Purpose

The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC).

Methods

HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function.

Results

EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation.

Conclusions

EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.

中文翻译：

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二十碳五烯酸 (EPA) 激活 PPARγ 信号传导，导致人类睑板腺上皮细胞 (hMGEC) 中的细胞周期退出、脂质积累和自噬。

目的

本研究的目的是了解天然 PPAR 激动剂二十碳五烯酸 (EPA) 激活 PPAR γ (γ) 信号传导导致人睑板腺上皮细胞 (hMGEC) 中睑板细胞分化的能力。

方法

HMGEC 单独暴露于 EPA，并与特定的 PPARγ 拮抗剂 T0070907 联合使用，以选择性阻断 PPARγ 信号传导。通过qPCR评估PPARγ反应基因的表达。使用 Ki-67 标记和蛋白质印迹评估对细胞周期的影响。在分化过程中，使用自噬串联传感器 (ATS) 和 LysoTracker 监测自噬。脂质积累的特点是受激拉曼散射显微镜 (SRS) 和中性脂质染色结合 ER-Tracker、LysoTracker 和 ATS。还使用蛋白质印迹研究了自噬。进行 Seahorse XF 分析以监测线粒体功能。

结果

EPA 通过减少细胞周期蛋白 D1 表达和增加 p21 和 p27 表达来特异性上调与脂质合成和诱导细胞周期退出相关的基因的表达。EPA 还通过特定的 PPARγ 信号传导以时间和剂量依赖性方式（P < 0.05）诱导脂滴的积累。脂质分析确定了脂质的从头合成和细胞外转运，以形成定位于 ER 的脂滴。PPARγ 信号传导还诱导 AMPK-ULK1 信号传导和自噬的激活，而自噬的抑制诱导线粒体危机，对脂质积累没有影响。

结论

EPA 通过 PPARγ 激活诱导 meibocyte 分化，其特征在于细胞周期退出、从头和转运脂质在 ER 中的积累以及自噬。