the control group (P 0.05). The level of ROS in HaCaT cells after incubation with DCFH-DA was compared with that of the control group (P0.05). The level of ROS in the pre-treatment of the red sandalwood was decreased (P0.05). The distribution of Nrf2/ Bach1 protein in the cytoplasm and nucleus of the Nrf2/ Bach1 protein was detected by Western Blot method, and the Nrf2 in the nucleus increased and Bach1 decreased. Phosphatase inhibitor Genistein was used to treat the HaCaT cells by Western Blot method, and the Bach1 in the nucleus was increased and Nrf2 was not changed. The changes of the activity of the phosphokinase inhibitor Genistein on the activity of HaCaT cells were detected by using the CCK-8 method. In comparison with the control group, the activity of the phosphatase inhibitor Genistein on the HaCaT cells was not affected (P0.05), and the activity of the phosphatase inhibitor Genistein on the HCaT cells was decreased (0.05). Nrf2-shRNA was transfected into HaCaT cells by lentivirus, and the inhibition rate of Nrf2 protein was 80%. HaCaT cells were analyzed by qRT-PCR, and the expression of bach1, bcl2l1 1, ftl, hmga2, hmox1, mafg, mapi, mp13, mp9, sqstm1, tfe3, vrno1-1 and vrno1-2 was increased (P0.05), and the Nrf2KD cells were analyzed by qRT-PCR. The expression of gclc, gclm, hmga2, hmox1, itpr2, me1, and vrnal-genes decreased (P0.05), and the expression of mafg, mapi, and vegf genes increased (P0.05). [Conclusion] 1. The effect of red sandalwood on the acute light injury of HaCaT cells induced by UVA/ UVB has a protective effect; The protective mechanism of the red sandalwood on the acute light loss of the HaCaT cells induced by UVA/ UVB may be the activation of the anti-oxidation signal pathway, increase the cell activity, and reduce the DNA damage; Bach1 plays an important role in the prevention of acute light damage of HaCaT cells induced by UVA/ UVB, which may be the expression of gclc, gclm, hmga2, hmox1, itpr2, me1, and vrno1-2 by co-regulation with Nrf2, while the expression of mafg, mapi and vegf genes is upregulated.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.1
本文编号：2504816
[Abstract]:[Study Background] The skin is the largest organ of the human body, which is covered on the body surface, and protects the human body from damage caused by various environmental factors, in particular ultraviolet (UV) irradiation, and the like. The UV wavelength can be divided into three types: long-wave ultraviolet (UVB,320-400 nm), medium-wave ultraviolet (UVB,280-320 nm) and short-wave ultraviolet (ultraviolet C, UVC,200-280 nm). Long-term or large amounts of UV radiation may cause skin lesions, such as acute sunburn, chronic actinic dermatitis, photoaging, and skin cancer. With the increasing of environmental pollution, the skin diseases caused by UV are becoming more and more serious. In the present study, the mechanism of UV-induced skin-light damage is complex, and in addition to the direct injury of the DNA double-chain and the generation of photogenic products, it is more important that oxidative stress causes an excess of reactive oxygen species (ROS) to accumulate, so that the proteins, DNA, The lipid and the like can cause damage and induce a plurality of light-sensitive skin diseases. How to remove excess ROS, enhance the activity of antioxidant enzymes, elucidate the defense mechanism of oxidative stress of the skin, and find a scientific and effective UV protective agent become the current research hotspot. The nuclear factor E2-related factor 2 (Nrf2) signal pathway is one of the most important anti-oxidative stress pathways. When an excess of ROS is produced, Nrf2 is coupled with the Kelch-like-Ech-associated-protein 1, Keap1, to the cell nucleus, and the transcription factor BTB-CNC homologue-1 (BTB CNC homology 1, Bach1), which is bound to the Maf recognition element (MAREs), is de-coupled. Bach1 in the nucleus of the cell is indexed to the cytoplasm, and the Nrf2 and MAREs form heterodimers and then recognize and bind to the anti-oxidation reaction element (ARE) to control the expression of the downstream II-phase detoxification enzyme and the anti-oxidation protein to play an anti-oxidation effect. Bach1 was previously thought to be a binding site to the Nrf2 competition, and a negative regulatory effect on the expression of the anti-oxidation gene. Recent studies have found that Bach1 can directly regulate the expression of related target genes such as redox and cell cycle, so as to play an important regulatory role in oxidative stress reaction and various diseases, and may be a new therapeutic target for oxidative stress-related diseases. The anti-oxidation effect of various natural plant components, such as rosewood, tea polyphenol, curcumin, lycium barbarum polysaccharide, leanthionane, and aloe, is shown at home and abroad, and the anti-oxidation effect can be exerted by adjusting the Nrf2 signal pathway in a variety of tumor cells. However, it is not clear whether the red sandalwood has a protective effect on the acute light loss caused by UV irradiation and the possible regulatory mechanism. Therefore, this study is to explore the protective effect of red sandalwood on the acute light damage of human immortalized keratinocytes (HaCaT) and the regulation of Bach1, and provide a theoretical basis for the development of novel photoprotective agent. [Objective] 1. To investigate the protective effect of red sandalwood on the acute light injury of HaCaT cells induced by UVA/ UVB. Objective To study the possible protective mechanism of red sandalwood on the acute light damage of HaCaT cells induced by UVA/ UVB. To study the mechanism of Bach1 in the treatment of acute light damage to HaCaT cells induced by UVB and UVA/ UVB. [Method] 1. HaCaT cells were irradiated with UVA (30 J/ cm2) and UVB (300 mJ/ cm2) doses, and the cell activity and cell morphology after irradiation were examined by MTT method and inverted microscope to prepare the acute light damage model of HaCaT cells. The non-cytotoxic effect of red sandalwood was observed by CCK-8 and inverted microscope. The cell activity and DNA damage before and after the irradiation of HaCaT cells with UVA/ UVB were detected by MTT and comet assay. The changes of SOD activity and ROS level in red sandalwood were detected by WST-1 method and DCFH-DA method before and after the irradiation of HaCaT cells with UVA/ UVB. The distribution of Nrf2/ Bach1 protein in cytoplasm and nucleus was detected by Western Blot method. The distribution of the Nrf2/ Bach1 protein in the cytoplasm and the nucleus of the phosphokinase inhibitor Genistein was detected by Western Blot. The changes of cell activity before and after the incubation of HaCaT cells with a phosphokinase inhibitor Genistein for UVA/ UVB were detected by the CCK-8 method. The expression of the lentiviral vector with short hairpin RNA (shRNA) was constructed, and the Nrf2 in the HaCaT cell was knocked down to obtain the Nrf2KD cell;9. The related target genes of Bach1 in HaCaT cells and Nrf2KD cells were analyzed by qRT-PCR. [Results] 1. After irradiation of HaCaT cells with UVB dose of 30 J/ cm2 and UVB dose of 300 mJ/ cm2, the cell activity decreased (P0.05). The non-cytotoxic effect of red sandalwood was 5. mu.M.3 by CCK-8 method and inverted microscope. The cell activity before and after the irradiation of HaCaT cells with UVA/ UVB was detected by MTT method, and the cell activity decreased after the irradiation with UVA/ UVB (P0.05); and the cell activity increased after the pre-treatment of the red sandalwood (P 0.05). The DNA damage before and after the irradiation of HaCaT cells with UVA/ UVB was detected by the comet assay, and the tail length, the tail moment, the tail DNA content and the tail DNA percentage of the nucleus were significantly increased (P 0.05), and the tail length, the tail moment and the tail DNA content of the nucleus after the pretreatment of the red sandalwood were significantly increased (P 0.05). The percentage of tail DNA was decreased (P0.05). The activity of SOD in red sandalwood was detected by WST-1 method before and after the irradiation of HaCaT cells with UVA/ UVB. Compared with the control group, the activity of SOD increased (P 0.05), and the activity of SOD in the treatment of the red sandalwood was higher than that of the control group (P 0.05). The level of ROS in HaCaT cells after incubation with DCFH-DA was compared with that of the control group (P0.05). The level of ROS in the pre-treatment of the red sandalwood was decreased (P0.05). The distribution of Nrf2/ Bach1 protein in the cytoplasm and nucleus of the Nrf2/ Bach1 protein was detected by Western Blot method, and the Nrf2 in the nucleus increased and Bach1 decreased. Phosphatase inhibitor Genistein was used to treat the HaCaT cells by Western Blot method, and the Bach1 in the nucleus was increased and Nrf2 was not changed. The changes of the activity of the phosphokinase inhibitor Genistein on the activity of HaCaT cells were detected by using the CCK-8 method. In comparison with the control group, the activity of the phosphatase inhibitor Genistein on the HaCaT cells was not affected (P0.05), and the activity of the phosphatase inhibitor Genistein on the HCaT cells was decreased (0.05). Nrf2-shRNA was transfected into HaCaT cells by lentivirus, and the inhibition rate of Nrf2 protein was 80%. HaCaT cells were analyzed by qRT-PCR, and the expression of bach1, bcl2l1 1, ftl, hmga2, hmox1, mafg, mapi, mp13, mp9, sqstm1, tfe3, vrno1-1 and vrno1-2 was increased (P0.05), and the Nrf2KD cells were analyzed by qRT-PCR. The expression of gclc, gclm, hmga2, hmox1, itpr2, me1, and vrnal-genes decreased (P0.05), and the expression of mafg, mapi, and vegf genes increased (P0.05). [Conclusion] 1. The effect of red sandalwood on the acute light injury of HaCaT cells induced by UVA/ UVB has a protective effect; The protective mechanism of the red sandalwood on the acute light loss of the HaCaT cells induced by UVA/ UVB may be the activation of the anti-oxidation signal pathway, increase the cell activity, and reduce the DNA damage; Bach1 plays an important role in the prevention of acute light damage of HaCaT cells induced by UVA/ UVB, which may be the expression of gclc, gclm, hmga2, hmox1, itpr2, me1, and vrno1-2 by co-regulation with Nrf2, while the expression of mafg, mapi and vegf genes is upregulated.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.1
【参考文献】
相关期刊论文 前2条
1 刘清;江娜;朱慧兰;;植物来源的抗氧化剂及其在皮肤科的应用[J];皮肤性病诊疗学杂志;2015年04期
2 彭世光;闫言;;紫外线对皮肤成纤维细胞蛋白差异表达的影响[J];中国生物美容;2009年04期
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