|关键词||驱虫斑鸠菊 白癜风 黑素合成 机理|
人类白癜风是一种顽固性皮肤病。主要表现为皮肤呈斑块状或片状脱色变白。形态学上可见病损皮肤色素颗粒减少，黑素细胞破坏、数量减少乃至消失。白癜风的确切发病机制尚未清楚，根据目前的实验证据人们普遍认同自身免疫病是其主要发病机制。目前、西药对白癜风的治疗效果并不理想因此研究者们利用传统医学的方法对白癜风开展了特殊治疗、达到了很好的治疗效果。Kaliziri（驱虫斑鸠菊）注射液是利用科学方法从新疆特有的菊科植物—驱虫斑鸠菊的种子中提炼而成的，其主要成分为黄酮类化合物。经多年临床应用显示，该药对白癜风具有明显治疗效果。因此应用体外细胞培养技术研究Kaliziri注射液对酪氨酸酶活性的影响，并利用酶学测定方法等研究Kaliziri注射液对黑素瘤细胞分泌酪氨酸酶活性、黑素瘤细胞生长状况、黑素分泌活性等的影响；应用Western blotting 和Real Time -PCR技术研究了Kaliziri注射液对黑素瘤细胞蛋白表达和mRNA表达的影响，从酶学、细胞、分子三个不同层次探讨了Kaliziri注射液对酪氨酸酶和黑素合成等并进行了较为系统的研究。此项研究为探讨白癜风的发病机制奠定了分子基础。同时，本研究还对Kaliziri黄酮类化合物和4-二甲氨基-4′-甲氧基查尔酮进行了抗白癜风活性评价并对4-二甲氨基-4′-甲氧基查尔酮进行了机理研究。
Abstract Vitiligo is an acquired, progressive, multifactorial, depigmentation disorder characterized by the appearance of circumscribed white macules in the skin caused by chronic, progressive loss of functional melanocytes in the epidermis. The etiology of vitiligo is poorly understood. There appears to be a genetic predisposition in a non-Mendelian pattern, with a polygenic and multi factorial inheritance. The Traditional Uyghur Medicine-Kaliziri [Vernonia anthelmintica (L.)Willd. ] injection was extracted from Kaliziri seeds by a scientific method (Pharmacopoeia of the People’s Republic of China, Uyghur Medicine volume, product with code number approved by SFDA:Z20063652) . Its main components are flavonoids. Clinical application over many years has shown that this medicine has a significant therapeutic effect on vitiligo. It is capable of regulating abnormal balgham by promoting blood circulation and coloring by increasing melanin cell function. Nonetheless, its effect and underlying mechanisms in melanogenesis are not very clear. The aim of this study was to clarify the effect of Kaliziri injection and its molecular mechanism in melanin biosynthesis in B16 melanoma cells. 1 Effect of Kaliziri injection on tyrosinase activity and melanin synthesis in B16 cells The effect of Kaliziri injection on the viability of B16 melanoma cells was examined using the MTT assay. Kaliziri injection showed very small cytotoxic effects on B16 cells.The effect of Kaliziri injection on tyrosinase was measured by L-DOPA oxidation. Treatment with Kaliziri injection at 0–40μg/ml resulted in a dose-dependent increase in tyrosinase activity in B16 cells. In the melanin content assay, we found that melanin levels increased in a dose-dependent manner by Kaliziri injection treatment in B16 cells. At 40μg/ml of Kaliziri injection, the melanin content only slightly increased, so 20μg/ml was chosen as an effective concentration of Kaliziri injection for further experiments. 2 Effect of Kaliziri injection on MITF 、TYR 、TRP-1 and TRP-2 protein expression in B16 cells Because Kaliziri injection increased tyrosinase activity and melanin synthesis, we further explored whether Kaliziri injection affects the expression of MITF, which plays a critical role in TYR gene expression and melanogenesis. We examined the MITF levels after Kaliziri injection treatment. Our data showed that MITF protein expression was significantly enhanced 24h after Kaliziri injection treatment of B16 cells and the level of TYR protein expression was up regulated by Kaliziri injection treatment in a time-dependent manner. To elucidate whether Kaliziri injection can affect melanogenic protein expression, western blotting was carried out using lysates of B16 murine melanoma cells treated with Kaliziri injection. TRP-1 and TRP-2 protein expression was up regulated by Kaliziri injection in a time-dependent manner in B16 melanoma cells. 3 Anti-Vitiligo activity of Kaliziri flavonoids We isolate Butin、Butein、Isocarthamidin and Liquirtigeninis from Kaliziri seeds and examine the cytoxicity of four flavonoids by MTT assay; Investigate their effects on tyrosinase activity and melanin content on B16 melanoma cells. The results showed that: Butin、Butein and Liquirtigeninis show dose-dependent activity in B16 melanoma cells. 4 Anti vitiligo activity and mechanism of 4 - dimethylamino - 4'-methoxy chalcone 4 - dimethylamino - 4' - methoxy chalcone subsequent significantly enhanced tyrosinase activity and melanin content in vitro on B16 melanoma cells, so in addition to investigated affects of 4 - dimethylamino - 4' - methoxy chalcone on the expression of TYR, TRP-1, TRP-2 and MITF and expression levels of nucleic acid, we study on the signaling pathways of melanogenesis regulation. We examined the effects of on CREB and p-CREB (AC / cAMP / PKA / CREB pathway); p38MAPK、p-p38MAPK、ERK and p-ERK (mitogen-activated protein kinases (MAPKs) pathway); β-catinin (Wnt/β-catinin pathway) and SOX-10 (SWI / SNF pathway) protein expression by western-botting analysis. The results show that 4 - dimethylamino - 4'-methoxy chalcone achieved the role of promoting melanin synthesis by upregulate the p-CREB, p-p38MAPK and β-catinin protein expression.
|阿迪拉·吐尔逊塔依. 维药驱虫斑鸠及相关化合物活性及分子机理研究[D]. 北京. 中国科学院大学,2014.|
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