Department of Endocrinology, Yangpu Hospital, Tongji University School of Medicine, 450 Tengyue Road, Yangpu, Shanghai, 200090, China.
Department of Cardiac Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, 200072, China.
Inflamm Res. 2021 May;70(5):591-603. doi: 10.1007/s00011-021-01463-0. Epub 2021 Apr 23.
FOXO3a is a widely studied transcription factor and plays an important role in a variety of biology. The purpose of this study was to explore the role and potential mechanism of FOXO3a on lipid accumulation and adipocyte inflammation in adipocytes through regulation of autophagy.
The obese mouse model was successfully induced by high-fat diet. SiRNA targeting FOXO3a was transfected into differentiation of 3T3-L1 adipocytes to reduce the expression of FOXO3a. The culture medium of RAW264.7 cells was added to the differentiated 3T3-L1 adipocytes to form a co-culture system. Subsequently, ELISA or AdipoRed assay was performed to measure the expression of triglyceride (TG) and cholesterol (TC) in mouse adipose tissue or differentiation of 3T3-L1 adipocytes. Adipocyte differentiation was detected by Oil Red O-staining. Ad-mCherry-GFP-LC3II was used to detect the level of autophagy in differentiation of 3T3-L1 adipocytes. Western blotting or qRT-PCR was used to detect the expression of FOXO3a, autophagy-related proteins (beclin 1, CEBPβ, PPARγ, ACC1 and KLF4), inflammatory cytokines (TNF-α, IL-1β, IL-6 and MCP1), NF-κB signal pathway-related proteins or adipokines (Adiponectin, AdipoR1 and resistin) in differentiated 3T3-L1 or RAW264.7 cells.
The expression of FOXO3a and autophagy levels were significantly increased in visceral adipose tissue of obese mice and differentiation of 3T3-L1 adipocytes. Downregulation of FOXO3a significantly inhibited the autophagy and lipid accumulation in differentiation of 3T3-L1 adipocytes. In addition, FOXO3a knockdown significantly reduced Lipopolysaccharide (LPS)-induced inflammation and adipokines release in RAW264.7 cells treated with the culture medium of 3T3-L1 adipocytes. These above activity changes could be reversed by autophagy inducer rapamycin.
FOXO3a could promote lipid accumulation and inflammation in differentiated 3T3-L1 adipocytes by targeting autophagy. Our results provide a new theoretical basis for FOXO3a to regulate obesity.
FOXO3a 是一种广泛研究的转录因子，在各种生物学中发挥重要作用。本研究旨在通过调节自噬来探索 FOXO3a 对脂肪细胞中脂质积累和脂肪细胞炎症的作用和潜在机制。
通过高脂肪饮食成功诱导肥胖小鼠模型。将靶向 FOXO3a 的 siRNA 转染至 3T3-L1 脂肪细胞分化中，以降低 FOXO3a 的表达。将 RAW264.7 细胞的培养基加入分化的 3T3-L1 脂肪细胞中，形成共培养系统。随后，通过 ELISA 或 AdipoRed 测定法测定小鼠脂肪组织或分化的 3T3-L1 脂肪细胞中甘油三酯（TG）和胆固醇（TC）的表达。通过油红 O 染色检测脂肪细胞分化。使用 Ad-mCherry-GFP-LC3II 检测分化的 3T3-L1 脂肪细胞中的自噬水平。通过 Western blot 或 qRT-PCR 检测分化的 3T3-L1 或 RAW264.7 细胞中 FOXO3a、自噬相关蛋白（beclin 1、CEBPβ、PPARγ、ACC1 和 KLF4）、炎症细胞因子（TNF-α、IL-1β、IL-6 和 MCP1）、NF-κB 信号通路相关蛋白或脂肪因子（脂联素、AdipoR1 和抵抗素）的表达。
肥胖小鼠内脏脂肪组织和 3T3-L1 脂肪细胞分化中 FOXO3a 的表达和自噬水平均显著升高。下调 FOXO3a 可显著抑制 3T3-L1 脂肪细胞分化中的自噬和脂质积累。此外，FOXO3a 敲低可显著降低 LPS 诱导的培养基处理的 RAW264.7 细胞中的炎症和脂肪因子释放。这些活性变化可被自噬诱导剂雷帕霉素逆转。
FOXO3a 可通过靶向自噬促进分化的 3T3-L1 脂肪细胞中的脂质积累和炎症。我们的结果为 FOXO3a 调节肥胖提供了新的理论依据。
