空气污染对皮肤角质层蛋白羰基化水平的影响及粉红胡椒木提取物和脂质混合物对皮肤损伤的防护作用

目的 研究空气污染对皮肤角质层蛋白羰基化水平的影响，评估粉红胡椒木提取物和脂质混合物对皮肤损伤的防护作用。方法 预实验分析影响因素后，荧光标记法检测34例健康受试者不同部位皮肤角质层蛋白羰基化水平。利用定制的污染模拟箱，以香烟烟雾模拟污染物， 将15名健康受试者前臂屈侧向上暴露于污染模拟箱中，分别于暴露后0、1、2、4、5 h用D?squame胶片采集角质层样本。选14例健康受试者，在单侧前臂屈侧选择相邻的3个区域分别外用1%粉红胡椒木提取物的水溶液（胡椒木组）、去离子水（对照组）或不涂任何样品（空白组），然后将手臂在污染模拟箱中暴露5 h，暴露前后采集各区域角质层样本。另选16例健康受试者，在单侧前臂屈侧选取3处区域分别外用含5% 脂质混合物的乳液（脂质混合物组）、不含脂质混合物的安慰剂乳液（对照组）或不涂任何样品（空白组），然后将手臂在污染模拟箱中暴露5 h，暴露前后采集各区域角质层样本。纳入20例健康受试者进行双盲半脸临床测试，即随机选择半脸外用含1%粉红胡椒木提取物的乳液，另外半脸外用安慰剂乳液，于产品使用前和使用56 d后，采用D?Squame胶片在受试者的面颊部分别采集角质层样本。采用荧光标记法检测上述角质层样本中皮肤蛋白羰基化水平。结果 对34名受试者检测显示，人体不同部位蛋白羰基化水平（平均荧光强度）存在显著差异（P < 0.001），其中，面颊（26.3 ± 7.1）和额部（22.9 ± 7.9）显著高于手臂（14.7 ± 4.9）和腰背部（12.6 ± 4.2）（均P < 0.001）,且手臂蛋白羰基化水平显著高于腰背部（P = 0.046）。短期模拟加速暴露实验中，蛋白羰基化水平随污染暴露时间的增加而持续升高（R2 = 0.995 9），暴露5 h后，胡椒木组和脂质混合物组皮肤角质层蛋白羰基化水平升高值分别为9.7 ± 5.2和5.8 ± 4.9，低于各自的空白组（19.0 ± 10.0、17.4 ± 8.8，均P < 0.005）和对照组（18.5 ± 7.3、15.9 ± 6.4，均P < 0.005），差异均有统计学意义。长期人体测试中，20名受试者使用含1%粉红胡椒木提取物的乳液8周后，与安慰剂侧相比，面部蛋白羰基化水平显著降低。结论 空气污染加剧皮肤角质层蛋白羰基化损伤，粉红胡椒木提取物和脂质混合物能有效降低蛋白羰基化水平。

Effect of air pollution on carbonylated protein level in the stratum corneum and protective effect of pink pepper tree extracts and lipid mixtures on skin damage

Jiang Yueming, Zhao Xiaomin, Qu XinAshland China Holdings Co., Ltd, Shanghai 200233, ChinaCorresponding author: Zhao Xiaomin, Email: rzhao@ashland.com【Abstract】 Objective To evaluate the effect of air pollution on carbonylated protein level in the stratum corneum, and to assess the protective effect of pink pepper tree extracts and lipid mixtures on skin damage. Methods After the investigation of influencing factors in the preliminary experiment, fluorescence labelling assay was performed to detect the carbonylated protein level in the skin stratum corneum at different body sites of 34 healthy testees. Cigarette smoke was used to simulate pollutants, and the forearms of 15 healthy testees were exposed in the customized pollution simulation chamber with the flexor aspects facing upwards. After 0, 1, 2, 4, 5 hours of exposure, stratum corneum samples were collected by using D-squame tapes. In each of 14 selected healthy testees, 3 adjacent areas on the flexor aspect of unilateral forearm were divided into 3 groups: pink pepper tree group treated with 1% aqueous solution of pink pepper tree extracts, control group treated with deionized water, and blank group receiving no treatment. Then, the forearms of the 14 testees were exposed in the pollution simulation chamber for 5 hours, and stratum corneum samples were collected from the 3 areas before and after the exposure. Another 16 healthy testees were included, and 3 adjacent areas on the flexor aspect of their unilateral forearms were divided into 3 groups: lipid mixture group treated with 5% lipid mixture emulsion, control group treated with lipid mixture-free placebo emulsion, and blank group receiving no treatment. Then, the forearms of the 16 testees were exposed in the pollution simulation chamber for 5 hours, and stratum corneum samples were collected from the 3 areas before and after the exposure. Moreover, twenty healthy testees were enrolled into the double-blind split-face clinical trial. That is, one half of the face was randomly chosen to be treated with 1% emulsion of pink pepper tree extracts, and the other facial side was treated with placebo emulsion. Before and after 56-day treatment, stratum corneum samples were collected from the cheeks of testees by using D-squame tapes. Fluorescence labelling assay was conducted to detect the carbonylated protein level in the above stratum corneum samples. Results The analysis of 34 testees showed that carbonylated protein levels （average fluorescence intensity） significantly differed among different body sites （P < 0.001）, and the carbonylated protein levels were significantly higher in the cheeks （26.3 ± 7.1） and forehead （22.9 ± 7.9） than in the forearm （14.7 ± 4.9） and waist and back （12.6 ± 4.2）（P < 0.001）, and higher in the forearm than in the waist and back （P = 0.046）. In the short-term simulated accelerated exposure experiment, the carbonylated protein level increased along with the increase of the duration of exposure to pollution （R2 = 0.995 9）. After 5-hour exposure, the pink pepper tree group and lipid mixture group both showed significantly lower elevated levels of carbonylated protein in the stratum corneum （9.7 ± 5.2, 5.8 ± 4.9） compared with the corresponding blank groups （19.0 ± 10.0, 17.4 ± 8.8; P < 0.005） and control groups （18.5 ± 7.3, 15.9 ± 6.4; P < 0.005）. In the long-term human trial, the carbonylated protein levels significantly decreased in the facial side treated with 1% emulsion of pink pepper tree extracts for 8 weeks compared with the placebo-treated facial side. Conclusion Air pollution aggravates skin damage induced by protein carbonylation in the stratum corneum, and pink pepper tree extracts and lipid mixtures can effectively reduce the carbonylated protein level.