芹黃素對(duì)氧化應(yīng)激引起的人黑素細(xì)胞凋亡的影響
發(fā)布時(shí)間：2018-07-29 06:23
【摘要】：白癜風(fēng)是一種獲得性的進(jìn)行性的色素脫失性皮膚病,以局部表皮功能性黑素細(xì)胞缺失為主要特征。組織學(xué)的表現(xiàn)提示凋亡是引起黑素細(xì)胞缺失的主要原因。盡管白癜風(fēng)確切的發(fā)病機(jī)制尚不明確,越來越多的證據(jù)顯示,氧化應(yīng)激在白癜風(fēng)發(fā)病機(jī)制中起著重要作用。多巴胺是氧化應(yīng)激的誘導(dǎo)劑,體外可誘導(dǎo)黑素細(xì)胞氧化應(yīng)激及凋亡。在白癜風(fēng)患者中多巴胺的水平明顯升高。體外多巴胺誘導(dǎo)的黑素細(xì)胞凋亡模型可用于篩選可能對(duì)白癜風(fēng)治療有效的抗氧化劑。傳統(tǒng)中藥大多屬于植物藥,很多有效成分具有抗氧化、抗凋亡作用。從中藥有效成分中尋找對(duì)白癜風(fēng)治療可能有效的抗氧化劑,是目前研究的一個(gè)新方向。 目的:1.建立體外多巴胺誘導(dǎo)的黑素細(xì)胞氧化應(yīng)激凋亡模型。2.對(duì)文獻(xiàn)報(bào)道的6種具有抗氧化抗凋亡的中藥成分,利用多巴胺誘導(dǎo)黑素細(xì)胞凋亡的體外模型,檢測(cè)其對(duì)氧化應(yīng)激引起的黑素細(xì)胞凋亡的保護(hù)作用。3.探討其中對(duì)黑素細(xì)胞有保護(hù)作用的中藥成分(芹黃素)對(duì)多巴胺引起的氧化應(yīng)激及相關(guān)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)信號(hào)轉(zhuǎn)導(dǎo)通路的影響,初步探討其作用機(jī)制。 方法:1.采用健康兒童包皮組織原代培養(yǎng)獲得黑素細(xì)胞,免疫細(xì)胞化學(xué)法檢測(cè)S-100蛋白,進(jìn)行細(xì)胞鑒定。MTT法檢測(cè)不同濃度及作用時(shí)間的多巴胺對(duì)黑素細(xì)胞活力的影響。通過Annexin-V/PI雙染流式細(xì)胞術(shù)凋亡細(xì)胞比例檢測(cè)及Western Blot法檢測(cè)caspase3和多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)的活化,觀察多巴胺對(duì)黑素細(xì)胞凋亡的影響。DCFH-DA(2’,7’-dichloro?uorescin diacetate)法流式細(xì)胞術(shù)檢測(cè)多巴胺對(duì)黑素細(xì)胞活性氧分子(ROS)生成的影響。2. MTT法檢測(cè)芹黃素等6種中藥成分預(yù)處理對(duì)多巴胺引起的黑素細(xì)胞活力降低的影響。采用Annexin-V/PI雙染流式細(xì)胞術(shù)檢測(cè)這6種中藥成分對(duì)多巴胺誘導(dǎo)的黑素細(xì)胞凋亡的影響。3. Western Blot法檢測(cè)篩選出的有效中藥成分(芹黃素)對(duì)多巴胺引起的caspase3和PARP的活化的影響。DCFH-DA法流式細(xì)胞術(shù)檢測(cè)芹黃素對(duì)多巴胺誘導(dǎo)的黑素細(xì)胞活性氧分子(ROS)生成的影響。4. Western Blot法檢測(cè)多巴胺對(duì)黑素細(xì)胞的p38、JNK及Akt信號(hào)通路的影響。Annexin-V/PI雙染流式細(xì)胞術(shù)檢測(cè)p38抑制劑SB203580、JNK抑制劑SP600125、Akt抑制劑LY294002預(yù)處理黑素細(xì)胞對(duì)多巴胺促凋亡作用的影響。Western Blot法檢測(cè)芹黃素預(yù)處理對(duì)多巴胺激活的p38、JNK及Akt信號(hào)通路的影響。 結(jié)果: 1.多巴胺誘導(dǎo)的體外黑素細(xì)胞氧化應(yīng)激凋亡模型的建立 原代培養(yǎng)純化的黑素細(xì)胞,顯現(xiàn)出黑素細(xì)胞的明顯特征, S-100免疫細(xì)胞化學(xué)鑒定證實(shí)為高純度黑素細(xì)胞。多巴胺可呈濃度依賴性和時(shí)間依賴性抑制黑素細(xì)胞活力。500μM多巴胺作用于黑素細(xì)胞12h,細(xì)胞活力下降約50%。Annexin-V/PI雙染流式細(xì)胞術(shù)結(jié)果顯示,不同濃度多巴胺作用于黑素細(xì)胞12h,可呈濃度依賴性增加凋亡細(xì)胞比例。Western Blot結(jié)果顯示,500μM多巴胺作用黑素細(xì)胞6h或12h,caspase3和PARP出現(xiàn)明顯活化,12h組活化的PARP和caspase 3蛋白含量高于6h組。DCFH-DA法流式細(xì)胞術(shù)結(jié)果顯示,多巴胺可明顯增加黑素細(xì)胞ROS含量。以上結(jié)果表明500μM多巴胺作用于黑素細(xì)胞12h可明顯誘導(dǎo)黑素細(xì)胞氧化應(yīng)激和凋亡。 2.抑制黑素細(xì)胞凋亡的抗氧化中藥成分篩選 根據(jù)文獻(xiàn)報(bào)道選出具有抗氧化和抗凋亡作用的芹黃素、芍藥苷、葛根素、麥角甾苷、人參皂甙Rb1和姜黃素6味中藥成分。結(jié)果發(fā)現(xiàn),不同濃度的6味中藥成分預(yù)處理黑素細(xì)胞后,芹黃素可明顯抑制多巴胺誘導(dǎo)的黑素細(xì)胞活力降低,減少多巴胺誘導(dǎo)的黑素細(xì)胞凋亡。其他中藥成分在實(shí)驗(yàn)濃度下對(duì)黑素細(xì)胞活力和凋亡均無明顯影響。 3.芹黃素對(duì)多巴胺誘導(dǎo)的黑素細(xì)胞氧化應(yīng)激及凋亡的影響 0.3~10μM的芹黃素可呈濃度依賴性的抑制多巴胺引起的黑素細(xì)胞活力降低的作用,減少凋亡細(xì)胞比例。芹黃素預(yù)處理可降低多巴胺對(duì)PARP和caspase 3的活化,抑制多巴胺誘導(dǎo)的ROS生成。 4.芹黃素對(duì)黑素細(xì)胞氧化應(yīng)激相關(guān)的凋亡信號(hào)通路的影響 500μM多巴胺作用于黑素細(xì)胞,p38、JNK及Akt的磷酸化蛋白含量均呈時(shí)間依賴性增加。p38抑制劑SB203580、JNK抑制劑SP600125、Akt抑制劑LY294002預(yù)處理,可明顯減少多巴胺誘導(dǎo)的Annexin-V陽性細(xì)胞比例,證實(shí)多巴胺的促黑素細(xì)胞凋亡機(jī)制有賴于p38、JNK及Akt信號(hào)通路。而芹黃素預(yù)處理可明顯抑制多巴胺激活的p38、JNK及Akt的磷酸化,說明p38、JNK、Akt信號(hào)通路參與了芹黃素的抗多巴胺誘導(dǎo)的黑素細(xì)胞凋亡的機(jī)制。 結(jié)論: 1. 500μM多巴胺作用于黑素細(xì)胞12h可明顯誘導(dǎo)黑素細(xì)胞氧化應(yīng)激和凋亡。 2.芹黃素可抑制多巴胺誘導(dǎo)的黑素細(xì)胞ROS生成,減少黑素細(xì)胞凋亡。芍藥苷、葛根素、麥角甾苷、人參皂甙Rb1、姜黃素在實(shí)驗(yàn)濃度范圍內(nèi)對(duì)多巴胺誘導(dǎo)的黑素細(xì)胞活力降低和凋亡無明顯保護(hù)作用。 3.多巴胺的促黑素細(xì)胞凋亡作用有賴于p38、JNK、Akt信號(hào)通路的激活。p38、JNK、Akt信號(hào)通路參與了芹黃素的抗多巴胺誘導(dǎo)的黑素細(xì)胞凋亡的機(jī)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.41;R285.5
本文編號(hào)：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.41;R285.5
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