蒿秦化斑方对紫外线损伤角质形成细胞黏附分子及PI3K/AKTAKT/GSK-3β通路的影响

王思艺 孙丽蕴

摘要 目的：觀察蒿秦化斑方对HaCat细胞紫外线B（UVB）急性损伤模型细胞黏附分子及PI3K/AKT/GSK-3β通路的影响，探讨该方治疗光敏感性皮肤病的作用机制。方法：采用300 mJ/cm2 UVB建立HaCat细胞急性光损伤模型，使用不同剂量蒿秦化斑方干预，分为空白对照组、UVB模型组、及蒿秦化斑方高、中、低剂量组。采用RT-PCR法检测细胞黏附分子ICAM-1、ELAM-1、VCAM-1相对表达量，采用蛋白质免疫印迹法分析PI3K/AKT/GSK-3β通路相关节点p-AKT（Ser-473）、p-AKT（Thr-308）、GSK-3β、PI3K相对表达量。结果：UVB急性光损伤可有效减少HaCat细胞ICAM-1的表达（P<0.01），对VCAM-1及ELAM-1表达影响不显著;蒿秦化斑方高剂量组能有效抑制HaCat细胞3种细胞黏附分子的表达（P<0.05）;蒿秦化斑方中剂量组能有效抑制HaCat细胞内皮细胞白细胞黏附分子-1（ELAM-1）及血管细胞黏附分子-1（VCAM-1）的表达（P<0.01）。UVB急性光损伤对HaCat细胞AKT磷酸化、PI3K表达影响不显著，蒿秦化斑方低剂量处理对HaCat细胞AKT磷酸化具有促进作用（P<0.05）。UVB急性光损伤使HaCat细胞糖原合成酶激酶-3β（GSK-3β）表达水平升高（P<0.01），蒿秦化斑方高、中、低剂量处理均对HaCat细胞GSK-3β表达水平有所抑制（P<0.01）。结论：蒿秦化斑方能降低由UVB急性损伤造成的HaCat细胞ICAM-1、ELAM-1、VCAM-1等黏附分子的表达进而缓解局部炎症和迟发型超敏反应的发生，并且能够激活HaCat细胞PI3K/AKT/GSK-3β通路，在UVB造成的急性光损伤中起到抗氧化、抗凋亡和降低炎症反应的作用。

关键词 光敏感性皮肤病;蒿秦化斑方;蛋白激酶B;糖原合成酶激酶-3β;细胞黏附分子;人永生化角质形成细胞;紫外线B;紫外线损伤

Abstract Objective：To observe the effects of Haoqin-Huaban on the relative adhesion factors and the PI3K/AKT/GSK-3β pathway of an acute UVB light HaCat-cell injury model in order to further explore the mechanism of the treatment for photosensitive skin disorders.Methods：UVB light（300 mJ/cm2） was used to establish an acute light HaCat-cell injury model.The intervention was performed by using Haoqin-Huaban in various concentrations.The groups were divided into：a blank control group（C）; an UVB model group（M）; a high Haoqin-Huaban dosage group（G）; medium Haoqin-Huaban dosage group（Z）; low Haoqin-Huaban dosage group（D）.Real-time PCR was conducted to detect the relative expressions of 3 cell adhesion molecules（CAMs），i.e.，ICAM-1，ELAM-1 and VCAM-1.Western Blotting was implemented to analyze the relative expressions of p-AKT（Ser-473），p-AKT（Thr-308），GSK-3β，and PI3K，which were all related nodes of the PI3K/AKT/GSK-3β pathway.Western Blot method was used to analyze the relative expression of p-AKT（Ser-473），p-AKT（Thr-308），GSK-3β and PI3K related nodes in the PI3K/AKT/GSK-3β pathway.Results：UVB acute light injury reduced ICAM-1 expression in the HaCat cell model and had no significant effect on the expressions of VCAM-1 and ELAM-1（P<0.01）.The expressions of the three CAMs in the HaCat cell model were effectively inhibited in the G group（P<0.05），while those of ELAM-1 and VCAM-1 were considerably impeded in the Z group（P<0.01）.It was also found that acute UVB light injury had no significant effect on AKT phosphorylation and PI3K expression in the HaCat cell model.The low-concentration treatment of Haoqin Huaban Forumla can promote the phosphorylation of AKT in HaCat cells（P<0.05）.Acute UVB light damage increased the GSK-3β expression level of HaCat cells（P<0.01）.The high，medium and low concentration treatments of Haoqin Huaban Formula inhibited the GSK-3β expression level of HaCat cells（P<0.01）.Conclusion：Haoqin-Huaban Formula can reduce the expression of adhesion molecules such as ICAM-1，ELAM-1 and VCAM-1 in HaCat cells caused by acute UVB damage，thereby alleviating the occurrence of local inflammation and delayed-type hypersensitivity，and can activate HaCat cells PI3K/AKT/GSK-3β pathway and plays a role in anti-oxidation，anti-apoptosis and reducing inflammation in the acute light damage caused by UVB.

Keywords Photosensitive skin disease; Haoqin-Huaban Formula; AKT; GSK-3β; Cell adhesion molecule; HaCat cells; Ultraviolet B; Ultraviolet injury

中图分类号：R289.5;R275.9文献标识码：Adoi：10.3969/j.issn.1673-7202.2021.19.012

光敏感性皮肤病是由紫外线照射造成皮肤多种病理反应的一系列疾病，其包括急性光损伤、慢性光老化、光变态反应（如多形性日光疹）等，甚至能够诱发皮肤肿瘤，其临床表现通常以日光暴露部位出现红斑、水肿及多形性皮损，患者自觉灼热瘙痒为主[1]。……