二乙基二硫代氨基甲酸通过调控Perilipin 5改善肝细胞脂毒性损伤

摘要/Abstract

摘要： 目的探讨二乙基二硫代氨基甲酸(DDC)改善脂肪酸诱导肝细胞脂毒性损伤的分子机制。方法应用0.5 mmol棕榈酸(PA)诱导建立LO2细胞脂毒性损伤模型(PA组),同时加入300 μmol DDC处理(DDC+PA组),并设立相应对照组。Real-time PCR检测TNF-α、CHOP、ATF4和BAX基因表达;JC-1染色观察线粒体膜电位;Real-time PCR和Western blot检测脂滴蛋白Perilipin5(Plin5)基因与蛋白表达;脂滴和线粒体染色观察脂滴含量、线粒体-脂滴相互作用。应用siRNA干扰Plin5表达,观察Plin5敲低后DDC对线粒体-脂滴相互作用、炎症、内质网应激、凋亡相关基因及线粒体膜电位的影响。结果与对照组相比,PA组TNF-α、CHOP、ATF4及BAX表达均显著升高[(1.436 ± 0.447)比(37.762 ± 9.715)、 (1.005 ± 0.024) 比(20.892 ± 2.721)、(1.467 ± 0.332) 比(15.184 ± 7.006)、(2.095 ± 0.814 ) 比(29.480 ± 13.564)](P<0.05);DDC+PA组TNF-α、CHOP、ATF4及BAX表达较PA组降低[(37.762 ± 9.715) 比(2.890 ± 0.939)、(20.892 ± 2.721) 比(12.56 ± 4.175)、(15.184 ± 7.006) 比(3.41 ± 1.159)、(29.480 ± 13.564) 比(8.462 ± 5.441)](P<0.05)。与对照组相比,PA组线粒体膜电位降低[(2.341 ± 0.491) 比(0.805 ± 0.214)](P<0.05);DDC+PA组线粒体膜电位较PA组升高[(0.805 ± 0.214) 比(1.507 ± 0.292)](P<0.05)。与PA组相比,DDC+PA组Plin5表达升高[(1.567 ± 0.152) 比(3.807 ± 0.579)](P<0.05),脂滴含量及线粒体-脂滴相互接触面积显著增加[(3.123 ± 1.102) 比(9.795 ± 1.740)、(0.362 ± 0.100) 比(2.550 ± 0.521)](P<0.05)。敲低Plin5可阻断DDC对LO2细胞中线粒体-脂滴相互作用、TNF-α、CHOP、ATF4、BAX以及线粒体膜电位的作用(P<0.05)。结论 DDC通过上调Plin5促进线粒体-脂滴相互作用,改善PA诱导的肝细胞脂毒性损伤。

关键词: 二乙基二硫代氨基甲酸, 脂毒性, Perilipin 5, 线粒体, 代谢相关脂肪性肝病

Abstract: Objective To investigate the molecular mechanism of diethyldithiocarbamate (DDC) ameliorating fatty acid-induced lipotoxic injury of hepatocytes. Methods Lipotoxic model of LO2 cells was induced by 0.5mM palmitic acid (PA) (PA group), and treated with 300μM DDC (DDC+PA group) at the same time, while a corresponding control group was established. Quantitative real-time PCR was performed to measure the mRNA levels of TNF-α, CHOP, ATF4 and BAX. Mitochondrial membrane potential was assessed using JC-1 staining. Real-time PCR and Western blot were used to detect the expression of Perilipin5 (Plin5) gene and protein. The content of lipid droplets and their interaction with mitochondria were visualized through staining of lipid droplets and mitochondria. SiRNA interference was used to knockdown Plin5. The effects of DDC on mitochondrial-lipid droplet interaction, inflammation, endoplasmic reticulum stress and apoptosis-related genes, mitochondrial membrane potential, were observed after Plin5 knockdown. Results Compared with the control group, the expression of TNF-α, CHOP, ATF4 and BAX were increased in PA group [(1.436 ± 0.447) vs (37.762 ± 9.715), (1.005 ± 0.024) vs (20.892 ± 2.721), (1.467 ± 0.332) vs (15.184 ± 7.006), (2.095 ± 0.814 ) vs (29.480 ± 13.564)] (P<0.05). Compared with the PA group, the expression of TNF-α, CHOP, ATF4 and BAX were decreased in DDC+PA group [(37.762 ± 9.715) vs (2.890 ± 0.939), (20.892 ± 2.721) vs (12.56 ± 4.175), (15.184 ± 7.006) vs (3.41 ± 1.159), (29.480 ± 13.564) vs (8.462 ± 5.441)] (P<0.05). Compared with the control group, the mitochondrial membrane potential (MP) was decreased in PA group [(2.341 ± 0.491) vs (0.805 ± 0.214)] (P<0.05), whereas increased significantly in DDC+PA group compared with the PA group [(0.805 ± 0.214) vs (1.507 ± 0.292)] (P<0.05). Compared with the PA group, the expression of Plin5 was increasedin DDC+PA group [(1.567 ± 0.152) vs (3.807 ± 0.579)] (P<0.05). Compared with the PA group, the content of lipid droplet and the contact between mitochondria and lipid droplets were increased in the DDC+PA group [(3.123 ± 1.102) vs (9.795 ± 1.740), (0.362 ± 0.100) vs (2.550 ± 0.521)] (P<0.05). Plin5 knockdown blocked the effects of DDC on the interaction of mitochondria-lipid droplet, TNF-α, CHOP, ATF4, BAX, and mitochondrial membrane potential in LO2 cells (P<0.05). Conclusion DDC promotes mitochondria-lipid droplet interaction, and improves PA-induced lipotoxic injury of hepatocytes through regulating Plin5.

Key words: Diethyldithiocarbamate, Lipotoxicity, Perilipin 5, Mitochondrion, Metabolic dysfunction associated fatty liver disease

孙祥云, 于庆红, 齐一菲, 白世锦, 刘天会. 二乙基二硫代氨基甲酸通过调控Perilipin 5改善肝细胞脂毒性损伤[J]. 肝脏, 2025, 30(4): 537-541.

SUN Xiang-yun, YU Qing-hong, QI Yi-fei, BAI Shi-jin, LIU Tian-hui. Diethyldithiocarbamate improves lipotoxic injury of hepatocytes by regulating Perilipin 5[J]. Chinese Hepatolgy, 2025, 30(4): 537-541.

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