Department of Pharmacology and Pharmaceutical Sciences, USC Alfred E. Mann School of Pharmacy, Los Angeles, California, United States.
Department of Ophthalmology, Roski Eye Institute, Keck School of Medicine of USC, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2024 Jun 3;65(6):1. doi: 10.1167/iovs.65.6.1.
Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model.
LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old, ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy.
Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites.
Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.
泪腺（LG）丧失功能会导致与年龄相关的干眼症，因为 LG 产生水状的泪膜。为了更好地理解这种恶化，我们评估了衰老模型中 LG 脂质代谢和炎症的变化。
从小鼠 LG 组织切片中鉴定与脂质代谢相关的基因和蛋白的表达差异，使用油红 O 或甲苯胺蓝染色检测脂质，用定量实时聚合酶链反应（qRT-PCR）分析和 LG 裂解物的 Western 印迹分析。使用 LG 切片的光漂白方案来淬灭与年龄相关的自发荧光，并用共聚焦荧光显微镜评估与 NPC1、NPC2、CTSL 和巨噬细胞（F4/80、CD11b）相关的免疫荧光随年龄的变化。
老年 LG 中可见明显的脂质增加，在腺泡细胞的基底聚集物和腺外部位尤为明显。LG 中编码脂质代谢相关蛋白的 Npc1、Npc2、Lipa 和 Mcoln2 的基因表达随年龄增加而增加。Western 印迹也显示 NPC1 在老年 LG 中显著增加。在光漂白的 LG 切片中，NPC1、NPC2 和 CTSL 的免疫荧光共聚焦荧光显微镜成像显示，巨噬细胞标记物 F4/80 检测到的巨噬细胞与年龄相关的丰度增加。尽管在所有年龄段的 LG 中都可以检测到单核巨噬细胞，但这种新型的多核巨噬细胞群体富含 NPC1、NPC2 和 CTSL，并富含 F4/80 和一些 CD11b，在腺外部位随年龄增加而增加。
富含 F4/80 阳性多核巨噬细胞的脂质代谢蛋白在与腺泡中脂质沉积相邻的老年 LG 中增加。
