白桦脂醇通过ER/MAPK信号通路对A375细胞黑素合成及机制

Melanin Synthesis and Mechanism of A375 Cells by Betulin via ER/MAPK Signaling Pathway

目的：研究植物雌激素成分白桦脂醇及加入受体阻断剂（ICI182780）后对A375细胞的黑素合成及机制的影响。方法：将1μmol·L^（-1）白桦脂醇作用于A375黑素细胞,实验分为空白组（DMEM完全培养液）,雌二醇组（1×10^（-3）μmol·L^（-1））,白桦脂醇组（1μmol·L^（-1））,白桦脂醇＋ICI182780组（1μmol·L^（-1）＋1μmol·L^（-1））。用Na OH裂解法检测黑素含量、蛋白免疫印迹法（Western blot）和实时荧光定量PCR法（Real-time PCR）测定雌激素受体/丝裂原激活的蛋白激酶（ER/MAPK）通路中关键蛋白激酶p38 MAPK,ERβ,c-Jun氨基末端激酶（JNK）,细胞外信号调节激酶2（ERK2）和小眼畸形相关转录因子（MITF）,酪氨酸酶（TYR）,酪氨酸酶相关蛋白-1（TRP-1）,酪氨酸酶相关蛋白-2（TRP-2）的蛋白及其mRNA表达。结果：与空白组比较,白桦脂醇组和白桦脂醇＋ICI182780组对A375细胞黑素合成有显著抑制作用（P〈0.01）;1μmol·L^（-1）的白桦脂醇对A375细胞中上述蛋白表达有不同程度的抑制作用（P〈0.05,P〈0.01）,对ERK2,MITF,TRP-1 mRNA的表达也有不同程度的抑制作用（P〈0.05,P〈0.01）。与白桦脂醇组比较,ICI182780可以逆转白桦脂醇对A375细胞黑素合成的抑制作用（P〈0.01）;ICI182780可以逆转白桦脂醇对A375细胞中上述蛋白表达的抑制作用及对ERK2,MITF,TRP-1 mRNA的表达的抑制作用（P〈0.05,P〈0.01）。结论：白桦脂醇能够通过ER/MAPK信号通路下调MITF,TYR,TRP-1及TRP-2蛋白表达量,从而减少黑素合成。

Objective： To study the effect of phytoestrogenic components of betulin and the addition of receptor blocker（ ICI182780） on melanin synthesis and its mechanism on A375 cells. Method： 1 μmol·L^（-1） betulin was applied to A375 melanocytes and then the experimental cells were divided into blank group（ DMEM complete culture fluid）,estradiol group（ 1 × 10^（-3）μmol·L^（-1））,betulin group（ 1 μmol·L^（-1））,and betulin ＋ICI182780 group（ 1 μmol·L^（-1）＋ 1 μmol·L^（-1））. Melanin content was determined by Na OH lysis; Western blot,and real-time fluorescent quantitative polymerase chain reaction（ Real-time PCR） were used to determine the protein content and mRNA expression of key protein kinase mitogen activated protein kinase（ MAPK） p38 in estrogen receptor（ ER）/mitogen activated protein kinase（ MAPK） pathway,c-Jun N-terminal kinase（ JNK）,extracellular signal-regulated kinase 2（ ERK2） and micrometamorphic-related transcription factor（ MITF）,dopa oxidation assay for tyrosinase（ TYR）,tyrosinase-related protein^（-1）（ TRP-1）,and tyrosinase-related protein-2（ TRP-2）. Result： As compared with the blank group,betulin group and betulin ＋ ICI182780 group had significant inhibition on melanin synthesis in A375 cells（ P〈0. 01）; 1 μmol·L^（-1） betulin had different inhibitory effects on expression of the above proteins in A375 cells（ P〈0. 05,P〈0. 01）,as well as on ERK2,MITF,and TRP-1（ P〈0. 05,P〈0. 01） mRNA expression（ P〈0. 05,P〈0. 01）. As compared with the betulin group,ICI182780 can reverse the inhibitory effect of betulin on melanin synthesis of A375 cells（ P〈0. 01）; ICI182780 can reverse the inhibitory effect of betulin on the above protein expression in A375 cells as well as mRNA expression of ERK2,MITF,and TRP-1（ P〈0. 05,P〈0. 01）. Conclusion： Betulin can down-regulate the expression levels of MITF,TYR,TRP-1 and TRP-2 proteins through ER-MAPK signaling pathway,thus reducing melanin synthesis.