目的 探討壬二酸和水楊酸對(duì)痤瘡丙酸桿菌Propionibacterium acnes誘導(dǎo)的炎癥因子及Toll樣受體4（TLR4）表達(dá)的影響。方法 采用CCK-8法檢測(cè)藥物對(duì)人角質(zhì)形成細(xì)胞HaCaT和人單核細(xì)胞THP-1增殖活力的影響，酶聯(lián)免疫吸附法（ELISA）檢測(cè)細(xì)胞培養(yǎng)上清中白細(xì)胞介素（IL）-8、IL-1β和腫瘤壞死因子-α（TNF-α）濃度，利用分子對(duì)接技術(shù)分析壬二酸、水楊酸與TLR4蛋白的分子間相互作用，并通過細(xì)胞免疫熒光和熒光強(qiáng)度分析驗(yàn)證其對(duì)HaCaT細(xì)胞表面TLR4蛋白表達(dá)的影響。結(jié)果 經(jīng)不同濃度的壬二酸或水楊酸處理48 h后，在HaCaT細(xì)胞實(shí)驗(yàn)中，壬二酸的最大實(shí)驗(yàn)質(zhì)量濃度為62.50 μg/mL，水楊酸為31.25 μg/mL；THP-1細(xì)胞實(shí)驗(yàn)中壬二酸的最大實(shí)驗(yàn)質(zhì)量濃度為1 000.00 μg/mL，水楊酸為500.00 μg/mL。壬二酸和水楊酸對(duì)P. acnes胞壁成分肽聚糖（PGN）＋脂磷壁酸（LTA）或熱滅活P. acnes誘導(dǎo)后HaCaT細(xì)胞產(chǎn)生IL-8以及對(duì)佛波酯（PMA）＋細(xì)菌脂多糖（LPS）或熱滅活P. acnes誘導(dǎo)后的THP-1細(xì)胞產(chǎn)生IL-1β和TNF-α均表現(xiàn)出一定的抑制作用，呈劑量相關(guān)性。分子對(duì)接結(jié)果表明，壬二酸、水楊酸均能與TLR4蛋白受體有效地結(jié)合。與對(duì)照組相比，PGN＋LTA或熱滅活P. acnes刺激后HaCaT細(xì)胞表面TLR4熒光強(qiáng)度明顯增高（P＜0.01），經(jīng)62.50 μg/mL壬二酸預(yù)處理后PGN＋LTA刺激的HaCaT細(xì)胞表面TLR4蛋白表達(dá)明顯降低（P＜0.01），31.25 μg/mL水楊酸預(yù)處理后能明顯降低熱滅活P. acnes誘導(dǎo)的細(xì)胞表面TLR4蛋白表達(dá)（P＜0.05）。結(jié)論 壬二酸和水楊酸能抑制P. acnes引起的角質(zhì)形成細(xì)胞和單核巨噬細(xì)胞中促炎因子的產(chǎn)生，同時(shí)還能減少角質(zhì)形成細(xì)胞表面TLR4蛋白的表達(dá)，從而發(fā)揮治療痤瘡的作用。

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

中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程項(xiàng)目（CAMS-2017-I2M-1-011）