基于MAPK途径介导的自噬探讨金水六君煎对雌性皮肤老化模型小鼠色素沉着作用的机制

Facial appearance is regarded as a typical index of aging .Skin hyperpigmented spots on account of aging,influence severely appearance,in addition to a strong association with a number of malignant diseases on skin, which lead to deterioration in the quality of life. Autophagy is a cellular degradation process for cellular aggregates and unneeded cellular compartments including melanosome. Recent evidences have suggested that aging is often associated with a reduced autophagic potential,and autophagy affects skin color determination by the regulation of melanin degradation in keratinocytes,which has been implicated in the development of pigmentation. TCM thinks that although the pigmented spot appears on the skin, the etiology is closely related with dysfunction of organs inside the body, obstruction of the channels and collaterals, of phlegm obstruction,stagnation of qi and blood stasis. The TCM etiology of this disease are mainly deficiency of the Shen,Gan＆Pi organs,obstruction of phlegm and blood stasis. Clinical practices show that ‘Adjust Shen,Gan＆Pi and Huatan Quyu’method in treatment of skin aging can yield definite therapeutic effects. JinShuiLiuJunJian (JSLJJ) which can nourish YIN-Shen＆Gan,supplement Pi,dissipate phlegmatic hygrosis and remove blood stasis, reflect the therapeutical method, is known as classics prescription ,but the further mechanism of action is unclear until now. Based on our previous study,the project aims to explore the protective and therapeutical mechanisms of JSLJJ in pigmentation through autophagy via MAPK signaling pathway, in order to provide experimental basis for this drug research. We establish mice model of skin aging,and a-MSH-treated B16 mouse melanoma cell line and mouse Melan-α-melanocytes cells are purchased. In this study, ultrastructural analyse ,immunohistochemistry and western blot will be used to detect accumulations of melanosome,Microphthalmia-associated transcription factor (MITF),tyrosinase(TYR) and tyrosinase-related protein 1/2 (TRP1/2) expression in vivo and in vitro.To observe the effect of JSLJJ on autophagy-lysosomal pathway, autophagic associated makers, ultrastructural analyse will be detected. Then autophagic inhibitors and siRNA ATG5 will be used to study the targets of JSLJJ in autophagy-lysosomal pathway and explore the mechanism of JSLJJ in pigmentation.

色素沉着是皮肤老化的重要表现之一，与某些皮肤恶性病变存在联系。研究发现，自噬功能伴增龄而衰退；其调节黑色素降解，影响皮肤颜色，在色素沉着中的作用机制已成为研究热点。中医认为，女性皮肤色素沉着与五脏虚衰尤其是肾肝脾亏虚和痰浊瘀阻相关。临床实践表明调补肾肝脾、化痰祛瘀法是延缓皮肤衰老的有效治法。金水六君煎具有补肾肝之阴、健脾化痰祛瘀的特点，是体现该治法的经方，但作用机制有待深入。本项目拟在前期研究基础上，通过局部紫外线照射并皮下注射D-gal复制内外源性皮肤衰老小鼠模型，并结合体外α-MSH处理的B16黑色素瘤细胞及Melan-α-黑素细胞模型，采用组织病理学及免疫印迹等技术检测自噬途径及黑素生成途径多种指标，观察金水六君煎对自噬溶酶体途径及黑素生成途径的影响。以自噬抑制剂为工具药，研究金水六君煎的作用靶点，探讨金水六君煎干预色素沉着的分子机制，为金水六君煎的临床应用提供科学思路及实验依据。

色素沉着是增龄性皮肤的表象之一，因此延缓皮肤老化成为防治增龄性色素沉着的关键所在。细胞自噬参与黑素体降解，影响皮肤颜色。目前自噬溶酶体途径在金水六君煎干预色素沉着的作用尚未见报道。我们通过整理古今文献，基于中医衰老理论，分析了金水六君煎延缓皮肤老化减少色素沉着的中医机制。通过整体和细胞两个层面，结合自噬和黑素生成的多种指标检测，观察金水六君煎对色素沉着的影响。我们发现以局部紫外线照射并皮下注射D-gal复制的皮肤老化小鼠模型，皮肤组织出现光老化的病理学改变且基底层出现大量阳性黑色素细胞；角质形成细胞中黑素体和自噬体大量增加；TYR、TRP1/2、MITF、p-P38，p-JNK等蛋白表达上调，同时出现细胞内自噬相关蛋白LC3Ⅱ、Beclin1的高表达。提示色素沉着的发生与黑色素生成和降解相关的途径被过度激活有关。造模同时金水六君煎干预可改善该模型小鼠皮肤的组织学表现，减少皮肤组织TYR、TRP1/2、MITF、p-P38，p-JNK蛋白表达和黑素体数目；并降低皮肤组织LC3Ⅱ、Beclin1表达和减少自噬体数目，改善自噬状态。我们还发现预防或治疗性给予金水六君煎也能改善模型小鼠的黑素生成和自噬状态。采用LC-MS/MS液质联用法发现金水六君煎水提物灌胃后有11种成分进入血液。体外结果表明，金水六君煎含药血清可减少a-MSH诱导的B16黑色素瘤细胞及Melan-α-黑素细胞内黑色素含量，可抑制MAPK信号通路下游MITF水平，下调TYR、TRP1/2表达，且明显增加LC3Ⅱ/LC3Ⅰ、Beclin1、LAMP1 与cathepsin D表达。用自噬抑制剂(Bafilomycin A1、ATG5-siRNA)预处理能明显减轻金水六君煎的抗黑素生成效应。提示金水六君煎可直接作用自噬溶酶体途径，参与抗黑素生成的过程。本研究为金水六君煎的临床应用提供理论基础和实验依据。