n-3多不饱和脂肪酸对SKH-1小鼠皮肤急性光损伤模型的保护作用研究

Protective effect of n-3 polyunsaturated fatty acids on acute skin photodamage in SKH-1 mice

目的 探讨膳食中n-3多不饱和脂肪酸（PUFA）对紫外线所致SKH-1无毛小鼠皮肤急性光损伤的保护作用及机制。方法 将50只SKH-1无毛小鼠等分成5组，定制5种特殊饲料［n-3 PUFA占总脂肪酸的比例分别为0（对照组）、12.5%、25%、50%、100%］分别喂养。喂养至第8周当日，采用日光紫外线模拟器照射上述5组小鼠背部，剂量为2个最小红斑量（MED），建立急性光损伤模型。24 h后肉眼及皮肤镜观察小鼠背部皮肤反应，取皮肤组织行HE染色观察表皮结构、细胞间水肿、炎症细胞浸润程度以及晒伤细胞情况，行免疫组化染色观察炎症细胞因子白细胞介素1β（IL-1β）、IL-6、肿瘤坏死因子α（TNF-α）表达情况，并采用ELISA检测组织匀浆中上述3种因子蛋白表达水平。结果 25%、50%、100% n-3 PUFA组小鼠皮肤急性光损伤反应程度以及表皮增厚、细胞间水肿、炎症细胞浸润程度较对照组、12.5% n-3 PUFA组轻；对照组、12.5% n-3 PUFA组小鼠表皮每100倍视野中晒伤细胞数（17.50 ± 4.93、14.25 ± 1.71）多于25%、50%、100% n-3 PUFA组（6.50 ± 1.73、4.75 ± 2.06、4.50 ± 1.73），组间差异有统计学意义（F = 19.1，P 〈 0.001）。免疫组化结果显示，紫外线照射后24 h IL-1β、IL-6、TNF-α在各组小鼠表皮和真皮有不同程度表达，与对照组、12.5% n-3 PUFA组比较，25%、50%、100% n-3 PUFA组3种炎症因子表达降低（P 〈 0.001）。ELISA检测结果表明，25%、50%、100% n-3 PUFA组小鼠皮肤组织中IL-1β、IL-6和TNF-α含量明显低于对照组、12.5% n-3 PUFA组（P 〈 0.05）。结论 膳食中n-3 PUFA含量达到总脂肪酸的25%以上时对紫外线引起的急性光损伤具有保护作用，且含量越高，保护作用越强。推测n-3 PUFA可能通过花生四烯酸通路抑制炎症反应。

Objective To evaluate the protective effect of dietary n-3 polyunsaturated fatty acids （PUFA） on ultraviolet-induced acute skin photodamage in SKH-1 hairless mice, and to explore its mechanism. Methods Totally, 50 SKH-1 hairless mice were equally divided into 5 groups to be fed with forages with the ratios of n-3 PUFA to fatty acid being 0, 12.5%, 25%, 50% and 100% respectively （control group, 12.5%, 25%, 50% and 100% n-3 PUFA groups）. On day 8 after feeding, the back of mice in the 5 groups were irradiated by a solar ultraviolet simulator at 2 minimum erythema doses （MED） to establish an acute photodamage model. After 24 hours, cutaneous reactions on the back of mice were observed by naked eyes and dermoscopy, and skin biopsy specimens were subjected to hematoxylin and eosin （HE） staining for observing the epidermal structure, intercellular edema, inflammatory cell infiltration and photodamaged cells. Immunohistochemical staining was performed to determine the of inflammatory cytokines including interleukin-1 beta （IL-1β）, IL-6 and tumor necrosis factor alpha （TNF-α）, and enzyme-linked immunosorbent assay（ELISA） to measure the protein of the above 3 inflammatory cytokines in the tissue homogenate. Results Compared with the control group and 12.5% n-3 PUFA group, the 25%, 50% and 100% n-3 PUFA groups all showed a milder degree of acute skin photodamage, epidermis thickening, intercellular edema, and inflammatory cell infiltration. The number of photodamaged cells per high-power field （× 100） was significantly higher in the control group and 12.5% n-3 PUFA group （17.50 ± 4.93, 14.25 ± 1.71, respectively） than in the 25%, 50% and 100% n-3 PUFA groups （6.50 ± 1.73, 4.75 ± 2.06, 4.50 ± 1.73, respectively; F = 19.1, P 〈 0.001）. Immunohistochemical results showed that IL-1β, IL-6 and TNF-α were expressed to different extents in the epidermis and dermis among the 5 groups at 24 hours after ultraviolet radiation. Compared with the control group and 12.5% n-3 PUFA group, the 25%, 50% and 100% n-3 PUFA groups showed significantly lower of the above 3 inflammatory cytokines （P 〈 0.001）. ELISA revealed that the levels of IL-1β, IL-6 and TNF-α in the skin tissues of the mice were significantly lower in the 25%, 50% and 100% n-3 PUFA groups than in the control group and 12.5% n-3 PUFA group （P 〈 0.05）. Conclusions When the ratio of n-3 PUFA to fatty acid reaches more than 25%, dietary n-3 PUFA has protective effects against ultraviolet-induced acute photodamage,. Moreover, the higher the content of n-3 PUFA is, the stronger the protective effect is. It is suggested that n-3 PUFA may inhibit the inflammatory reaction through the arachidonic acid pathway.