【作者】 卜今；

【导师】 尹跃平； 马鹏程； 张国毅； 周武庆；

【作者基本信息】 中国协和医科大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 1本课题组先前的研究发现,中药单体丹皮酚具有较强的抑制黑素合成的活性。本文研究了丹皮酚对黑素合成限速酶-Tyr的活性、mRNA和蛋白表达的影响,及对Tyr启动子配体小眼畸形转录因子(MITF)和MITF启动子配体cAMP反应元件结合蛋白(CREB)磷酸化表达的影响。本研究首次表明,丹皮酚可以通过抑制CREB的磷酸化,使其不活化,抑制MITF的转录,进而下调Tyr的转录以及表达,最终影响黑素合成。本研究结果有助于理解丹皮酚抗黑素合成的作用机制,有利于研究和筛选抗黑素沉着异常的药物。2我们研究了丹皮酚抑制黑素合成的信号转导途径,以进一步探讨丹皮酚美白功效的分子机制。研究结果表明,JNK1/2/3特异性抑制剂SP600125能够拮抗丹皮酚对黑素合成的抑制作用,包括黑素含量、Tyr活性及其mRNA和蛋白表达等多个指标;同时发现丹皮酚能够上调磷酸化JNK/SAPK的表达,而对磷酸化ERK和p38MAPK均无影响。表明在B16F10细胞内,丹皮酚可以激活MAPK家族的JNK/SAPK信号转导途径,从而减少黑素合成。而该家族的ERK1/2以及p38MAPK信号转导途径未参与介导丹皮酚的作用。根据上述结果,我们推测丹皮酚的作用机制是:丹皮酚激活了JNK/SAPK激酶,活化了JNK/SAPK途径,该途径的活化直接或间接激活了某种磷酸化酶,从而使磷酸化的CREB去磷酸化,下调MITF表达,进而减少Tyr表达,抑制黑素合成。在B16F10小鼠黑素瘤细胞系内,有关JNK/SAPK参与介导化学物质对黑素合成的调节作用尚未见报道。另外,我们的研究表明,cAMP/PKA特异性激动剂IBMX和抑制剂H89能分别拮抗和加剧丹皮酚对黑素合成的抑制作用。提示cAMP/PKA途径可能也参与了介导丹皮酚的抑制黑素合成作用。由于cAMP/PKA途径的激活本身对黑素合成具有上调作用,所以对该途径在丹皮酚确切作用机制有待进一步研究。对丹皮酚抑制黑素合成以及相关信号转导途径的研究有助于理解丹皮酚的作用机制,包括丹皮酚在其它疾病中的治疗机制。并为其用于治疗某些色素沉着异常性疾病提供了实验依据,有助于更全面地理解黑素合成机制以及色素异常性疾病的发病机制。3本课题组的前期研究发现丹皮酚在单独的MC以及KC-MC共培养模型中的抗黑素合成作用有明显差异,对共培养模型中黑素合成的抑制作用弱于对MC细胞中抑制作用,提示丹皮酚可能通过KC细胞间接影响MC细胞,从而削弱了其对黑素合成的抑制作用。因此我们利用单独KC、MC培养以及KC-MC共培养模型进一步研究这种差异的可能原因。KC细胞主要通过其分泌的细胞因子调节(主要包括ET-1和sSCF等)调节MC的黑素合成。在本研究中,我们利用已知的能够影响黑素合成的药物(HQ、丹皮酚、8-MOP、L-酪氨酸、熊果苷和烟酰胺)作用于单独KC、MC培养以及KC-MC共培养模型,检测不同药物作用后,单独培养的KC细胞模型和KC-MC共培养模型中ET-1和sSCF水平的变化,从而探讨MC单细胞模型和KC—MC共培养模型对药物作用产生不同反应的可能原因,并为探讨黑素合成反馈机制提供基础。研究结果表明,正是由于KC的存在,使得干预物对单独MC模型和KC-MC共培养模型中黑素合成的作用出现了明显的差异。药物作用72h后检测发现,在KC-MC共培养模型中,HQ可以同时显著减少KC合成ET-1和sSCF;而熊果苷虽然在抑制KC合成sSCF上和HQ作用相当,但是其对ET-1的抑制作用不如HQ;而丹皮酚对模型合成ET-1和sSCF则均无影响。同样作为促黑素合成干预物,酪氨酸可以促进细胞因子ET-1分泌,而8-MOP反而抑制了共培养模型合成ET-1。以上结果提示在共培养模型中,KC在一定程度上影响着药物对黑素合成的作用,而这种作用可以是拮抗的也可以是协同的,很大程度上取决于药物在共培养模型中对KC的影响,这种影响可能和药物本身的生物活性相关。说明在研究、筛选和开发抗色素异常性疾病的治疗药物时,选择KC-MC共培养模型比单一MC培养模型更能反映被选化合物的活性。该结果对指导新药研发具有一定意义。同时,通过对单独KC模型和KC-MC模型的比较,我们发现了一个有趣的现象:8-MOP对共培养模型中ET-1合成有抑制作用,但是在单独培养的KC细胞模型中,8-MOP作用72h时ET-1和sSCF均无明显改变(p＞0.05),提示8-MOP对共培养模型中ET-1的影响不是通过直接抑制KC完成的,而可能是通过其对MC的作用间接影响ET-1。为进一步研究黑素沉着中的反馈机制提供了思路。4筛选了10、20和50μM浓度的8-MOP作为正相的干扰信息(增加黑素合成)。筛选10和20μM浓度的PTU作为负相的干扰信息(减少黑素合成),利用此两种药物作为干扰信息,结合KC-MC共培养模型我们构建了一个闭环系统,检测并分析了72h后模型内黑素含量(输出变量)的改变和模型内细胞因子ET-1和sSCF(反馈信息)变化的关系。结果表明,变量黑素的改变影响了体系内ET-1的水平,各组的ET-1水平和黑素含量形成明显负相关(r=-0.858),并证明ET-1能够促进黑素合成,结果论证了KC-MC共培养体系中反馈调节机制的存在。本研究得出了关于黑素沉着的新的机制,即由MC、KC、黑素以及KC源性细胞因子构成的一个完整的反馈体系:KC通过自身分泌的细胞因子调节黑素沉着,当环境刺激(干扰信息)作用于MC(控制系统)时,MC合成并转运到KC内的黑素的量(输出变量)发生改变,KC(受控系统)感受到了这种改变,产生或减少细胞因子ET-1的合成分泌(反馈信息),反馈信息作用于MC,从而调节黑素合成。由此可推测,在表皮内的表皮黑素单位中,MC合成黑素并转运到KC,而KC通过自身分泌的细胞因子调节MC,MC通过所合成的黑素调节KC分泌细胞因子,以达到局部内环境的平衡。这种调控应答外界环境及干扰物对黑素沉着的调节。当某种外界因素(在一定的范围之内)作用于表皮黑素单位时,机体启动反馈调节机制,使黑素沉着保持在一定的水平。当刺激过强时,超出反馈控制系统的调节能力,原有的平衡被打破,黑素沉着产生异常。本研究提出并论证了黑素沉着中反馈控制系统及反馈调节机制的存在,对黑素沉着整体机制形成了必要的补充,有助于更全面地了解皮肤生理及色素异常性皮肤病的发病机理;并对临床用药有一定的指导意义。更多还原

【Abstract】 1 Paeonol,which is Chinese traditional medicine,has been proved to have a new function:inhibition of melanogenesis.Tyrosinase and its transcriptional regulator microphthalmia-associated transcription factor(MITF) play critical roles in regulation of melanogenesis,and are required for environmental cues or agents in modulation of melanin synthesis.In this report,we discovered that paeonol down-regulated melanin production via decreasing MITF expression and consequent mRNA and protein levels of tyrosinase.We also found that paeonol reduced phosphorylation of a cAMP responsive element binding protein (phospho-CREB),which binds and activates MITF.This research contributes to understanding the mechanism of inhibition by paeonol in B16F10 melamoma ceils.2 Identifying the signals mediating the effects of paeonol on melanogenesis is crucial to understanding how pigmentation responds to paeonol.Resultes showed that a selective inhibitor of c-jun N-terminal or stress-activated protein kinases(JNK/SAPK)-SP600125 significantly reversed paeonol-induced down-regulation of melanin synthesis.Inhibition of cAMP/PKA pathway intensified the hypopigmentation response to paeonol.These results identify a mechanism in which paeonol induces the down-regulation of melanogenesis through inhibition of CREB phosphorylation,leading to the expression reduction of MITF and subsequently tyrosinase.The key kinase mediating the effects of paeonol on melanogenesis in B16F10 cells is JNK/SAPK.Additionally,the cAMP/PKA pathway may take part in this process.Base on the two results,we conclude that melanogenesis inhibited by paeonol may be mediated by an unknown phosphorylase activated by JNK/SAPK pathway,and inhibiting phosphorylation of CREB and expression of MITF, consequent level of tyrosinase and melanin synthesis.The mediation of JNK/SAPK in pigmentation induced by chemicals has not been reported.The study on the key proteins,enzymes and signaling transduction pathways mediating the inhibition of melanogenesis by paeonol conduces to understanding the mechanism of paeonal on hypopigmentation,and contributes to the apply of the paeonol on pigmentary disorders in future.And it is also useful for the research on mechanism of pigmentation.3 In our prior researches,significant difference was found in the levels of inhibition on pigmentation by paeonol in MC culture and co-culture model.And the decreasing of melanogenesis in co-culture model was more obvious than that in MC culture cells.The phenomenon suggests that except for the direct effect on MC,paeonol may also act on KC,then influence on the melanogenesis indirectly. It is already kown that KC play a key role in regluating epidermal pigmentation by cytokines such as endothelin-1(ET-1) and stem cell factor(SCF) in paracrine way.Both of the two cytokines play important roles in melanin synthesis and proliferation of MC.We detected the levels of ET-1 and sSCF in KC and co-culture models after treated with classic regulators including melanogenesis agonists like HQ,and antagonists such as 8-MOP,in order to analysis the mechanism of different reactions to stimulators in single cell culture and co-culture model.Results showed that the vary effects on KC by different regulators is the main reason of different status of melanogenesis in two kinds of models.The effects on KC could be synergetic or antagonistic,which mainly dependes on the activity and/or dose of regulators.It shows that the KC-MC coculture model is more suitable for screening,studying the medicines applyed to clinical treatment.4 At the same time,significant difference was found in the levels of cytokines ET-1 and sSCF under stimulation of interferents in two models,although the number of cells were the same.For example,8-MOP in co-culture model inhibited the level of ET-1 with no effect on the level of ET-1 in KC model.We know 8-MOP is the antagonist of melanogenesis,and the finding suggests that the inhibition of ET-1 may result from the increasing of melanin induced by 8-MOP.What kind of interaction is it between the two cytes? Is it regulated by feedback mechanism?Because of close to the physiological environment of skin,the co-culture model is the suitable subject for us to study the interaction between KC and MC. Using co-culture model and specific regulators which affect melanin synthesis without effect on cytokines paracrined by KC,we set up an close loop system-feedback system,which is necessary for studying feedback mechanism. Negative correlation was proved between the melanin content and the concentration of ET-1 in co-culture model.And ET-1 is able to improve the melanin synthesis simultaneously.Base on the two results,we initially demonstrated that the feedback mechanism exists between MC and KC and regulates the pigmenattion to some extent.Summarily,the KC,MC,melnin and cytokines consist the total feedback system,in which the KC regulates the pigmentation by cytokines,when the MC is stimulated by environmental factors,the content of melanin transferred into KC changes,then KC regulates its cytokines’ synthesis and secretion to adjust the pigmentation.Furthermore it suggests that in epidermal melanin unit,KC regulates the melanin synthesis by cytokines and MC regulates the cytokines secreted by melanogenesis,and it is an balancing feedback system.When there is a strong stimulators which exceeds the regulation of system,the feedback has no fuctions, so the balance is broken,abnormal pigmentation happens consequently.It is the first time to initiate and demonstrate the feedback mechanism existing in co-culture model.This research is an important part of the complete pigmentation theory,and it can help us to understand the pathogenesis of pigmentary diseases and to find new targets for treatment.更多还原