补骨脂二氢黄酮甲醚对A375细胞黑素合成及ER/MAPK信号通路的影响

Effect of bavachinin on melanin synthesis of A375 cells and the signal pathway of ER/MAPK

目的初步阐明补骨脂二氢黄酮甲醚通过ER/MAPK信号通路干预人黑素瘤细胞(A375细胞)黑素合成的作用机制。方法将实验分为空白组、雌二醇组、补骨脂二氢黄酮甲醚组、雌激素受体(ER)阻断剂(ICI182780)+补骨脂二氢黄酮甲醚组、JNK抑制剂(SP600125)+补骨脂二氢黄酮甲醚组。MTT法测定细胞活性;RT-PCR法测定酪氨酸酶(TYR)、酪氨酸相关蛋白1/2(TRP-1/2)、细胞外信号调节激酶1/2(ERK1/2)、c-Jun N末端激酶(JNK)的m RNA表达;Western blot测定TYR和JNK的蛋白表达。结果与空白组比较,10-3μmol/L的雌二醇组对细胞活性无影响(P>0.05)。10、100μmol/L的补骨脂二氢黄酮甲醚组能显著抑制A375细胞增殖(P<0.01)。1μmol/L的补骨脂二氢黄酮甲醚组可抑制A375细胞黑素合成及TYR活性(P<0.01),并可抑制TRP-1/2、TYR、ERK1/2、JNK2m RNA的表达(P<0.01),抑制TYR和JNK2蛋白的表达(P<0.01)。ICI182780+骨脂二氢黄酮甲醚组和SP600125+补骨脂二氢黄酮甲醚组均能逆转补骨脂二氢黄酮甲醚组对黑素合成、TYR活性和TRP-1/2、TYR、ERK1/2、JNK2 m RNA表达和TYR、JNK2蛋白表达的抑制作用(P<0.05或P<0.01)。结论补骨脂二氢黄酮甲醚能够抑制A375细胞黑素合成,其机制可能是通过植物雌激素与ER结合,启动第二信使激活ER-MAPK信号通路,降低ERK、JNK的表达,从而抑制TYR活性和酶的量,最终达到抑制黑素合成的目的。

Objective To preliminary clarify the mechanism of bavachinin in intervening melanin synthesis of human melanoma cells （A375） through ER/MAPK signal pathways. Methods The experiment was divided into blank group, estra- diol group, bavaehinin group, estrogen receptor （ER） blockers （ICI182780） ＋bavachinin group, JNK inhibitors （SP600125） ＋ havachinin group. Cell viability was determined by MTT method. Tyrosinase （TYR）, tyrosinase-related protein kinas- es 1/2 （TRP-1/2）, extracellular signal regulating kinase 112 （ERKI/2）, e-Jun N terminal kinase （JNK） mRNA expressions were determined by RT-PCR;TYR and JNK protein expressions were determined by Western blot method. Results Compared with blank group, 10-3 μmol/L of estradiol group had no influence to cellular activity （P 〉 0.05）. 10, 100 μmol/L of bavachinin group could significantly inhibit the A375 cell activity （P 〈 0.01）. 1 μmol/L of bavachinin group could inhibit melanin synthesis and TYR activities （P 〈 0.01）, restrained the mRNA expressions of TRP-1/2, TYR, ERK1/2, JNK2 （P 〈 0.01）, inhibited the expressions of TYR and JNK2 protein （P 〈 0.01）. ICI182780 ＋ bavachinin group and SP600125 ＋ bava- ehinin group eould reverse the inhibiting effeet of bavaehinin group for melanin synthesis, TYR aetivities and TRP-1/2, TYR, ERK1/2, JNK2 mRNA expressions and TYR, JNK2 protein expressions （P 〈 0.05 or P 〈 0.01）. Conclusion Bava- ehinin can inhibit the melanin synthesis of A375 cells and its mechanism is likely to be the eombination of phytoestrogensand ER, to lower the expression of ERK and JNK by start- ing a second messenger activation MAPK signaling path- ways, so as to inhibit the TYR activity and the amount of enzyme, and to ultimately inhibit melanin synthesis.