目的 探讨壬二酸和水杨酸对痤疮丙酸杆菌Propionibacterium acnes诱导的炎症因子及Toll样受体4（TLR4）表达的影响。方法 采用CCK-8法检测药物对人角质形成细胞HaCaT和人单核细胞THP-1增殖活力的影响，酶联免疫吸附法（ELISA）检测细胞培养上清中白细胞介素（IL）-8、IL-1β和肿瘤坏死因子-α（TNF-α）浓度，利用分子对接技术分析壬二酸、水杨酸与TLR4蛋白的分子间相互作用，并通过细胞免疫荧光和荧光强度分析验证其对HaCaT细胞表面TLR4蛋白表达的影响。结果 经不同浓度的壬二酸或水杨酸处理48 h后，在HaCaT细胞实验中，壬二酸的最大实验质量浓度为62.50 μg/mL，水杨酸为31.25 μg/mL；THP-1细胞实验中壬二酸的最大实验质量浓度为1 000.00 μg/mL，水杨酸为500.00 μg/mL。壬二酸和水杨酸对P. acnes胞壁成分肽聚糖（PGN）＋脂磷壁酸（LTA）或热灭活P. acnes诱导后HaCaT细胞产生IL-8以及对佛波酯（PMA）＋细菌脂多糖（LPS）或热灭活P. acnes诱导后的THP-1细胞产生IL-1β和TNF-α均表现出一定的抑制作用，呈剂量相关性。分子对接结果表明，壬二酸、水杨酸均能与TLR4蛋白受体有效地结合。与对照组相比，PGN＋LTA或热灭活P. acnes刺激后HaCaT细胞表面TLR4荧光强度明显增高（P＜0.01），经62.50 μg/mL壬二酸预处理后PGN＋LTA刺激的HaCaT细胞表面TLR4蛋白表达明显降低（P＜0.01），31.25 μg/mL水杨酸预处理后能明显降低热灭活P. acnes诱导的细胞表面TLR4蛋白表达（P＜0.05）。结论 壬二酸和水杨酸能抑制P. acnes引起的角质形成细胞和单核巨噬细胞中促炎因子的产生，同时还能减少角质形成细胞表面TLR4蛋白的表达，从而发挥治疗痤疮的作用。

Objective To investigate the effects of azelaic acid and salicylic acid on the expression of inflammatory factors and Toll-like receptor4 (TLR4) induced by Propionibacterium acnes. Methods The effect of drugs on cell proliferation was detected by CCK-8 method. The concentrations of interleukin (IL)-8, IL-1β and tumor necrosis factor-α (TNF-α) in the supernatant of cell culture were detected by enzyme linked immunosorbent assay (ELISA). The molecular interaction of azelaic acid, salicylic acid and TLR4 protein receptor was analyzed by molecular docking. The expression of TLR4 protein on the surface of HaCaT cells was verified by cellular immunofluorescence and fluorescence intensity analysis. Results After treated with azelaic acid or salicylic acid for 48 h, the safe concentrations of azelaic acid and salicylic acid in HaCaT cells were 62.50 μg/mL and 31.25 μg/mL, respectively. The maximum experimental mass concentration of azelaic acid and salicylic acid in THP-1 cells were 1 000.00 μg/mL and 500.00 μg/mL, respectively. Azelaic acid and salicylic acid inhibited IL-8 production in HaCaT cells induced by P. acnes cell wall component peptidoglycan (PGN) + lipoteichoic acid (LTA) or heat inactivated P. acnes. Furthermore, azelaic acid and salicylic acid could also inhibit IL-1β and TNF-α production in THP-1 cells induced by phorbol ester (PMA) + lipopolysaccharide (LPS) or heat inactivated P. acnes in a dose-dependent manner. Molecular docking showed that azelaic acid and salicylic acid could bind to TLR4 protein receptor effectively. Compared with the control group, the fluorescence intensity of TLR4 on the surface of HaCaT cells stimulated by PGN + LTA or heat inactivated P. acnes was significantly increased (P < 0.01). After treated with 62.50 μg/mL azelaic acid, the expression of TLR4 protein on the surface of HaCaT cells was significantly decreased (P < 0.01). Meanwhile, pretreatment with 31.25 μg/mL salicylic acid could significantly reduce the expression of TLR4 protein on the surface of HaCaT cells induced by heat inactivated P. acnes (P < 0.05).Conclusion Azelaic acid and salicylic acid can inhibit the production of pro-inflammatory factors in keratinocytes and mononuclear macrophages induced by P. acnes, and also reduce the expression of TLR4 protein on the surface of keratinocytes. Thus, azelaic acid and salicylic acid all play an important role in the treatment of acne.

R966

中国医学科学院医学与健康科技创新工程项目（CAMS-2017-I2M-1-011）