青刺果油对神经酰胺合成及神经酰胺酶表达的影响

Effects of Prinsepia utilis Royle oil on the synthesis of ceramide and expression of ceramidase

目的探讨青刺果油对神经酰胺（Cer）合成及酸性神经酰胺酶（ASH1）表达影响的研究，探讨其部分保湿机制及修复皮肤屏障机制。方法培养人角质形成细胞，设对照组及实验组，对照组加入不含青刺果油的K—SFM培养液，实验组加入含青刺果油K—SFM培养液，于0、3、8、24、48h分别取实验组及对照组的上清液，用ELISA方法测定上清液中Cer含量。将裸鼠背部皮肤分为受试区，基质区、空白对照区及阴性对照区，其中前3区用丙酮及乙醚破坏了裸鼠表皮屏障，受试区及基质区分别涂搽含青刺果油乳剂和基质，空白对照区不涂抹任何乳剂，用无创性皮肤测试方法分别于0、1、3、7d测定裸鼠皮肤经表皮水分流失（TEWL）、表皮含水量及皮脂含量，同时取裸鼠皮肤组织，用免疫组化方法观察ASHl表达。结果ELISA结果显示，实验组上清液中Cer含量随时问增加而增加，24hCer含量（1．3817±0．100）及48hCer含量（1．3737±0．047）与0h（0．7630±0．143）比较，差异有统计学意义（P〈0．05）；实验组与对照组比较，Cer含量高于对照组，24h、48h时差异有统计学意义（P〈0．05）。无创性皮肤测试显示，随时间增加，受试区、基质区，空白对照区TEWL值逐渐减少，表皮含水量、皮脂含量逐渐增加，受试区TEWL3d（10．85±0．64）、7d（8．01±0．58）时较0d（12．65±0．71）低，皮脂含量3d（29．14±O．40）、7d（31．30±0．88）时较0d（27．02±0．65）高，其1d（13．98±0．28）、3d（15．00±0．38）、7d（15．86±0．18）的表皮含水量较0d（11．74±0．62）高，差异有统计学意义（P〈0．05）。同一时间四区比较，7d时，受试区的TEWL值较基质区、空白对照区及阴性对照区低，表皮含水量及皮脂含量较基质区、空白对照区及阴性对照区高，差异有统计学意义（P〈0．05）。免疫组化结果显示，受试区、基质区，空白对照区ASI-II的表达7d较0d高，差异有统计学意义（P〈0．05）；7d时受试区ASHl的表达较基质区、空白对照区及阴性对照区高，差异有统计学意义（P〈0．05）。结论青刺果油的保湿及修复皮肤屏障的作用与其增加Cer含量及上调ASH1表达有关。

Objective To evaluate the effects of Pffnsepia utilis Royle oil （PURO） on the synthesis of ceramide and expression of acid ceramidase N-acylsphingosine amidohydrolase 1 （ASH1）, and to explore the mechanisms underlying its moisturizing and skin barrier-repairing effects. Methods Keratinocytes from human foreskin tissue were classified into 2 groups to be cultured in keratinocyte-serum free medium （K-SFM） with or without the presence of PURO. Enzyme linked immunosorbent assay （ELISA） was performed to measure the level of ceramide in the culture supernatant of keratinocytes at 0, 3, 8, 24 and 48 hours. The back of nude mice was divided into 4 areas, i.e., test area, matrix area, blank control area and negative control area. Acetone and ether were used to destroy the epidermal barrier in the test, matrix, and blank control areas, then, the former 2 areas were topically treated with emulsions containing 1% PURO and matrix, respectively, and the blank control area remained untreated. The epidermal barrier remained intact and untreated in the negative control area. Non- invasive methods were used to determine transepidermal water loss （TEWL）, epidermal moisture content and skin lipid content in these areas on day 0, 1, 3, and 7. Skin tissue was obtained from these areas on day 0 and 7 followed by an immunohistochemical study for the quantification of ASHI expression. Results The level of supernatant ceramide increased with time in the PURO-treated keratinocytes, which was significantly higher at 24 hours and 48 hours than at 0 hour （1.3817 ± 0.100 and 1.3737 ± 0.047 vs. 0.7630 ±0.143, both P〈 0.05）. The supernatant ceramide was also elevated in the PURO-treated keratinocytes compared with untreated keratinocytes at 24 and 48 hours （both P 〈 0.05）. Noninvasive skin tests showed a gradual decrease in the TEWL, but an increase in the epidermal moisture content and skin lipid content with time in the 3 epidermal barrier- destroyed areas. As far as the test area was concerned, TEWL value was significantly lower on day 3 and 7 than on day 0 （10.85 ± 0.64 and 8.01 ± 0.58 vs. 12.65 ± 0.71, both P 〈 0.05）, while a significant increment was observed in the skin lipid content on day 3 and 7 compared with day 0 （29.14 ± 0.40 and 31.30 ± 0.88 vs. 27.02 ± 0.65, both P 〈 0.05）, as well as in the epidermal moisture content on day 1, 3 and 7 compared with day 0 （13.98 ± 0.28, 15.00 ± 0.38 and 15.86 ± 0.18 vs. 11.74 ± 0.62, all P〈 0.05）. On day 7, there was a statisti- cal decline in TEWL value, but an elevation in epidermal moisture content, skin lipid content and ASH1 ex- pression in the test area compared with the matrix area and blank control area （all P 〈 0.05）. Also, the expres- sion of ASH1 was upregulated on day 7 compared with day 0 in the 3 barrier-destroyed areas （all P 〈 0.05）. Conclusion PURO may exert skin-moisturizing and barrier-repairing effects by enhancing the synthesis of ce- ramide and expression of acid ceramidase ASH1.