吡咯喹啉醌对小鼠年龄相关皮肤衰老的延缓作用及机制研究

Delaying effect of pyrroloquinoline quinone on age-related skin aging in mice and its mechanisms

目的研究吡咯喹啉醌对小鼠年龄相关皮肤衰老的作用及机制。方法将昆明种小鼠SPF级喂养, 分为3组, 每组10只, 年轻组用普通饮食喂养8个月, 年老组用普通饮食喂养20个月构建小鼠自然衰老模型, 吡咯喹啉醌组在每公斤普通饮食饲料中添加吡咯喹啉醌4 mg喂养20个月。喂养结束后, 取各组小鼠背部皮肤组织, HE染色检测皮肤表皮和真皮厚度;Masson染色检测皮肤总胶原变化;免疫组化检测增殖蛋白Ki67表达变化;透射电镜检测皮肤自噬体变化;Western印迹检测自噬相关蛋白Beclin1、LC3Ⅱ/LC3Ⅰ、p62表达变化, 各组间比较采用单因素方差分析, 组间两两比较采用LSD-t检验。结果 HE和Masson染色显示, 年老组表皮厚度[(15.67 ± 0.36)μm]和真皮厚度[(87.95 ± 11.86)μm]以及总胶原阳性百分率[(22.12 ± 1.72)%]均显著低于年轻组[(29.37 ± 0.25)μm、(264.93 ± 10.34)μm、(45.03 ± 1.54)%, 均P<0.05], 而吡咯喹啉醌组表皮厚度[(25.53 ± 0.47)μm]和真皮厚度[(145.01 ± 9.71)μm]以及总胶原阳性百分率[(31.17 ± 1.20)%]较年老组增加(均P<0.05)。免疫组化结果显示, 年老组皮肤增殖蛋白Ki67阳性细胞表达率[(13.74 ± 3.06)%]低于年轻组[(29.07 ± 2.79)%, P<0.05]和吡咯喹啉醌组[(21.20 ± 1.47)%, P<0.05]。透射电镜观察显示, 年老组与年轻组比较, 皮肤自噬体数量增加(P<0.05), 吡咯喹啉醌组自噬体数量较年老组减少(P<0.05)。Western印迹实验显示, 与年轻组比较, 年老组Beclin-1表达降低(P<0.05), LC3Ⅱ/LC3Ⅰ比值下降(P<0.05), p62表达升高(P<0.05);而吡咯喹啉醌组与年老组比较, Beclin1表达升高(P<0.05), LC3Ⅱ/LC3Ⅰ比值增加(P<0.05), p62表达降低(P<0.05)。结论吡咯喹啉醌能够延缓小鼠皮肤衰老, 其机制可能与增加小鼠皮肤细胞增殖能力和促进皮肤自噬水平相关。

Objective To investigate the effect of pyrroloquinoline quinone(PQQ)on age-related skin aging in mice and its mechanisms.Methods Thirty Kunming mice were fed in specific pathogen-free condition,and equally divided into 3 groups:young group was fed with a normal diet for 8 months,old group was fed with a normal diet for 20 months to establish a mouse model of natural aging,and PQQ group was fed with PQQ-containing forages(4 milligrams of PQQ per kilogram of normal forages)for 20 months.After feeding,the mouse dorsal skin tissues were obtained,hematoxylin and eosin(HE)staining was performed to measure the epidermal and dermal diickness,Masson staining to detect changes in total skin collagen,immunohistochemical study to detect changes in expression of the proliferation marker Ki67,transmission electron microscopy to detect changes in autophagosomes in the mouse skin,and Western blot analysis to determine the expression of autophagy-related proteins Beclinl,LC3 Ⅱ/LC3 Ⅰ,and p62.Statistical analysis was earned out by using one-way analysis of variance for intergroup comparisons followed by least significant difference(LSD)-t test for multiple comparisons.Results HE and Masson staining showed that the epidermal and dermal thickness and the percentage of area of dermis stained positive for collagen among the total area of dermis in the tested region were significantly lower in the old group(15.67±0.36 pm,87.95±11.86 μm,22.12%±1.72%,respectively)than in the young group(29.37±0.25 μm,264.93±10.34 μm,45.03%±1.54%,respectively,all P<0.05),and significantly higher in the PQQ group(25.53±0.47μm,145.01±9.71μm,31.17%±1.20%,respectively)than in the old group(all P<0.05).Immunohistochemical study revealed that the expression of Ki67 was significantly lower in the old group(13.74%±3.06%)than in the young group(29.07%±2.79%,P<0.05)and PQQ group(21.20%±1.47%,P<0.05).Transmission electron microscopy showed that the number of autophagosomes in the skin was significantly higher in the old group than in the young group(P<0.05),but significantly lower in the PQQ group than in the old group(P<0.05).As Western blot analysis revealed,the old group showed significantly decreased Beclinl expression and LC3 Ⅱ/LC3 Ⅰ ratio,but significantly increased p62 expression compared with the young group(all P<0.05);compared with the old group,the PQQ group showed significantly increased Beclinl expression and LC3 Ⅱ/LC3 Ⅰ ratio,but significantly decreased p62 expression(all P<0.05).Conclusion PQQ can delay the age-related skin aging in mice,likely by increasing the proliferative capacity of mouse skin cells and promoting skin autophagy.