薰衣草总黄酮对小鼠皮肤光损伤的防护作用机制

Protective Mechanism of Total Flavonoids of Lavender Against Skin Photoaging in Mice

目的:研究薰衣草总黄酮对中波紫外线(UVB)致小鼠皮肤光损伤的防护作用,并从核因子E_(2)相关因子2(Nrf2)抗氧化损伤通路探讨其作用机制。方法:将84只雌性KM小鼠随机分为7组:空白组,模型组,溶媒组,维生素E组(0.013 g·kg^(-1)),薰衣草总黄酮低、中、高剂量组(0.25,1.25,2.50 g·kg^(-1))。通过UVB辐照小鼠背部裸露皮肤建立光损伤模型,照射前30 min外涂薰衣草总黄酮,连续照射1周后,对小鼠背部皮肤进行评价并测得其水分和弹性;采用苏木素-伊红(HE)染色和苦味酸-酸性品红法(VG)染色考察薰衣草总黄酮对小鼠皮肤组织病理学改变的影响;生化比色法检测皮肤匀浆后丙二醛(MDA),超氧化物歧化酶(SOD),总抗氧化能力(T-AOC),一氧化氮合酶(NOS),谷胱甘肽过氧化物酶(GSH-Px)的水平;采用酶联免疫吸附测定法(ELISA)检测皮肤组织中炎症因子白细胞介素-1(IL-1),白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α);实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠Nrf2,Kelch样环氧氯丙烷相关蛋白1(Keap1),BTB-CNC异体同源蛋白1(Bach1),血红素加氧酶-1(HO-1),醌氧化还原酶1(NQO1),谷氨酸半胱氨酸合成酶催化亚基(GCLC)mRNA表达。结果:与空白组比较,模型组小鼠外观评分显著升高(P<0.01),皮肤水分和弹性显著降低(P<0.01);皮肤组织出现较为明显的病理学变化,表皮明显增厚且伴有结痂小脓肿,损伤程度严重;小鼠的MDA,NOS水平明显升高(P<0.05,P<0.01),SOD,T-AOC水平明显降低(P<0.05,P<0.01),炎症因子IL-1,IL-6和TNF-α的水平明显升高(P<0.05,P<0.01),Nrf2,Keap1,NQO1,GCLC mRNA表达明显降低(P<0.05,P<0.01),Bach1 mRNA的表达显著升高(P<0.01);与模型组比较,薰衣草总黄酮低、中、高剂量组的小鼠外观评分显著降低(P<0.01),皮肤水分和弹性显著升高(P<0.01),各给药组小鼠的MDA,NOS水平明显降低(P<0.05,P<0.01),SOD,T-AOC水平明显升高(P<0.05,P<0.01),炎症因子IL-1,IL-6和TNF-α的水平明显降低(P<0.05,P<0.01),Nrf2,Keap1,NQO1,HO-1,GCLC mRNA表达明显升高(P<0.05,P<0.01),Bach1 mRNA表达显著降低(P<0.01)。结论:薰衣草总黄酮对UVB所致的小鼠皮肤光损伤防护效果显著,其作用机制可能与激活Keap1/Nrf2/ARE信号通路,调控氧化应激反应,改善炎症反应有关。

Objective:To study the protective effect of total flavonoids of lavender on skin photoaging induced by ultraviolet B(UVB)in mice and to explore its mechanism from the perspective of nuclear factor E_(2)-related factor 2(Nrf2)antioxidant pathway.Method:Eighty-four female KM mice were randomly divided into seven groups,namely blank group,model group,solvent group,vitamin E(0.013 g·kg^(-1))group,as well as low-,middle-,and high-dose(0.25,1.25,2.50 g·kg^(-1))groups of total flavonoids of lavender.The naked skin on the back of mice was irradiated with UVB for inducing optical damage.Thirty minutes before irradiation,the skin was coated with the total flavonoids of lavender.After continuous irradiation for one week,the skin moisture and elasticity on the back of mice were evaluated,and the effects of total flavonoids of lavender on histopathological changes in mouse skin were investigated by hematoxylin-eosin(HE)and Van Gieson(VG)staining.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),total antioxidant capacity(TAOC),nitric oxide synthase(NOS),and glutathione peroxidase(GSH-Px)after skin homogenization were detected by colorimetry,the inflammatory factors interleukin-1(IL-1),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in skin tissue by enzyme-linked immunosorbent assay(ELISA),and the mRNA expression levels of Nrf2,Kelch-like epichlorohydrin-associated protein 1(Keap1),BTB-CNC homology 1(Bach1),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1),and glutamate-cysteine ligase catalytic subunit(GCLC)by real-time polymerase chain reaction(Real-time PCR).Result:Compared with the blank group,the model group exhibited significantly increased appearance score(P<0.01),reduced skin moisture and elasticity(P<0.01),pronounced pathological changes in the skin tissue like epidermal thickening,scabbing,small abscess,and severe injury,elevated MDA,NOS,IL-1,IL-6 and TNF-α(P<0.05,P<0.01),lowered SOD,T-AOC,Nrf2,Keap1,NQO1 and GCLC mRNA expression(P<0.05,P<0.01),and up-regulated Bach1 mRNA expression(P<0.01).Compared with the model group,the total flavonoids of lavender at the low,middle,and high doses all remarkably reduced the appearance score(P<0.01),enhanced the skin moisture and elasticity(P<0.01),diminished the MDA,NOS,IL-1,IL-6,and TNF-α(P<0.05,P<0.01),increased SOD,T-AOC,Nrf2,Keap1,NQO1,HO-1 and GCLC mRNA expression(P<0.05,P<0.01),and down-regulated the expression of Bach1 mRNA(P<0.01).Conclusion:The protective effect of the total flavonoids of lavender against skin photoaging in mice is significant,which may be related to its activation of Keap1/Nrf2/ARE signaling pathway,regulation of oxidative stress,and improvement of inflammatory response.