桑白皮多酚对B16细胞内黑色素生成的影响及其机制

Effect of polyphenol from Cortex Mori on melanogenesis of B16 cells and its mechanism

目的研究桑白皮多酚(polyphenol from Cortex Mori,CMP)对小鼠B16细胞内黑色素生成的影响,并探究其作用机制。方法体外培养小鼠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。CMP干预B16细胞,MTT法测定细胞活性;分别利用NaOH裂解法和L-Dopa氧化法,分析细胞内黑色素生成含量和酪氨酸酶活性的变化;Western blot和实时荧光定量PCR法分别测定B16细胞中酪氨酸酶(TYR)、酪氨酸酶相关蛋白-1(TRP-1)、酪氨酸酶相关蛋白-2(TRP-2)、小眼畸形相关转录因子(MITF)的蛋白质和mRNA水平。结果 CMP对α-MSH诱导的B16细胞内黑色素生成及酪氨酸酶活力均具有明显的抑制作用(P<0.05),且呈量效关系。当CMP浓度为20 mg·L^(-1)时,对细胞内黑色素生成及酪氨酸酶活性抑制率分别为52.95%、32.85%,阳性对照熊果苷(100 mg·L^(-1))的抑制率分别为17.29%、16.75%,表明CMP对黑色素生成的抑制效果强于熊果苷。与α-MSH模型组相比,CMP干预后细胞内TYR、TRP-1、TRP-2、MITF的mRNA和蛋白表达被明显抑制(P<0.05)。结论 CMP明显抑制α-MSH诱导黑色素的生成,其机制可能是通过调控TYR、TRP-1、TRP-2、MITF mRNA和蛋白表达,进而抑制酪氨酸酶活性实现的。

To investigate the inhibitory effect of polyphenol from Cortex Mori(CMP)on melanogenesis in mouse melanoma B16 cells and its possible mechanism.Methods Melanoma B16 cells with high expression melanin were induced byα-melanocyte-stimulating hormone(α-MSH)to establish cell model.Cell viability was detected by MTT assay.The melanin synthesis and tyrosinase activity were measured by NaOH and L-Dopa assays,respectively.The tyrosinase(TYR),tyrosinase-related protein-1(TRP-1),tyrosinase-related protein-2(TRP-2)and microphthalmia associated transcription factor(MITF)protein and mRNA levels were measured by Western blot and qRT-PCR,respectively.Results CMP could inhibit the melanin synthesis and tyrosinase activity inα-MSH stimulated B16 cells in a dose-dependent manner(P<0.05).The melanin content and tyrosinase activity significantly decreased by 52.95%,32.85%at 20 mg·L^-1 of CMP,respectively.Treatment of 100 mg·L^-1 of arbutin reduced the melanin content and tyrosinase activity by 17.29%,16.75%,respectively.Based on the results of this study,CMP showed a stronger anti-melanogenesis activity than that of positive control arbutin.After treated by CMP,the protein and mRNA levels of TYR,TRP-1,TRP-2 and MITF were significantly inhibited compared to theα-MSH group(P<0.05).Conclusions CMP could suppress the melanogenesis inα-MSH stimulated B16 cells,and its mechanism may be related to its regulation of the protein and mRNA expressions of TYR,TRP-1,TRP-2 and MITF,and the inhibition of tyrosinase activity.