丙戊酸通过Mitf通路促进企鹅珍珠贝黑色素合成

VALPROIC ACID ENHANCES MELANIN SYNTHESIS BY Mitf PATHWAY IN PTERIA PENGUIN

研究利用HPLC-MS/MS检测、酪氨酸酶活性分析和荧光定量PCR技术,探索丙戊酸(Valproic acid, VPA)作为企鹅珍珠贝[Pteria penguin (R?ding, 1798)]黑色素增强剂的生物毒性、有效性和作用途径。生物毒性分析显示,高浓度(100 mmol/L)丙戊酸作用24h即可显著降低存活率至83.3%(P<0.05),并存在明显的时间依赖效应, 10 mmol/L丙戊酸作用72h是最大的安全使用浓度和作用时间。丙戊酸的作用分析发现, 10和100 mmol/L丙戊酸作用72h可以显著提高企鹅珍珠贝黑色素含量43.7%(P<0.05)和47.1%(P<0.05),并显著提高酪氨酸酶(黑色素合成标志酶)活性59.7%(P<0.05)和136.1%(P<0.01),说明丙戊酸可以有效地促进企鹅珍珠贝的黑色素合成。对丙戊酸的作用途径分析发现,丙戊酸可以显著增加小眼相关转录因子(Melanogenesis associated transcription factor, Mitf)、酪氨酸酶(Tyrosinase, Tyr)、Cdk2 (Cyclin-dependent kinase 2)和Bcl2 (B-cell lymp homa2)等黑色素合成相关基因的表达,但对环磷腺苷效应元件结合蛋白(cAMP response element binding protein, Creb2)的表达无显著影响,说明丙戊酸是通过Mitf-Tyr-melanin途径参与企鹅珍珠贝黑色素合成的。研究为在生产上建立珍珠贝的色泽优化技术提供了重要数据。

The winged pearl oyster Pteria penguin(Röding,1798)is an important marine cultured species that can produce high-quality seawater pearl.Melanin is the main pigment in P.penguin and largely affects the color and value of the pearl.This study analyzed the toxicity,function and signal pathway of valproic acid(VPA).Toxicity detection showed that 100 mmol/L VPA significantly reduced the survival rate of experimental animals in a time-dependent manner,and 10 mmol/L VPA for 72h was identified as the maximal safe concentration and working time for P.penguin.The liquid chromatograph-tandem mass spectrometer(LC-MS/MS)analysis showed that the content of PDCA(pyrrole-2,3-dicarboxylic acid)and PTCA(pyrrole-2,3,5-tricarboxylic acid),main alkaline oxidation products of melanin,increased significantly by 43.7%(P<0.05)and 47.1%(P<0.05)after 10 and 100 mmol/L VPA for 72h.Tyrosinase activity,a marker of melanin synthesis,increased by 59.7%(P<0.05)and 136.1%(P<0.01),correspondingly.These results indicated that valproic acid can effectively accelerate the melanin synthesis in P.penguin.The qRT-PCR(quantitative real-time PCR)showed that valproic acid increased the expression of genes in melanin synthesis pathway,including Mitf(microphthalmia-associated transcription factor),Tyr(Tyrosinase),PpBcl2(B-cell lymphoma 2)and Cdk2(cyclindependent kinase 2),but had no effect on PpCreb2(cyclin-dependent kinase 2).This suggest that valproic acid can enhance melanin synthesis of P.penguin via Mitf-Tyr-melanin pathway.