补骨脂素对A375细胞黑素合成及ER/MAPK信号通路调控机制探讨

Mechanism of Psoralen on Melanin Synthesis and ER/MAPK Signaling Pathway in A375 Cells

目的：探讨补骨脂素对A375细胞黑素含量、酪氨酸酶活性的影响及作用机制。方法：以密度2×105个/孔的细胞数接种于6孔板中,将细胞分为空白组、雌二醇组、补骨脂素组、补骨脂素＋雌激素受体（ER）阻断剂组（ICI182780）（浓度分别为1×10-3,1,1μmol·L^（-1））。采用Na OH裂解法、多巴醌氧化法检测黑素含量及酪氨酸酶活性;蛋白免疫印迹法（Western blot）和实时荧光定量聚合酶链式反应（Real-time PCR）法测定ER/丝裂原激活的蛋白激酶（ER/MAPK）通路中关键蛋白激酶p38,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）,多巴氧化法检测酪氨酸酶（TYR）,酪氨酸酶相关蛋白-1（TRP-1）,酪氨酸酶相关蛋白-2（TRP-2）的蛋白含量及相应mRNA表达。结果：与空白组比较,补骨脂素组对A375细胞中的黑素合成及TYR活性具有显著抑制作用（P〈0.01）,对A375细胞中雌激素受体β（ERβ）蛋白的表达具有明显的抑制作用（P〈0.05）,对p38 MAPK,ERK2,JNK的蛋白表达极显著的抑制作用（P〈0.01）,对MITF,TYR,TRP-1,TRP-2的蛋白表达极显著的抑制作用（P〈0.01）,对ERK2,p38 MAPK,MITF,TRP-1 mRNA表达显著被抑制（P〈0.01）;与补骨脂素组比较,ICI182780能够逆转ERβ,ERK,MITF,TYR,TRP-1的蛋白表达（P〈0.01）及p38MAPK,JNK,TRP-2的蛋白表达（P〈0.05）,同时能显著阻断补骨脂素对ERK2,p38MAPK,MITF,TRP-1 mRNA表达作用（P〈0.01）。结论：补骨脂素通过ER/MAPK信号通路下调MITF和TYR,TRP-1,TRP-2的表达量,从而减少黑素合成。

Objective： To investigate the effect and mechanism of psoralen on melanin content and tyrosinase activity in A375 cells. Method： Cells were inoculated in 6-well plates at a cell density of 2 × 105 cells and then divided into blank group,estradiol group,psoralen group,psoralen ＋ estrogen receptor（ ER） receptor blocker group（ ICI182780）,with the concentrations of 1 × 10-3,1,1 μmol·L-1,respectively. The melanin content and tyrosinase activity were detected by Na OH lysis method and dopaquinone oxidation method; Western blot and real-time fluorescent quantitative PCR assays were used to determine key protein kinases p38,c-Jun N-terminal kinase（ JNK）,extracellular signal-regulated kinase 2（ ERK2） and ocular hypotrophy-associated transcription factors（ MITF） in the ER/mitogen-activated protein kinase（ MAPK） pathway. The protein contents and corresponding mRNA expressions of tyrosinase（ TYR）, tyrosinase-associated protein-1（ TRP-1）, and tyrosinase-associated protein-2（ TRP-2） were measured by dopa oxidation. Result： Compared with the blank group,the psoralen group had a significant inhibitory effect on melanin synthesis and TYR activity in A375 cells（ P 〈0. 01）,with a significant inhibitory effect on the expression of estrogen receptor β（ ERβ） protein in A375 cells（ P 〈0. 05）,p38 MAPK,ERK and JNK protein expressions（ P〈 0. 01）,protein expressions of MITF,TYR,TRP-1,and TRP-2（ P〈 0. 01）,and ERK2,p38 MAPK,MITF and TRP-1 mRNA expressions（ P〈 0. 01）; compared with psoralen group,ICI182780 reversed the protein expressions of ERβ,ERK,MITF,TYR,TRP-1（ P〈 0. 01） and the protein expressions of p38,JNK,and TRP-2（ P〈 0. 05）; at the same time,it can significantly block psoralen on ERK2,p38,MITF,TRP-1 mRNA expressions（ P〈 0. 01）. Conclusion： Psoralen down-regulates the expressions of MITF and TYR,TRP-1,and TRP-2 through ER/MAPK signaling pathway,so as to reduce melanin synthesis.