Sulfur mustard analog 2-chloroethyl ethyl sulfide increases triglycerides by activating DGAT1-dependent biogenesis and inhibiting PGC1ɑ-dependent fat catabolism in immortalized human bronchial epithelial cells

Using sulfur mustard analog 2-chloroethyl ethyl sulfide (CEES), we established an in vitro model by poisoning cultured immortalized human bronchial epithelial cells. Nile Red staining revealed lipids accumulated 24 h after a toxic dose of CEES (0.9 mM). Lipidomics analysis showed most of the increased lipids were triglycerides (TGs), and the increase in TGs was further confirmed using a Triglyceride-Glo™ Assay kit. Protein and mRNA levels of DGAT1, an important TG biogenesis enzyme, were increased following 0.4 mM CEES exposure. Under higher dose CEES (0.9 mM) exposure, protein and mRNA levels of PPARγ coactivator-1ɑ (PGC-1ɑ), a well-known transcription factor that regulates fatty acid oxidation, were decreased. Finally, application with DGAT1 inhibitor A 922500 or PGC1ɑ agonist ZLN005 was able to block the CEES-induced TGs increase. Overall, our dissection of CEES-induced TGs accumulation provides new insight into energy metabolism dysfunction upon vesicant exposure.HIGHLIGHTS
In CEES (0.9 mM)-injured cells:
Triglycerides (TGs) were abundant in the accumulated lipids.
Expression of DGAT1, not DGAT2, was increased.
Expression of PGC1ɑ, not PGC1β, was reduced.
DGAT1 inhibitor or PGC1ɑ agonist blocked the CEES-mediated increase in TGs.

In CEES (0.9 mM)-injured cells:
Triglycerides (TGs) were abundant in the accumulated lipids.
Expression of DGAT1, not DGAT2, was increased.
Expression of PGC1ɑ, not PGC1β, was reduced.
DGAT1 inhibitor or PGC1ɑ agonist blocked the CEES-mediated increase in TGs.

本研究以芥子气类似物2-氯乙基乙基硫醚（2-chloroethyl ethyl sulfide, CEES）为工具，通过染毒体外培养的永生化人支气管上皮细胞，构建了体外损伤模型。尼罗红（Nile Red）染色结果显示，给予0.9 mM毒性剂量的CEES处理24小时后，细胞内出现脂质蓄积。脂质组学分析表明，丰度升高的脂质以甘油三酯（triglycerides, TGs）为主，该结果通过Triglyceride-Glo™ Assay试剂盒得到进一步验证。经0.4 mM CEES暴露后，甘油三酯生物合成关键酶DGAT1的蛋白及mRNA表达水平均显著上调。而在更高剂量0.9 mM CEES暴露下，调控脂肪酸氧化的经典转录因子PPARγ辅激活因子1α（PPARγ coactivator-1ɑ, PGC-1ɑ）的蛋白及mRNA表达水平则显著下调。最后，使用DGAT1抑制剂A 922500或PGC-1ɑ激动剂ZLN005处理细胞，均可阻断CEES诱导的甘油三酯蓄积。综上，本研究对CEES诱导的甘油三酯蓄积机制的解析，为糜烂性毒剂暴露后能量代谢功能紊乱的研究提供了新的视角。

研究亮点：
1. 在0.9 mM CEES损伤的细胞中，蓄积脂质以甘油三酯（TGs）为主要成分；
2. 仅DGAT1的表达水平上调，而非DGAT2；
3. 仅PGC-1ɑ的表达水平下调，而非PGC1β；
4. DGAT1抑制剂或PGC-1ɑ激动剂可阻断CEES介导的甘油三酯蓄积。