中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (7): 509-512.

• 论著 • 上一篇    下一篇

人参皂苷Rb1对人表皮黑素细胞黑素生成的影响

卢珊珊1,李国艳1,赵丹1,张微1,林茂2,涂彩霞3   

  1. 1. 大连医科大学附属第二医院皮肤科
    2. 大连医科大学附属第二医院
    3. 大连医科大学第二临床学院皮肤科
  • 收稿日期:2011-08-16 修回日期:2012-03-08 出版日期:2012-07-15 发布日期:2012-07-02
  • 通讯作者: 涂彩霞 E-mail:tucx2010@sina.com
  • 基金资助:

    辽宁省教育厅创新团队项目;辽宁省科技厅社会发展攻关计划

Effects of ginsenoside Rb1 on melanogenesis in human epidermal melanocytes

  • Received:2011-08-16 Revised:2012-03-08 Online:2012-07-15 Published:2012-07-02
  • Contact: Cai-Xia TU E-mail:tucx2010@sina.com

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摘要：

目的 探讨人参皂苷Rb1对体外培养的人黑素细胞黑素生成的影响及其作用机制。方法 人表皮黑素细胞取自小儿包皮环切术标本，取第2 ～ 5代细胞进行实验。采用MTT法测定体外培养的人黑素细胞增殖情况，分光光度计法测定酪氨酸酶的多巴氧化酶活性，氢氧化钠裂解法测定黑素含量，免疫蛋白印记法测定黑素细胞酪氨酸酶、小眼畸形相关转录因子（MITF）的表达及cAMP应答元件结合蛋白（CREB）的磷酸化。结果 体外培养的人黑素细胞分别加入25、50、100 μmol/L人参皂苷Rb1 作用72 h后，黑素细胞存活率3组间差异无统计学意义（P ＞ 0.05），黑素含量呈浓度依赖性增加（与溶剂对照组相比的相对黑素含量分别为112.4% ± 5.7%、155.7% ± 6.3%、217.2% ± 11.7%），酪氨酸酶活性增加（相对酪氨酸酶活性分别为117.9% ± 5.7%、158.2% ± 9.6%、182.6% ± 10.0%）。100 μmol/L人参皂苷Rb1作用72 h，黑素细胞酪氨酸酶（225.4 %± 12.8%）、MITF（313.5% ± 16.7%）蛋白表达明显升高（与溶剂对照组相比，P值均 < 0.01），CREB磷酸化明显增加（322.5% ± 21.1%）（与溶剂对照组相比，P < 0.01）。100 μmol/L人参皂苷Rb1 作用黑素细胞8 h，MITF蛋白表达无明显变化；作用24 h后，MITF表达明显增加，与0时间点相比，P < 0.01。10 μmol/L的蛋白激酶A（PKA）抑制剂H-89预处理细胞，可明显抑制100 μmol/L人参皂苷Rb1引起的CREB激活、酪氨酸酶及MITF蛋白表达的增加，与100 μmol/L人参皂苷Rb1作用72 h比较，P值均 < 0.01，进而抑制人参皂苷Rb1引起的酪氨酸酶活性作用增强及黑素合成增加。 结论 人参皂苷Rb1可促进正常人黑素细胞的黑素合成和酪氨酸酶活性。PKA/CREB/MITF/酪氨酸酶信号通路可能参与了人参皂苷Rb1的促黑素生成机制。

关键词: 黑素生成

Abstract:

Objective To estimate the effects of ginsenoside Rb1 on melanogenesis in human melanocytes and underlying mechanisms. Methods Epidermal melanocytes were obtained from circumcision specimens of children, and subjected to primary culture. After 2 to 5 passages, the melanocytes were treated with different concentrations of ginsenoside Rb1, dimethyl sulfoxide （DMSO, vehicle control）, forskolin at 10 μmol/L （positive control） or remained untreated （blank control）. After additional culture for 72 hours, methyl thiazolyl tetrazolium （MTT） assay and NaOH lysis method were used to evaluate cell viability and melanin content in melanocytes respectively, spectrophotometer to determine dopa oxidase activity of tyrosinase, Western blot to quantify the protein level of tyrosinase, microphthalmia-associated transcription factor （MITF）, phosphorylated and total cAMP response element binding protein （p-CREB and t-CREB） in melanocytes. Results After treatment with ginsenoside Rb1 of 25, 50 and 100 μmol/L for 72 hours, the melanocytes experienced no significant changes in viability （P > 0.05）, but a significant dose-dependent increase in melanin content （112.4% ± 5.7%, 155.7% ± 6.3%, 217.2% ± 11.7% vs. 100%, P < 0.05 or 0.01） and tyrosinase activity（117.9% ± 5.7%, 158.2% ± 9.6%, 182.6% ± 10.0% vs. 100%, P < 0.05 or 0.01） compared with the vehicle control melanocytes. The protein expressions of tyrosinase, MITF and p-CREB were statistically higher in melanocytes treated with ginsenoside Rb1 of 100 μmol/L for 72 hours than in the vehicle control melanocytes （225.4% ± 12.8% vs. 100% ± 7.9%, 313.5% ± 16.7% vs. 100% ± 9.8%, 322.5% ± 21.1% vs. 100% ± 9.1%, all P < 0.01）. The increase in MITF protein expression was inapparent in melanocytes at 8 hours after the treatment with ginsenoside Rb1 of 100 μmol/L, but statistically significant at 24 hours compared with the melanocytes at baseline （P < 0.01）. The pretreatment with H-89 （a selective inhibitor of PKA） at 10 μmol/L, significantly suppressed the ginsenoside Rb1 （100 μmol/L for 72 hours） -induced phosphorylation of CREB, increase in MITF, tyrosinase expression, as well as tyrosinase activity and melanin content in melanocytes （all P < 0.01）. Conclusions Ginsenoside Rb1 could enhance the melanogenesis and tyrosinase activity in normal human melanocytes. The PKA/CREB/MITF/tyrosinase signaling pathway may contribute to the pro-melanogenic effect of ginsenoside Rb1.

Key words: Melanogenesis