目的 探讨1,25(OH)2D3对油酸（oleic acid, OA）诱导的肝细胞脂质蓄积的改善作用,并从调节Ca2+稳态、缓解内质网应激（endoplasmic reticulum stress, ERS）途径阐明分子机制。方法 将HepG2细胞分为3组,对照组给予普通培养基,模型组给予1.5 mmol/L OA,干预组给予1.5 mmol/L OA+0.1 μmol/L 1,25(OH)2D3,处理24 h;油红O染色检测细胞脂质蓄积,甘油三酯（triacylglycerol, TG）试剂盒检测细胞TG水平,Fluo-4荧光探针检测细胞Ca²⁺水平,免疫荧光染色检测GRP78的表达水平,蛋白质免疫印迹法检测蛋白激酶R样内质网激酶（protein kinase r-like endoplasmic reticulum kinase, PERK）、磷酸化蛋白激酶R样内质网激酶（phosphorylated protein kinase r-like endoplasmic reticulum kinase, p-PERK）、真核翻译起始因子2α亚基（eukaryotic translation initiation factor 2 subunit alpha ,eIF2α）、磷酸化真核翻译起始因子2α亚基（phosphorylated eukaryotic translation initiation factor 2α subunit ,p-eIF2α）、固醇调节元件结合蛋白1c（sterol regulatory element-binding protein 1c,SREBP-1c）、乙酰辅酶A羧化酶（acetyl-CoA carboxylase ,ACC）和脂肪酸合成酶（fatty acid synthase, FAS）蛋白表达水平。结果 与对照组相比,OA组细胞内出现大量脂滴,同时TG水平显著升高,而1,25(OH)2D3干预后显著改善;进一步的分子机制研究结果显示,与模型组相比,1,25(OH)2D3干预组细胞中Ca2+水平显著回升,GRP78表达水平显著下降,同时ERS相关PERK/eIF2α信号通路及其调控的SREBP-1c/ACC/FAS脂肪酸合成信号通路显著下调。结论 1,25(OH)2D3可能通过调节细胞内Ca2+稳态,缓解ERS及其引起的脂代谢紊乱,改善OA诱导的HepG2细胞脂质蓄积。
Abstract
Objective To investigate the effects of 1,25(OH)2D3 on oleic acid (OA)-induced lipid accumulation in hepatocytes and elucidate the underlying mechanism by focusing on the regulation of calcium ion balance and the alleviation of endoplasmic reticulum stress (ERS). Methods HepG2 cells were divided into three groups: a control group cultured in normal medium, an OA group treated with 1.5 mmol/L OA, and an OA+VD group treated with 1.5 mmol/L OA and 0.1 μmol/L 1,25(OH)2D3. All treatments were maintained for 24 hours. Lipid accumulation was evaluated using Oil Red O staining. Cellular triglyceride (TG) levels were measured using a commercial assay kit. Intracellular calcium ion levels were measured with Fluo-4 fluorescent probe. To evaluate the expression and cellular localization of GRP78, immunofluorescence staining was performed. The protein expression levels of protein kinase r-like endoplasmic reticulum kinase (PERK), phosphorylated protein kinase r-like endoplasmic reticulum kinase(p-PERK), eukaryotic translation initiation factor 2 subunit Alpha (eIF2α),phosphorylated eukaryotic translation initiation factor 2α subunit (p-eIF2α), sterol regulatory element-binding protein 1c (SREBP-1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) were measured by Western blot. Results Compared with the control group, the OA group exhibited a significant increase in lipid droplet accumulation and a marked elevation in TG content. These pathological changes were significantly ameliorated in the OA+VD group. Mechanistic investigations revealed that the OA+VD group had a notably restored intracellular calcium ion balance and a significantly downregulated expression of GRP78. Meanwhile, the ERS-related PERK/eIF2α signaling pathway and its downstream fatty acid synthesis pathway involving SREBP-1c, ACC and FAS were significantly suppressed. Conclusion 1,25(OH)2D3 can alleviate OA-induced fat accumulation in HepG2 cells. The underlying mechanism is associated with the restoration of intracellular calcium homeostasis and inhibition of ERS.
关键词
1,25(OH)2D3 /
HepG2细胞 /
油酸 /
脂质蓄积 /
内质网应激
Key words
1,25(OH)2D3 /
HepG2 cells /
oleic acid /
lipid accumulation /
endoplasmic reticulum stress
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基金
国家重点研发计划项目(No．2024YFF1106000); 山西省应用基础研究(No．202303021211124); 山西省高等教育＂百亿工程＂科技引导专项
