目的 探究二十碳五烯酸（eicosapentaenoic acid, EPA）和二十二碳六烯酸（docosahexaenoic acid, DHA）是否经肌源性白细胞介素-6（interleukin-6, IL-6）分别介导AMPK 和STAT3信号通路,改善3T3-L1脂肪细胞脂质代谢。方法 （1）用50、100、200、400 μmol/L EPA或DHA干预C2C12肌管细胞24 h。干预结束后,将肌管细胞与成熟脂肪细胞共培养24 h,RT-PCR检测肌管细胞IL-6、解整合素和金属蛋白酶10（A disintegrin and metalloproteinase 10,ADAM10）、解整合素和金属蛋白酶17（A disintegrin and metalloproteinase 17,ADAM17）、细胞因子信号转导抑制因子3（suppressors of cytokine signaling 3,SOCS3）mRNA表达,ELISA测定共培养液中IL-6分泌水平;（2）用IL-6 siRNA沉默肌管细胞,再用200 μmol/L的EPA或DHA干预肌管细胞,同时采用AMPK抑制剂Dorsomorphin（compound C）与 STAT3 抑制剂 FLLL32 预处理脂肪细胞4 h,再将处理后的肌管细胞和脂肪细胞共培养24 h。RT-PCR检测脂肪细胞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共激活物1α(PPARG coactivator 1 alpha, PGC-1α)、固醇调节元件结合蛋白-1c(sterol regulatory element binding protein-1c, SREBDP-1c)mRNA表达,Western blot检测脂肪细胞的IL-6、腺苷酸活化蛋白激酶（adenosine 5’-monophosphate-activatedproteinkinase,AMPK）和信号转导和转录激活因子3(signal transduction and activator of transcription 3, STAT3)蛋白的表达。结果 100、200、400 μmol/L EPA和200、400 μmol/L DHA可显著上调肌管细胞IL-6及ADAM10、ADAM17表达(P<0.01),仅DHA能提高SOCS3表达(P<0.05);除50 μmol/L DHA,其他浓度的EPA或DHA干预均显著升高共培养液IL-6的分泌(P<0.05或P<0.01);200 μmol/L EPA和DHA干预的肌管细胞分别显著提高脂肪细胞的p-STAT3/STAT3比值(P<0.05)和p-AMPK/AMPK比值(P<0.01);沉默肌管细胞IL-6后该效应消失(P>0.05);200 μmol/L EPA干预的肌管细胞显著上调脂肪细胞的ATGL、PGC-1α和SREBP-1c基因表达(P<0.05或P<0.01);沉默IL-6后,ATGL的上调被抑制(P<0.01),PGC-1α和SREBP-1c的表达无影响(P>0.05);单独使用FLLL32或联合使用IL-6 siRNA,脂肪细胞ATGL、PGC-1α和SREBP-1c的表达较干预组显著降低(P<0.05或P<0.01);200 μmol/L DHA干预的肌管细胞显著上调脂肪细胞的ATGL、HSL和PGC-1α基因表达(P<0.05或P<0.01),同时下调SREBP-1c表达(P<0.01);单独使用Compound C或联合使用IL-6 siRNA后,ATGL、HSL和PGC-1α表达有所降低(P<0.01),但SREBP-1c的表达较干预组显著升高(P<0.05或P<0.01)。结论 适宜浓度的EPA和DHA干预肌管细胞均显著促进肌源性IL-6分泌;EPA通过IL-6介导脂肪细胞STAT3通路促进脂质分解,DHA则通过IL-6激活AMPK通路促进脂质分解及氧化。
Abstract
Objective To investigate whether eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) improve lipid metabolism in 3T3-L1 adipocytes, by activating the AMPK and STAT3 signaling pathways via myogenic interleukin-6 (IL-6), respectively. Methods 1. C2C12 myotubes were treated with 50, 100, 200, or 400 μmol/L EPA or DHA for 24 h. After intervention, myotubes were co-cultured with mature adipocytes for 24 h. RT-PCR was used to detect mRNA expression of IL-6, ADAM10, ADAM17, and SOCS3 in myotubes. ELISA was performed to measure IL-6 content in the co-culture medium. 2. Myotubes were transfected with IL-6 siRNA, then treated with 200 μmol/L EPA or DHA. Meanwhile, adipocytes were pretreated for 4 h with the AMPK inhibitor dorsomorphin (compound C) or the STAT3 inhibitor FLLL32. The treated myotubes and adipocytes were then co-cultured for 24 h. RT-PCR was used to detect mRNA expression of ATGL, HSL, PGC-1α, and SREBP-1c in adipocytes. Western blot was performed to analyze protein expression of IL-6, AMPK, p-AMPK, STAT3, and p-STAT3 in adipocytes. Results EPA (100, 200, 400 μmol/L) and DHA (200, 400 μmol/L) significantly upregulated IL-6, ADAM10, and ADAM17 expression in myotubes (P<0.01). Only DHA increased SOCS3 mRNA expression (P<0.05). IL-6 secretion was significantly increased by all concentrations except 50 μmol/L DHA (P<0.05 or P<0.01). Myotubes treated with 200 μmol/L EPA or DHA specifically increased the p-STAT3/STAT3 ratio (P<0.05) and p-AMPK/AMPK ratio (P<0.01) in co-cultured adipocytes, respectively. This effect was abolished by IL-6 silencing (P>0.05). EPA-treated myotubes significantly upregulated ATGL, PGC-1α, and SREBP-1c expression in adipocytes (P<0.05 or P<0.01). IL-6 silencing inhibited ATGL upregulation (P<0.01) but did not affect the expression of PGC-1α and SREBP-1c. Inhibition with FLLL32 (alone or combined with IL-6 siRNA) significantly reduced the expression of ATGL, PGC-1α, and SREBP-1c compared to the EPA intervention group (P<0.05 or P<0.01). DHA-treated myotubes significantly upregulated ATGL, HSL, and PGC-1α (P<0.05 or P<0.01) while downregulating SREBP-1c (P<0.01) in adipocytes. Inhibition with compound C (alone or combined with IL-6 siRNA) significantly reduced ATGL, HSL, and PGC-1α expression (P<0.01) but increased SREBP-1c expression compared to the DHA intervention group (P<0.05 or P<0.01). Conclusion EPA and DHA at appropriate concentrations promote myogenic IL-6 secretion. EPA improves lipid metabolism primarily by activating the STAT3 pathway in adipocytes via IL-6, while DHA exerts its effects on lipid metabolism mainly through the IL-6-mediated AMPK pathway.
关键词
二十碳五烯酸 /
二十二碳六烯酸 /
脂质代谢 /
肌源性IL-6 /
AMPK/STAT3信号通路
Key words
eicosapentaenoic acid (EPA) /
docosahexaenoic acid (DHA) /
lipid metabolism /
myogenic IL-6 /
AMPK/STAT3 signaling pathway
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