苦水玫瑰精油对B16细胞中黑色素合成的影响及机制研究

Effects of Kushui rose(Rosa setate × Rosa rugosa) essential oil on melanin synthesis in B16 cells and its mechanism

本文研究苦水玫瑰精油对小鼠B16黑素瘤细胞中黑色素合成的影响,并探讨其作用机制,为苦水玫瑰精油作为天然活性物质的开发应用提供实验依据。采用气相色谱与质谱联用技术(GC-MS)分析苦水玫瑰精油中的挥发性物质成分,研究对象为体外培养的小鼠B16细胞,构建α-黑素细胞刺激素(α-MSH)诱导的黑色素高表达细胞模型;经不同质量浓度的苦水玫瑰精油干预后,对B16细胞进行台盼蓝染色观察,采用CCK8法测定苦水玫瑰精油对黑色素细胞活力的影响;分别采用酶联免疫吸附法(ELISA)和多巴氧化法分析B16细胞中的黑色素含量和酪氨酸酶(Tyr)活性的变化;利用质量浓度为2 g/L的苦水玫瑰精油分别处理B16细胞12,24和48 h,通过实时荧光定量PCR(quantitative Real-timePCR,qRT-PCR)以及蛋白免疫印迹检测(Westernblot)实验测定B16细胞内环磷酸腺苷(cyclicadenosine monophosphate,cAMP)的反应元件结合蛋白(CRE-bindingprotein,CREB)、p38丝裂原活化蛋白激酶(p38MAP kinase,p38MAPK)、细胞外调节蛋白激酶(extracellularsignal-regulatedkinases,ERK)与小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)的表达情况,探究苦水玫瑰精油对黑色素合成途径的影响。qRTPCR实验结果显示,苦水玫瑰精油处理B16细胞48 h后细胞内MITF基因的表达被显著抑制,MITF基因的表达下调了76%,CREB,p38 MAPK的表达也被显著抑制,其中抑制率分别达到54%,41%,而ERK1与ERK2的表达随时间延长而逐渐升高;Western-blot实验也得到相似结果,CREB,p38 MAPK蛋白与磷酸化表达水平的变化趋势与MITF相一致,随着时间增加表达量随之减少,而ERK蛋白表达水平随着处理时间的增加而逐渐升高。表明苦水玫瑰精油可以通过抑制上述三条信号途径下调MITF的表达,从而减少细胞内黑色素含量。

The effects of Kushui rose essential oil on melanin synthesis in mouse B16 melanoma cells were investigated and its mechanism of action was explored to provide an experimental basis for the development and application of Kushui rose essential oil as a natural active substance. The volatile components of the essential oil were analyzed by gas chromatography-mass spectrometry(GC-MS). The cell model with high melanin expression induced by α-melanocyte-stimulating hormone (α-MSH) was constructed using in vitro cultured mouse B16 cells. The effects of Kushui rose essential oil on melanocyte viability were determined by CCK8 method;the changes in melanin content and tyrosinase(Tyr) activity in B16 cells were analyzed by enzyme-linked immunosorbent assay(ELISA) and dopa oxidation method, respectively;the expression of cyclic adenosine monophosphate(cAMP)-responsive element-binding protein(CRE-binding protein, CREB), p38 MAP kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) in three signaling pathways was measured. The expression of CREB, p38 MAPK, ERK and microphthalmia-associated transcription factor(MITF) in B16 cells was determined by quantitative real-time PCR(qRT-PCR) and western blotting using Kushui rose essential oil at a mass concentration of 2 g/L for 12 h, 24 h and 48 h, respectively. qRT-PCR results showed that the expression of MITF gene was significantly inhibited after 48 h treatment of B16 cells with Kushui rose essential oil. The expression of MITF gene was down-regulated by 76%, and the expression of CREB and p38 MAPK was also significantly inhibited, in which the inhibition rates reached 54% and 41%, respectively, while the expression of ERK1 and ERK2 gradually increased with time;similar results were obtained by western blot, and the trend of the expression levels of CREB and p38 MAPK was consistent with the expression level of MITF, which decreased with time, while the expression level of ERK gradually increased with time. It demonstrates that the Kushui rose essential oil reduces intracellular melanin production through three signaling pathways: cAMP/PKA, p38 MAPK and ERK/MAPK, and thus inhibit the expression of MITF.