海参脑苷脂对前脂肪细胞分化的抑制作用及机制

Inhibitory Effect and Mechanism of Sea Cucumber Cerebroside on the Differentiation of Preadipocytes

目的:探究海参脑苷脂在3T3-L1小鼠脂肪细胞增殖以及分化过程中发挥的作用,并研究其具体机理。方法:运用噻唑蓝法探究海参脑苷脂在3T3-L1细胞正常生长过程中发挥的作用;并通过乳酸脱氢酶实验探究海参脑苷脂对前脂肪细胞是否存在毒性作用;诱导3T3-L1细胞为成熟细胞,采用油红O染色法以及测定成熟脂肪细胞中甘油三酯的含量来探究海参脑苷脂是否会改变脂肪细胞的脂质积累情况;反转录荧光实时定量聚合酶链式反应(reverse transcription real-time polymerase chain reaction,qRT-PCR)测定过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor gamma,PPARγ)、CCAAT/增强子结合蛋白α(CCAAT/enhancer binding protein alpha,C/EBPα)以及Wnt/β-catenin通路相关基因的表达水平。通过Western blotting法测定β-catenin蛋白向核内转移的情况。结果:海参脑苷脂能极显著抑制3T3-L1细胞的增殖以及分化(P<0.01),且对细胞无毒性作用。qRT-PCR结果显示,海参脑苷脂可极显著抑制脂肪细胞PPARγ和C/EBPα以及Wnt/β-catenin通路相关基因的表达(P<0.01)。Western blotting结果表明,海参脑苷脂可以极显著提高脂肪细胞核内β-catenin蛋白的表达水平(P<0.01)。结论:海参脑苷脂可以明显地抑制3T3-L1细胞的增殖以及分化,产生这种效果的机理和转录因子PPARγ、C/EBPα以及Wnt/β-catenin通路相关。

This study aimed to observe the effect of sea cucumber cerebroside on the proliferation and differentiation of 3T3- L1 preadipocytes and to explore the underlying mechanism. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to explore the effect of cerebroside on the proliferation of preadipocytes. 3T3-L1 preadipocytes were induced into mature adipocytes by the traditional cocktail method, and oil red O staining and triglyceride content of mature adipocytes were used to evaluate whether sea cucumber cerebroside could change lipid accumulation in adipocytes. The mRNA expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα) genes and those associated with the Wnt/β-catenin signaling pathway were measured by reverse transcription real-time polymerase chain reaction (qRT-PCR). The transfer of β-catenin protein to the nucleus was measured by Western blotting. Sea cucumber cerebroside could significantly inhibit the proliferation and differentiation of 3T3-L1 preadipocytes without cytotoxicity (P < 0.01). The results of qRT-PCR showed that the expression of PPARγ and C/EBPα genes and those associated with the Wnt/β-catenin signaling pathway were significantly inhibited by sea cucumber cerebroside. Western blotting results showed that sea cucumber cerebroside could significantly increase the content of β-catenin protein in nuclei (P < 0.01). In conclusion, sea cucumber cerebroside can inhibit the proliferation and differentiation of 3T3-L1 preadipocytes. The mechanism is related to the transcription factors PPARγ, C/EBPα and the Wnt/β-catenin signaling pathway.