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目的:探讨白桦脂醇治疗黄褐斑的内在作用机制。方法:选择对数生长期的A375细胞,培养24 h,弃掉液体,然后分组并加入不同浓度药液200μL,组别设定为空白组、雌二醇组(1×10~(-3)μmol·L~(-1)浓度的雌二醇)和白桦脂醇组(设定浓度的白桦脂醇),每组均设置6个复孔。用白桦脂醇干预A375细胞,噻唑蓝(MTT)法检测细胞活性;Na OH裂解法检测黑素量;多巴氧化法检测酪氨酸酶(TYR)活性;逆转录-聚合酶链式反应(RT-PCR)法检测TYR,酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)的表达,及丝裂原激活的蛋白激酶(MAPK)信号通路中关键蛋白激酶ERK1,ERK2,JNK2的mRNA表达。结果:与空白组比较,白桦脂醇在1,1×10~(-1),1×10~(-2),1×10~(-3)μmol·L~(-1)剂量下可明显抑制A375细胞黑素合成及TYR活性,1μmol·L~(-1)白桦脂醇可明显下调A375细胞TYR,TRP-1,TRP-2 mRNA表达,以及ERK1,ERK2,JNK2 mRNA表达(P<0.05,P<0.01)。结论:白桦脂醇可能是通过抑制TYR活性和/或下调TYR,TRP-1及TRP-2 mRNA表达,抑制了A375细胞中黑素的合成,并且这种作用与抑制ERK1,ERK2,JNK2的基因表达有关。
Objective: To investigate the intrinsic mechanism of betulin in the treatment of melasma. Methods: The A375 cells in logarithmic growth phase were selected and cultured for 24 h. The liquid was discarded and then divided into groups and added with 200 μL of different concentrations of drugs. The groups were set as blank group, estradiol group (1 × 10 -3) μmol·L -1 estradiol) and betulin (concentration betulin), six replicate wells were set in each group. A380 cells were treated with betulin, cell viability was assayed by MTT assay, melanin content was assayed by Na OH cleavage assay, tyrosinase (TYR) activity was detected by the dopa oxidation assay, and transcriptase-polymerase chain reaction RT-PCR) was used to detect the expression of TYR, TRP-1 and TRP-2 and the mitogen-activated protein kinase (MAPK) signal Pathway of key protein kinase ERK1, ERK2, JNK2 mRNA expression. Results: Compared with the blank group, the betal concentration of 1 × 10 ~ (-1), 1 × 10 ~ (-2), 1 × 10 ~ (-3) μmol·L ~ (-1) Significantly inhibited the melanin synthesis and TYR activity of A375 cells. 1μmol·L -1 betulin could down-regulate the expression of TYR, TRP-1 and TRP-2 mRNA as well as ERK1, ERK2 and JNK2 mRNA in A375 cells (P < 0.05, P <0.01). CONCLUSION: Betulin inhibits the synthesis of melanin in A375 cells possibly by inhibiting TYR activity and / or down-regulating the expression of TYR, TRP-1 and TRP-2 mRNA, and this effect is related to the inhibition of ERK1, ERK2 and JNK2 genes Expression related.