目的 探讨二十二碳五烯酸（eicosapentaenoic acid, EPA）或二十碳六烯酸（docosahexaenoic acid, DHA）干预的C2C12肌管细胞对脂肪细胞脂质代谢功能的改善作用。方法 用50、100、200、400 μmol/L EPA或DHA干预肌管细胞24 h,同时设立对照组,干预结束后,撤掉干预液,将干预后的肌管细胞在transwell小室中与成熟的脂肪细胞共培养24 h。用油红O定量脂肪细胞内的脂质蓄积情况,用PCR法测定脂肪细胞的脂肪甘油三酯脂肪酶（adipose triglyceride lipase, Atgl）、激素敏感性脂肪酶（hormone-sensitive lipase, Hsl）、过氧化物酶体增殖物激活受体γ的共激活物 1α（PPARG coactivator 1 alpha, Pgc-1α）、解偶联蛋白-1（uncoupling protein-1, Ucp-1）和固醇调节元件结合蛋白-1c（sterol regulatory element binding protein-1c, Srebp-1c）基因mRNA表达水平,用Western blot检测脂肪细胞腺苷酸活化蛋白激酶（adenosine 5’-monophosphate (AMP)-activated protein kinase, AMPK）与信号转导和转录激活因子3（signal transduction and activator of transcription 3, STAT3）信号通路的蛋白表达。结果 经100、200、400 μmol/L EPA干预的肌管细胞可以显著降低脂肪细胞的脂质蓄积（P<0.05或P<0.001）,而DHA干预后的肌管细胞则没有显著的改善脂质蓄积的作用;经一定浓度的EPA或DHA干预的肌管细胞可以显著上调脂肪细胞脂肪分解基因Atgl和Hsl的表达（P<0.05或P<0.01）;经一定浓度的EPA或DHA干预的肌管细胞可以显著提高脂质氧化基因Ucp-1和Pgc-1α的表达（P<0.05或P<0.01）;经一定浓度EPA干预的肌管细胞可以显著提升脂质合成基因Srebp-1c的表达（P<0.05或P<0.01）,而一定浓度DHA干预的肌管细胞则是可以显著降低脂质合成基因Srebp-1c的表达（P<0.01）;经所有浓度EPA干预的肌管细胞可以显著提高脂肪细胞p-STAT3/STAT3的比值（P<0.01）,对p-AMPK/AMPK比值变化的影响则没有显著性;经200、400 μmol/L DHA干预的肌管细胞可以显著提高脂肪细胞p-AMPK/AMPK的比值（P<0.01）,但是p-STAT3/STAT3比值的变化没有显著性意义。结论 EPA和DHA在适宜浓度均可以促进肌管细胞提高脂肪细胞的脂质分解和脂质氧化,EPA干预的肌管细胞还可以一定程度提高脂质合成,DHA干预的肌管细胞可以一定程度降低脂质合成;EPA干预后的肌管细胞可能通过STAT3通路改善脂质代谢,DHA干预后的肌管细胞可能通过AMPK通路改善脂质代谢。
Abstract
Objective To explore the improvement effects of C2C12 myotube cells pre-treated with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on lipid metabolism of 3T3-L1 adipocytes. Methods Myotube cells were pre-treated with 50, 100, 200, 400 μmol/L EPA or DHA for 24 hours, and a control group was set up at the same time. Myotube cells were co-cultured with mature adipocytes in the transwell chamber for 24 hours after the treatment. Oil red O staining was used to quantify lipid accumulation in the adipocytes, and PCR was used to determine the expressions of lipid metabolism genes adipose triglyceride lipase (Atgl), hormone-sensitive lipase (Hsl), PPARG coactivator 1 alpha (Pgc-1α), Uncoupling protein-1 (Ucp-1) and Sterol regulatory element binding protein-1c (Srebp-1c). Western blot was used to detect the protein expression of adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) and signal transduction and activator of transcription 3 (STAT3) signaling pathways. Results Myotube cells pre-treated with 100, 200, and 400 μmol/L EPA significantly reduced lipid accumulation in the adipocytes (P<0.05 or P<0.001), while myotube cells pre-treated with DHA showed no improvement of lipid accumulation. EPA or DHA at certain concentrations can significantly upregulated the expressions of adipocyte lipolysis genes Atgl and Hsl and lipid oxidation genes Ucp-1 and Pgc-1α (P<0.05 or P<0.01). Myotube cells pre-treated with certain concentrations of EPA significantly increased the expression of the lipid synthesis gene Srebp-1c (P<0.05 or P<0.01), while myotube cells pre-treated with certain concentrations of DHA significantly reduced the expression of the lipid synthesis gene Srebp-1c(P<0.01 or P<0.001). Myotube cells pre-treated with EPA significantly increased the p-STAT3/STAT3 ratio, but had no significant effect on the p-AMPK/AMPK ratio. Myotube cells pre-treated with 200 and 400 μmol/L DHA significantly increased the p-AMPK/AMPK ratio, but had no significant effect on the p-STAT3/STAT3 ratio. Conclusion At appropriate concentrations, both EPA and DHA can activate myotube cells to improve lipid decomposition and lipid oxidation of the adipocytes. Myotube cells pre-treated with EPA can increase lipid synthesis to a certain extent through the STAT3 pathway, and those pre-treated with DHA can reduce lipid synthesis to a certain extent through the AMPK pathway.
关键词
二十碳五烯酸 /
二十二碳六烯酸 /
C2C12肌管细胞 /
3T3-L1脂肪细胞 /
脂质代谢 /
STAT3/AMPK信号通路 /
细胞共培养
Key words
eicosapentaenoic acid (EPA) /
docosahexaenoic acid (DHA) /
C2C12 myotubes /
3T3-L1 adipocytes /
lipid metabolism /
STAT3/AMPK signaling pathway /
co-culture
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