Supplementary Material for: Free fatty acids induce lipid accumulation, autophagy and apoptosis in human sebocytes

Background: A disruption of sebocyte differentiation and lipogenesis has fatal consequences and can cause a wide spectrum of skin diseases, from acne vulgaris to sebaceous carcinoma, however, the relevant molecular mechanisms have not been fully clarified. Objectives: The induction of autophagy and apoptosis in human sebocytes in response to biologically relevant fatty acids was investigated. Methods: Free fatty acids (arachidonic acid, linoleic acid, palmitic acid and palmitoleic acid) and the pan-caspase inhibitor QVD-Oph were added in the supernatant of cultured human SZ95 sebocytes. Individual relevant proteins were analysed by Western blotting. Apoptosis and cell viability were determined, and typical autophagy structures were detected through electron microscopy. To obtain cell growth curves, cell confluence was continuously monitored by real-time cell analysis. Results: Fatty acids induced the development of intracellular lipid droplets with subsequent apoptosis, whereas arachidonic acid caused the most rapid effect. Cleavage products of caspase-3 were only detected in arachidonic acid-induced apoptosis. The high basal apoptotic rate of cultured SZ95 sebocytes was strongly suppressed by QVD-Oph. Fatty acid-induced apoptosis was also markedly inhibited by QVD-Oph, whereas intracellular lipid droplets further accumulated. While cell viability after incubation with linoleic acid, palmitic acid or palmitoleic acid and QVD-Oph was comparable with non-treated controls, arachidonic acid significantly reduced cell viability and cell density despite the concomitant pan-caspase inhibitor treatment. Using electron microscopy, typical autophagy structures were detected, such as autophagosomes and autolysosomes, at the basal level, which became more pronounced after treatment with fatty acids. Conclusions: Our findings contribute to a better understanding of the inflammation-associated mechanisms of lipogenesis and cell death induction in human sebocytes and may help to unveil the effects of fatty acid-rich human nutrition.

背景：皮脂腺细胞（sebocyte）分化与脂质生成过程的紊乱会引发严重后果，并可诱发涵盖从寻常痤疮（acne vulgaris）到皮脂腺癌（sebaceous carcinoma）在内的多种皮肤疾病，但相关分子机制尚未完全阐明。目的：本研究旨在探究具有生物学相关性的脂肪酸对人皮脂腺细胞自噬与细胞凋亡的诱导作用。方法：将游离脂肪酸（花生四烯酸、亚油酸、棕榈酸及棕榈油酸）与泛半胱天冬酶抑制剂QVD-Oph加入培养的人SZ95皮脂腺细胞的上清液中。通过蛋白质免疫印迹（Western blotting）分析相关蛋白表达，检测细胞凋亡与细胞活力，并通过电子显微镜（electron microscopy）观察典型自噬结构。为绘制细胞生长曲线，通过实时细胞分析（real-time cell analysis）持续监测细胞汇合度。结果：脂肪酸可诱导细胞内脂滴形成并后续引发细胞凋亡，其中花生四烯酸的诱导效应最为迅速。仅在花生四烯酸诱导的细胞凋亡中检测到半胱天冬酶-3（caspase-3）的裂解产物。QVD-Oph可显著抑制培养的SZ95皮脂腺细胞的高基础凋亡率；同时，脂肪酸诱导的细胞凋亡也被QVD-Oph显著阻断，但细胞内脂滴进一步蓄积。与未经处理的对照组相比，经亚油酸、棕榈酸或棕榈油酸联合QVD-Oph孵育后的细胞活力无明显差异，但即便联合泛半胱天冬酶抑制剂处理，花生四烯酸仍可显著降低细胞活力与细胞密度。电子显微镜观察显示，基础状态下即可检测到自噬体与自噬溶酶体等典型自噬结构，经脂肪酸处理后此类结构更为显著。结论：本研究结果有助于更深入地阐明人皮脂腺细胞中脂质生成与细胞死亡诱导的炎症相关分子机制，或可为揭示富含脂肪酸的人类饮食的相关影响提供新的思路。