【作者】 姜泽群；

【导师】 何光源； 杨广笑；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 白发是由于黑色素的生成受阻所致,受阻的原因包括:黑素细胞、黑素体较少,酪氨酸酶活性降低等。临床上中医治疗白发有大量方剂,这些是研究白发治疗的宝贵资源,其中何首乌和黑芝麻在乌发方剂中频繁出现,说明它们具有很好的促黑素生成的效果。虽然有关何首乌和黑芝麻乌发的研究很多,但对其乌发有效成分、乌发机理的研究却仍不深入。本文除了对何首乌和黑芝麻的致色素原理作了研究外,还阐明了其中具有显著增色作用的活性成分和其详细作用机理,佐证了其传统乌发功效的现代科学含义。现将本文的具体研究内容概括如下:1.何首乌和黑芝麻水提物均有激活蘑菇酪氨酸酶和促进黑素生成的作用,增色效果显著。采用蘑菇酪氨酸酶多巴速率氧化法和NaOH裂解法研究了何首乌和黑芝麻水提物或醇提物对蘑菇酪氨酸酶活性和黑素生成的影响。结果发现:何首乌的水提物和黑芝麻的水提物在体外对蘑菇酪氨酸酶的激活及促进黑素生成方而都有较强的作用,而醇提物的效果则并不明显。2.何首乌和黑芝麻水提物都能显著增强B16黑素瘤细胞中的黑色素生成,对细胞增殖、黑素生成和酪氨酸酶活性均有激活作用,并可促进黑素生成相关基因的转录。采用B16黑素瘤细胞系为实验对象,检测了何首乌水提物和黑芝麻水提物对细胞内黑素含量和酪氨酸酶活性的影响,同时利用RT-PCR和Western-blot方法检测了其对黑素生成相关基因表达的影响。结果显示:在B16黑素瘤细胞内,何首乌水提物和黑芝麻水提物都能浓度依赖性地增加酪氨酸酶活性和黑素生成量,并促进酪氨酸酶和小眼相关转录因子(Microphthalmia-associtated transcription factor,MITF)在转录和翻译水平上的表达。3.何首乌的有效成分二苯乙烯苷可通过激活有丝分裂原活化蛋白激酶p38MAPK和转录因子MITF,促进酪氨酸酶基因的表达,增强酪氨酸酶的活性,从而上调B16黑素瘤细胞中黑素的生成。用L-多巴染色法检测了二苯乙烯苷对酪氨酸酶活性的影响,发现二苯乙烯苷促进黑素生成的机理之一是可显著增加酪氨酸酶的活性。RT-PCR以及Western-blot检测结果显示二苯乙烯苷能促进酪氨酸酶基因在转录和翻译水平上的表达。利用Western-blot方法还发现二苯乙烯苷可以导致酪氨酸酶转录因子MITF的激活,并同时诱导环磷腺苷反应元件结合蛋白CREB的磷酸化表达水平升高,说明MITF的激活可能是依赖于cAMP的。另外,Westem blot法检测二苯乙烯苷对不同MAPK途径关键激酶磷酸化蛋白表达的调节作用,发现它可以上调磷酸化p38MAPK的表达,MAPK信号通路抑制剂实验则从反向证明了二苯乙烯苷能通过激活p38 MAPK信号通路来上调酪氨酸酶和MITF基因的表达,从而促进了B16细胞中黑素的生成。4.阐明了芝麻中具有显著增色作用的有效成分是芝麻素,芝麻素可通过激活有丝分裂原活化蛋白激酶p38 MAPK和cAMP依赖的蛋白激酶A(PKA)信号通路来上调酪氨酸酶和MITF基因的表达,从而促进B16黑素瘤细胞中黑素的生成。芝麻素对黑色素的生成和细胞形态的黑素化表现出剂量依赖性的促进作用。研究发现,芝麻素促进黑素生成的机理之一是增加酪氨酸酶的活性并上调酪氨酸酶基因的表达。另外,芝麻素可以促进p38 MAPK和环磷腺苷反应元件结合蛋白CREB的磷酸化表达水平升高,并引起MITF的持续激活,说明MAPK和PKA信号通路可能参与了芝麻素诱导的黑素生成过程。还发现芝麻素可以导致PKA活性的增加,诱导CRE启动子的激活,并使细胞内cAMP的含量显著升高,进一步证明了芝麻素对cAMP-PKA信号通路的激活作用。信号通路抑制剂实验则从反向证明了芝麻素能通过激活p38 MAPK和cAMP-PKA信号途径来上调酪氨酸酶和MITF基因的表达,从而促进了B16细胞中黑素的生成。更多还原

【Abstract】 The main reason of gray hair is the decrease of melanins, resulting from the deficiency of melanocytes, melanosomes and tyrosinase activity. There are many Chinese herbs used for hair graying treatment, of them Polygonum multiflorum Thunb and Semen Sesami Nigrum are the two most frequently used herbs, indicating their potential in stimulating melanogenesis. Although there are many reports about the two traditional herbs on gray hair, little is known about their effective components and related mechanism on melanogenesis. The present study is to clarify their effect, mechanism and to find out the effective component for melanogenesis. The results above indicated the relationship between the two traditional pigmentation herbs and melanogenensis. The major results are summarized as follows:1. The water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum was found to have a strong pigmentation effect by increasing mushroom tyrosinase activity and melanin content, suggesting their potential for effective components development. The mushroom tyrosinase activity and melanin synthesis were measured by dopaoxidase and NaOH methods respectively. The results showed that the water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum increased the mushroom tyrosinase activity and melanin content significantly in a dose-dependent manner, while their ethanol extract had no significant effect.2. The water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum induces melanogenesis by increasing cell viability, melanin content and tyrosinase activity, as well as the expression of melanogenesis key enzymes. The tyrosinase activity and melanin synthesis were measured in B16 melanoma cells by dopaoxidase and NaOH methods respectively. The protein and mRNA expression of melanogenesis key enzymes were investigated by western blot and RT-PCR. The results showed that the water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum could increase the tyrosinase activity and melanin content significantly in a dose-dependent manner in B16 cells. The increasing mRNA and protein expression of TYR and MITF (microphthalmia-associated transcription factor) were also observed in a dose-dependent manner.3. The 2, 3, 5, 4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a water-soluble component extracted from dried tuber root of Polygonum multiflorum Thunb, was found to induce pigmentation in B16 cells by MAP kinase activation and tyrosinase upregulation. L-Dopa zymography was used to study the tyrosinase activity and NaOH method was used for melanin content assay. We found that THSG could increase the melanin content, tyrosinase activity and tyrosinase expression in a concentration-dependent manner. We then investigated whether THSG influences the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. THSG was found to induce sustained MITF up-regulation and cAMP response element (CRE) binding protein (CREB) activation, suggesting that THSG-mediated MITF activation may be cAMP dependent. Furthermore, Western blot analysis revealed that THSG could elevate the level of phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) significantly; a p38 MAPK inhibitor, SB203580, almost completely attenuated the THSG-mediated up-regulation of melanin synthesis and induction of MITF and tyrosinase expression.4. Sesamin, an active lignan of Semen Sesami Nigrum, was found to have a strong pigmentation effect in BI6 melanoma cells via activation of protein Kinase A and p38 MAP kinase. Treatment of B16 cells with sesamin increased the melanin content, tyrosinase activity and tyrosinase expression in a dose-dependent manner. Moreover, result of Western blot showed that sesamin induced sustained MITF (microphthalmia-associated transcription factor) activation, CRE-binding protein phosphorylation, p38 MAPK up-regulation but ERK down-regulation in a time-dependent manner. These results indicate that the protein Kinase A (PKA) and MAP kinase (MAPK) signaling pathways are both involved in sesamin-induced melanogenesis. Furthermore, intracellular cAMP concentration and CRE promoter activity were significantly increased by sesamin. Additionaly, we found that sesamin could induce protein kinase A (PKA) activation in B16 cells. All these results suggest the involvement of cAMP-PKA pathway in sesamin-induced melanogenesis. Consistent with this, inhibitors of PKA and p38 MAPK almost completely attenuated sesamin-inediated up-regulation of melanin synthesis and induction of MITF and tyrosinase. These data suggest that sesamin induces melanogenesis via the cAMP-PKA and MAPK signaling pathways.更多还原