黄芩苷调控细胞自噬改善皮肤光老化的作用机制研究

Recent studies find that abnormally reduced autophagy is one of the important factors that cause aging of the skin and the whole body. More than 40 proteins are required to participate in the procedure of autophagy. Our preliminary studies found that 1) Baicalin protected human skin fibroblasts from ultraviolet radiation-induced photoaging; 2)the autophagy activity was significantly reduced in photoaged skin cells; 3) autophagy activator can reverse many performance of skin photoaging; 4) the protein expressions of AMPK and Beclin-1 confirmed by western blot were significantly increased in the baicalin-treated cells. In this condition, we propose the hypothesis: baicalin may participate in the development of skin photoaging through regulation of autophagy. The present project intends to observed the impact of baicalin and autophagy on established skin photoaging cells and animal models. On the basis of verify the roles of baicalin in autophagy, clarify the exact mechnisms of baicalin regulation of photoaging. Our expeirments will provide an experimental basis of specific mechanisms of new targets for the prevention and treatment of skin photodamage.

新近发现细胞自噬异常减少是导致机体老化的重要因素之一；发生细胞自噬需要40余种蛋白参与。前期研究发现：黄芩苷对紫外线诱导的光损伤有防护作用；皮肤光老化细胞的自噬活性显著降低，提高自噬水平可逆转皮肤光老化诸多表现；免疫印迹检测证实黄芩苷可增强皮肤光老化模型中AMPK及Beclin-1的表达。为此提出假说：黄芩苷可能通过调控细胞自噬而参与皮肤光老化的发生发展。本项目拟通过已建立的皮肤老化细胞及动物模型，观察黄芩苷与自噬对皮肤光老化的影响，验证黄芩苷通过调控自噬改善老化的具体机制。在确证“黄芩苷-细胞自噬-皮肤光老化”调控环节的基础上，明确黄芩苷调控光老化的具体机制，为防治皮肤光老化明确新靶点提供理论基础和实验参考。

新近发现细胞自噬异常减少是导致机体老化的重要因素之一；发生细胞自噬需要40余种蛋白参与。前期研究发现：黄芩苷对紫外线诱导的光损伤有防护作用；皮肤光老化细胞的自噬活性显著降低，提高自噬水平可逆转皮肤光老化诸多表现。为此提出假说：黄芩苷可能通过调控细胞自噬而参与皮肤光老化的发生发展。.我们分别用不同浓度黄芩苷作用于人皮肤成纤维细胞，通过CCK8实验证实25ng/ml黄芩苷对细胞无明显光毒性。通过流式检测发现，与UVB组相比，UVB+黄芩苷组caspase3、PARP和BAX的蛋白表达量下降，Bcl-2蛋白表达上调，显示黄芩苷可改善UVB辐射诱导的细胞凋亡。与UVB组相比，UVB+黄芩苷组LC3II/LC3I、Beclin1表达明显上升。加入自噬抑制剂Bafilomycin A1后，自噬过程并无明显改变，进一步说明黄芩苷可增加UVB辐射所致的自噬水平增高。表明黄芩苷可增强光损伤皮肤成纤维细胞的自噬水平。免疫印迹结果显示与UVB+黄芩苷组相比，UVB+黄芩苷+3-MA组细胞LC3II/LC3I、Beclin1表达水平明显下降。表明自噬抑制剂3-MA可降低黄芩苷诱导自噬，改善凋亡的作用。与空白对照组相比，UVB组p-AMPK蛋白表达量增加，p-mTOR蛋白表达量下降。与UVB组相比，UVB+黄芩苷组p-AMPK蛋白表达量增加，p-mTOR蛋白表达量下降，并且这种差异可被AMPK抑制物compound C所逆转。免疫印迹结果显示与UVB+黄芩苷组相比，UVB+黄芩苷+compound C组细胞LC3II/LC3I、Beclin1表达水平明显下降。这些结果表明黄芩苷通过AMPK-mTOR通路调控光损伤成纤维细胞的自噬和凋亡。.总之，本研究证实了以下假说：黄芩苷→AMPK高表达→mTOR低表达→自噬能力增强→老化和受损的细胞器被清除→皮肤光损伤表现减轻。观察到黄芩苷与自噬对皮肤光损伤的影响，验证黄芩苷通过调控自噬改善光损伤的具体机制。在确证“黄芩苷-细胞自噬-皮肤光损伤”调控环节的基础上，明确黄芩苷调控光损伤的具体机制，为防治皮肤光老化明确新靶点提供理论基础和实验参考。但本研究只涉及到了体外实验，后期还应进一步在体内行相关验证。而光损伤的发病机制复杂，除了上述机制外，还可能存在其他的调控通路或靶点，值得我们今后进一步探索。