芹黄素对氧化应激引起的人黑素细胞凋亡的影响
发布时间：2018-07-29 06:23
【摘要】：白癜风是一种获得性的进行性的色素脱失性皮肤病,以局部表皮功能性黑素细胞缺失为主要特征。组织学的表现提示凋亡是引起黑素细胞缺失的主要原因。尽管白癜风确切的发病机制尚不明确,越来越多的证据显示,氧化应激在白癜风发病机制中起着重要作用。多巴胺是氧化应激的诱导剂,体外可诱导黑素细胞氧化应激及凋亡。在白癜风患者中多巴胺的水平明显升高。体外多巴胺诱导的黑素细胞凋亡模型可用于筛选可能对白癜风治疗有效的抗氧化剂。传统中药大多属于植物药,很多有效成分具有抗氧化、抗凋亡作用。从中药有效成分中寻找对白癜风治疗可能有效的抗氧化剂,是目前研究的一个新方向。 目的:1.建立体外多巴胺诱导的黑素细胞氧化应激凋亡模型。2.对文献报道的6种具有抗氧化抗凋亡的中药成分,利用多巴胺诱导黑素细胞凋亡的体外模型,检测其对氧化应激引起的黑素细胞凋亡的保护作用。3.探讨其中对黑素细胞有保护作用的中药成分(芹黄素)对多巴胺引起的氧化应激及相关的c-Jun氨基端激酶(c-Jun N-terminal Kinase,JNK)、p38和磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)/Akt(v-akt murine thymoma viral oncogene homolog)信号转导通路的影响,初步探讨其作用机制。 方法:1.采用健康儿童包皮组织原代培养获得黑素细胞,免疫细胞化学法检测S-100蛋白,进行细胞鉴定。MTT法检测不同浓度及作用时间的多巴胺对黑素细胞活力的影响。通过Annexin-V/PI双染流式细胞术凋亡细胞比例检测及Western Blot法检测caspase3和多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)的活化,观察多巴胺对黑素细胞凋亡的影响。DCFH-DA(2’,7’-dichloro?uorescin diacetate)法流式细胞术检测多巴胺对黑素细胞活性氧分子(ROS)生成的影响。2. MTT法检测芹黄素等6种中药成分预处理对多巴胺引起的黑素细胞活力降低的影响。采用Annexin-V/PI双染流式细胞术检测这6种中药成分对多巴胺诱导的黑素细胞凋亡的影响。3. Western Blot法检测筛选出的有效中药成分(芹黄素)对多巴胺引起的caspase3和PARP的活化的影响。DCFH-DA法流式细胞术检测芹黄素对多巴胺诱导的黑素细胞活性氧分子(ROS)生成的影响。4. Western Blot法检测多巴胺对黑素细胞的p38、JNK及Akt信号通路的影响。Annexin-V/PI双染流式细胞术检测p38抑制剂SB203580、JNK抑制剂SP600125、Akt抑制剂LY294002预处理黑素细胞对多巴胺促凋亡作用的影响。Western Blot法检测芹黄素预处理对多巴胺激活的p38、JNK及Akt信号通路的影响。 结果: 1.多巴胺诱导的体外黑素细胞氧化应激凋亡模型的建立 原代培养纯化的黑素细胞,显现出黑素细胞的明显特征, S-100免疫细胞化学鉴定证实为高纯度黑素细胞。多巴胺可呈浓度依赖性和时间依赖性抑制黑素细胞活力。500μM多巴胺作用于黑素细胞12h,细胞活力下降约50%。Annexin-V/PI双染流式细胞术结果显示,不同浓度多巴胺作用于黑素细胞12h,可呈浓度依赖性增加凋亡细胞比例。Western Blot结果显示,500μM多巴胺作用黑素细胞6h或12h,caspase3和PARP出现明显活化,12h组活化的PARP和caspase 3蛋白含量高于6h组。DCFH-DA法流式细胞术结果显示,多巴胺可明显增加黑素细胞ROS含量。以上结果表明500μM多巴胺作用于黑素细胞12h可明显诱导黑素细胞氧化应激和凋亡。 2.抑制黑素细胞凋亡的抗氧化中药成分筛选 根据文献报道选出具有抗氧化和抗凋亡作用的芹黄素、芍药苷、葛根素、麦角甾苷、人参皂甙Rb1和姜黄素6味中药成分。结果发现,不同浓度的6味中药成分预处理黑素细胞后,芹黄素可明显抑制多巴胺诱导的黑素细胞活力降低,减少多巴胺诱导的黑素细胞凋亡。其他中药成分在实验浓度下对黑素细胞活力和凋亡均无明显影响。 3.芹黄素对多巴胺诱导的黑素细胞氧化应激及凋亡的影响 0.3~10μM的芹黄素可呈浓度依赖性的抑制多巴胺引起的黑素细胞活力降低的作用,减少凋亡细胞比例。芹黄素预处理可降低多巴胺对PARP和caspase 3的活化,抑制多巴胺诱导的ROS生成。 4.芹黄素对黑素细胞氧化应激相关的凋亡信号通路的影响 500μM多巴胺作用于黑素细胞,p38、JNK及Akt的磷酸化蛋白含量均呈时间依赖性增加。p38抑制剂SB203580、JNK抑制剂SP600125、Akt抑制剂LY294002预处理,可明显减少多巴胺诱导的Annexin-V阳性细胞比例,证实多巴胺的促黑素细胞凋亡机制有赖于p38、JNK及Akt信号通路。而芹黄素预处理可明显抑制多巴胺激活的p38、JNK及Akt的磷酸化,说明p38、JNK、Akt信号通路参与了芹黄素的抗多巴胺诱导的黑素细胞凋亡的机制。 结论: 1. 500μM多巴胺作用于黑素细胞12h可明显诱导黑素细胞氧化应激和凋亡。 2.芹黄素可抑制多巴胺诱导的黑素细胞ROS生成,减少黑素细胞凋亡。芍药苷、葛根素、麦角甾苷、人参皂甙Rb1、姜黄素在实验浓度范围内对多巴胺诱导的黑素细胞活力降低和凋亡无明显保护作用。 3.多巴胺的促黑素细胞凋亡作用有赖于p38、JNK、Akt信号通路的激活。p38、JNK、Akt信号通路参与了芹黄素的抗多巴胺诱导的黑素细胞凋亡的机制。
[Abstract]:Vitiligo is an acquired progressive pigmented dermatosis with local epidermal functional melanocyte depletion as the main feature. Histologic findings suggest that apoptosis is the main cause of melanocyte depletion. Although the exact pathogenesis of vitiligo is not clear, the more evidences show that oxidative stress is in vitiligo It plays an important role in the pathogenesis. Dopamine is an inducer of oxidative stress. In vitro, it can induce oxidative stress and apoptosis in melanocytes. The level of dopamine in patients with vitiligo is significantly increased. In vitro, dopamine induced melanocyte apoptosis model can be used to screen the effective antioxidants for the treatment of vitiligo. Many effective components are antioxidant and anti apoptotic. It is a new research direction to find effective antioxidants for the treatment of vitiligo.
Objective: 1. to establish a dopamine induced melanocyte oxidative stress apoptosis model.2. in vitro, 6 kinds of traditional Chinese medicine with antioxidant and anti apoptosis, using dopamine to induce the apoptosis of melanocyte in vitro, and to detect the protective effect of.3. on melanocyte apoptosis induced by oxidative stress The effect of the protective effect of Chinese herbal composition (apigenin) on the oxidative stress induced by dopamine and the related c-Jun amino terminal kinase (c-Jun N-terminal Kinase, JNK), p38 and phosphatidylinositol 3- kinase (phosphatidylinositol 3-kinase, PI3K) /Akt (phosphatidylinositol 3-kinase, PI3K) signal transduction pathway System.
Methods: 1. the melanocytes were obtained by primary culture of healthy children's prepuce tissue, S-100 protein was detected by immunocytochemical method, and the effect of dopamine on the activity of melanocytes was detected by.MTT method, and the proportion of apoptotic cells in Annexin-V/PI double dye flow cytometry and the detection of Ca by Western Blot method. Activation of spase3 and ADP- poly ADP-ribose polymerase (PARP), the effect of dopamine on the apoptosis of melanocytes,.DCFH-DA (2 ', 7' -dichloro? Uorescin diacetate) method of flow cytometry to detect the effect of dopamine on the production of reactive oxygen molecules (ROS) in melanocytes, 6 kinds of herbal ingredients, such as apigenin, were detected by.2. Effects of treatment on the degradation of melanocyte activity induced by dopamine. The effect of Annexin-V/PI double dye flow cytometry on the apoptosis of melanocytes induced by dopamine, the effect of.3. Western Blot method on the activation of Caspase3 and PARP induced by dopamine (apigenin) was determined by Annexin-V/PI Blot method. The effect of apigenin on the production of ROS in melanocytes induced by dopamine by DA method of flow cytometry the effects of.4. Western Blot on the p38, JNK and Akt signaling pathways of melanocytes were detected by.Annexin-V/PI dual dye flow cytometry for the detection of p38 inhibitor SB203580, JNK inhibitors The effects of melanocytes on dopamine induced apoptosis were examined by.Western Blot assay. The effects of apigenin pretreatment on dopamine activated p38, JNK and Akt signaling pathways were detected.
Result:
1. dopamine induced apoptosis model of melanocyte oxidative stress in vitro
The primary culture of purified melanocytes showed obvious characteristics of melanocytes. S-100 immunocytochemical identification proved to be highly purified melanocytes. Dopamine could be dependent on the concentration dependent and time dependent inhibitory activity of melanocyte.500 M dopamine on melanocyte 12h, and the cell viability decreased by about 50%.Annexin-V/PI double dye flow cytometry The results showed that different concentrations of dopamine acted on the melanocyte 12h and increased the proportion of apoptotic cells in a concentration dependent manner.Western Blot. The results showed that 500 uh M dopamine acted as 6h or 12h, Caspase3 and PARP were obviously activated. The content of PARP and caspase 3 protein activated in the 12h group was higher than that of the 6h group flow cytometry. The results showed that dopamine significantly increased the content of ROS in melanocytes. The above results showed that the action of 500 mu M dopamine on melanocyte 12h could induce oxidative stress and apoptosis in melanocytes.
2. screening of antioxidant components for inhibiting melanocyte apoptosis
According to the literature, we selected the antioxidant and anti apoptotic effect of apigenin, paeoniflorin, puerarin, ergosterin, ginsenoside Rb1 and curcumin 6 ingredients. The results showed that after treating melanocytes with different concentrations of 6 ingredients, apigenin could obviously inhibit the decrease of melanocyte activity induced by dopamine and reduce dopamine Apoptosis of melanocytes was induced. Other Chinese medicine components had no significant effect on melanocyte viability and apoptosis under experimental concentration.
3. effects of apigenin on oxidative stress and apoptosis induced by dopamine in melanocytes
Apigenin of 0.3~10 mu M can decrease the activity of melanocyte activity in a concentration dependent inhibition of dopamine and reduce the proportion of apoptotic cells. Apigenin preconditioning can reduce the activation of dopamine to PARP and caspase 3 and inhibit the formation of ROS induced by dopamine.
4. effects of apigenin on oxidative stress related signaling pathways in melanocytes
The phosphorylated protein content of 500 M dopamine in melanocytes, p38, JNK and Akt showed a time dependent increase of.P38 inhibitor SB203580, JNK inhibitor SP600125, and Akt inhibitor LY294002 pretreatment, which could significantly reduce the proportion of dopamine induced Annexin-V positive cells, and confirmed that the mechanism of dopamine melanocyte apoptosis depends on p38, and Akt signaling pathway. Apigenin preconditioning can significantly inhibit the phosphorylation of dopamine activated p38, JNK and Akt, indicating that p38, JNK, Akt signaling pathway participates in the mechanism of apigenin induced melanocyte apoptosis.
Conclusion:
The action of 1.500 mu M dopamine on melanocytes 12h can induce the oxidative stress and apoptosis of melanocytes.
2. apigenin could inhibit the ROS formation of melanocytes induced by dopamine and reduce the apoptosis of melanocytes. Paeoniflorin, puerarin, ergosterin, ginsenoside Rb1 and curcumin have no obvious protective effect on the decrease of dopamine induced melanocyte activity and apoptosis in the experimental concentration range.
3. the apoptosis of dopamine melanocytes depends on the activation of p38, JNK, Akt signaling pathway and.P38, and the JNK, Akt signaling pathway participates in the mechanism of apigenin induced melanocyte apoptosis.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.41;R285.5
本文编号：2151811
[Abstract]:Vitiligo is an acquired progressive pigmented dermatosis with local epidermal functional melanocyte depletion as the main feature. Histologic findings suggest that apoptosis is the main cause of melanocyte depletion. Although the exact pathogenesis of vitiligo is not clear, the more evidences show that oxidative stress is in vitiligo It plays an important role in the pathogenesis. Dopamine is an inducer of oxidative stress. In vitro, it can induce oxidative stress and apoptosis in melanocytes. The level of dopamine in patients with vitiligo is significantly increased. In vitro, dopamine induced melanocyte apoptosis model can be used to screen the effective antioxidants for the treatment of vitiligo. Many effective components are antioxidant and anti apoptotic. It is a new research direction to find effective antioxidants for the treatment of vitiligo.
Objective: 1. to establish a dopamine induced melanocyte oxidative stress apoptosis model.2. in vitro, 6 kinds of traditional Chinese medicine with antioxidant and anti apoptosis, using dopamine to induce the apoptosis of melanocyte in vitro, and to detect the protective effect of.3. on melanocyte apoptosis induced by oxidative stress The effect of the protective effect of Chinese herbal composition (apigenin) on the oxidative stress induced by dopamine and the related c-Jun amino terminal kinase (c-Jun N-terminal Kinase, JNK), p38 and phosphatidylinositol 3- kinase (phosphatidylinositol 3-kinase, PI3K) /Akt (phosphatidylinositol 3-kinase, PI3K) signal transduction pathway System.
Methods: 1. the melanocytes were obtained by primary culture of healthy children's prepuce tissue, S-100 protein was detected by immunocytochemical method, and the effect of dopamine on the activity of melanocytes was detected by.MTT method, and the proportion of apoptotic cells in Annexin-V/PI double dye flow cytometry and the detection of Ca by Western Blot method. Activation of spase3 and ADP- poly ADP-ribose polymerase (PARP), the effect of dopamine on the apoptosis of melanocytes,.DCFH-DA (2 ', 7' -dichloro? Uorescin diacetate) method of flow cytometry to detect the effect of dopamine on the production of reactive oxygen molecules (ROS) in melanocytes, 6 kinds of herbal ingredients, such as apigenin, were detected by.2. Effects of treatment on the degradation of melanocyte activity induced by dopamine. The effect of Annexin-V/PI double dye flow cytometry on the apoptosis of melanocytes induced by dopamine, the effect of.3. Western Blot method on the activation of Caspase3 and PARP induced by dopamine (apigenin) was determined by Annexin-V/PI Blot method. The effect of apigenin on the production of ROS in melanocytes induced by dopamine by DA method of flow cytometry the effects of.4. Western Blot on the p38, JNK and Akt signaling pathways of melanocytes were detected by.Annexin-V/PI dual dye flow cytometry for the detection of p38 inhibitor SB203580, JNK inhibitors The effects of melanocytes on dopamine induced apoptosis were examined by.Western Blot assay. The effects of apigenin pretreatment on dopamine activated p38, JNK and Akt signaling pathways were detected.
Result:
1. dopamine induced apoptosis model of melanocyte oxidative stress in vitro
The primary culture of purified melanocytes showed obvious characteristics of melanocytes. S-100 immunocytochemical identification proved to be highly purified melanocytes. Dopamine could be dependent on the concentration dependent and time dependent inhibitory activity of melanocyte.500 M dopamine on melanocyte 12h, and the cell viability decreased by about 50%.Annexin-V/PI double dye flow cytometry The results showed that different concentrations of dopamine acted on the melanocyte 12h and increased the proportion of apoptotic cells in a concentration dependent manner.Western Blot. The results showed that 500 uh M dopamine acted as 6h or 12h, Caspase3 and PARP were obviously activated. The content of PARP and caspase 3 protein activated in the 12h group was higher than that of the 6h group flow cytometry. The results showed that dopamine significantly increased the content of ROS in melanocytes. The above results showed that the action of 500 mu M dopamine on melanocyte 12h could induce oxidative stress and apoptosis in melanocytes.
2. screening of antioxidant components for inhibiting melanocyte apoptosis
According to the literature, we selected the antioxidant and anti apoptotic effect of apigenin, paeoniflorin, puerarin, ergosterin, ginsenoside Rb1 and curcumin 6 ingredients. The results showed that after treating melanocytes with different concentrations of 6 ingredients, apigenin could obviously inhibit the decrease of melanocyte activity induced by dopamine and reduce dopamine Apoptosis of melanocytes was induced. Other Chinese medicine components had no significant effect on melanocyte viability and apoptosis under experimental concentration.
3. effects of apigenin on oxidative stress and apoptosis induced by dopamine in melanocytes
Apigenin of 0.3~10 mu M can decrease the activity of melanocyte activity in a concentration dependent inhibition of dopamine and reduce the proportion of apoptotic cells. Apigenin preconditioning can reduce the activation of dopamine to PARP and caspase 3 and inhibit the formation of ROS induced by dopamine.
4. effects of apigenin on oxidative stress related signaling pathways in melanocytes
The phosphorylated protein content of 500 M dopamine in melanocytes, p38, JNK and Akt showed a time dependent increase of.P38 inhibitor SB203580, JNK inhibitor SP600125, and Akt inhibitor LY294002 pretreatment, which could significantly reduce the proportion of dopamine induced Annexin-V positive cells, and confirmed that the mechanism of dopamine melanocyte apoptosis depends on p38, and Akt signaling pathway. Apigenin preconditioning can significantly inhibit the phosphorylation of dopamine activated p38, JNK and Akt, indicating that p38, JNK, Akt signaling pathway participates in the mechanism of apigenin induced melanocyte apoptosis.
Conclusion:
The action of 1.500 mu M dopamine on melanocytes 12h can induce the oxidative stress and apoptosis of melanocytes.
2. apigenin could inhibit the ROS formation of melanocytes induced by dopamine and reduce the apoptosis of melanocytes. Paeoniflorin, puerarin, ergosterin, ginsenoside Rb1 and curcumin have no obvious protective effect on the decrease of dopamine induced melanocyte activity and apoptosis in the experimental concentration range.
3. the apoptosis of dopamine melanocytes depends on the activation of p38, JNK, Akt signaling pathway and.P38, and the JNK, Akt signaling pathway participates in the mechanism of apigenin induced melanocyte apoptosis.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.41;R285.5
【参考文献】
相关期刊论文 前3条
1 谭榀新;叶涛;刘湘新;贺建华;;植物提取物抗氧化成分及机理研究进展[J];食品科学;2010年15期
2 孙秀琴;李荣华;唐涛;;芹菜素抗氧化作用研究进展[J];中国现代医生;2009年27期
3 郑瑞生;封辉;戴聪杰;洪小斌;宁秋蓉;;植物中抗氧化活性成分研究进展[J];中国农学通报;2010年09期
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