七组中药醇提物通过HaCaT细胞对黑素细胞株B10BR增殖和黑素合成的影响

Effects of extracts from seven kinds of traditional Chinese medicine on the proliferation and melanogenesis of a melanocyte cell line B10BR by interaction with HaCaT cells

目的研究黑胡椒等中药醇提物通过角质形成细胞对黑素细胞增殖及黑素合成的间接作用,探讨其外用治疗白癜风的可能机理。方法选取七组中药的醇提物,其中Ⅰ号:透骨草、浮萍各等份;Ⅱ号:白芷、蛇床子各等份;Ⅲ号:补骨脂;Ⅳ号:黑胡椒;Ⅴ号:黑胡椒、补骨脂各等份;Ⅵ号:女贞子;Ⅶ号:复方卡力孜然酊,加入到HaCaT细胞培养基中,采用ELISA法测定上述中药干预后HaCaT细胞上清液中内皮素-1和碱性成纤维细胞生长因子(bFGF)的含量;MTT法测定经中药干预后HaCaT细胞上清液对水生化黑素细胞株B10BR增殖的影响,多巴氧化法检测酪氨酸酶活性,氧氧化钠裂解法测定黑素含量。结果Ⅱ号和Ⅴ号中药能明显刺激HaCaT细胞分泌内皮素-1(P<0.01及P<0.05).Ⅲ号、Ⅳ号和Ⅶ号可促进HaCaT细胞分泌bFGF(P<0.01,P<0.05及P<0.05)。经各组中药处理后的HaCaT细胞培养上清液,Ⅲ号、Ⅵ号能显著促进B10BR的增殖(P<0.01),Ⅰ号、Ⅱ号和Ⅲ号能明显提高B10BR酪氨酸酶活性(P<0.01);Ⅰ号、Ⅱ号、Ⅵ号、Ⅶ号能明显促进B10BR黑素的合成(P<0.01或<0.05)。结论部分中药醇提物治疗白癜风有效可能与中药促进了角质形成细胞分泌细胞因子、改变表皮的微环境有关。

Objective To investigate the indirect effects of extracts from seven kinds of traditional Chinese medicine （ TCM ） on melanocyte proliferation and melanogenesis by interaction with keratinocytes, and to study their possible mechanism in the treatment of vitiligo. Methods Seven kinds of TCM, including No.l, a mixture of equal amount of tougucao and fuping, No.2, a mixture of equal amount of baizhi and shechuangzi, No.3, buguzhi, No.4, heihujiao, No.5, a mixture of equal amount of heihujiao and buguzhi, No.6, nuzhenzi, No.7, a mixture of quchongbanjiuju, buguzhi, heshouwu, danggui, fangfeng, shechuangzi, baixianpi, wumei, baijieji, dingxiang, were selected and extracted by alcohol. HaCaT cells were cultured in vitro and treated with the above extracts, respectively, for 24 h. Then, the supematants in the culture of these treated HaCaT cells were used to treat B10BR cells. The concentrations of endothelin-1 （ ET-1 ） and basic fibroblast growth factor （ bFGF ） in the supemanant of HaCaT cells treated with TCM were determined by ELISA. MTT method, DOPA-oxidation and NaOH-assay were applied to detect the proliferation, tyrosinase activity and melanogenesis of B10BR, respectively. Results The level of ET-1 in the supernatants of HaCaT cells was significantly increased with the treatment of TCM No. 2 and 5 （ P 〈 0.01, P 〈 0.05, respectively ）. TCM No. 3, 4 and 7 significantly elevated the concentration of bFGF in the supernatant of HaCaT cells （ P 〈 0.01, P 〈 0.05, P 〈 0.05, respectively ）. The proliferation of B10BR was significantly enhanced by the supernatant of cultured HaCaT cells treated with TCM No. 3 and 6 （ both P 〈 0.01 ）. All the supernatants in the HaCaT cells treated with TCM No. 1, 2 and 3 could significantly improve the activity of tyrosinase （ all P 〈 0.01 ）, and those treated with No.1, 2, 6 and 7 could promote the melanogenesis of B10BR （ P 〈 0.01, P 〈 0.01, P 〈 0.05, P 〈 0.01, respectively ）. Conclusions Our results suggest that the efficacy of some TCM in the treatment ofvitiligo may be related to their involvement in the secretion of cytokines and alteration of epidermal microenvironment by keratinocytes.