芦丁促进3T3-L1前脂肪细胞棕色化及其机制

Rutin Promotes Browning of 3T3-L1 Preadipocytes and Its Mechanism

目的:观察芦丁对3T3-L1前脂肪细胞棕色化效应的影响,并探讨其机制。方法:细胞增殖与活性检测-8(CCK-8)法检测不同浓度芦丁(3.125、6.25、12.5、25、50、100、200μmol·L^(-1))对3T3-L1细胞活性影响,蛋白免疫印迹法(Western blot)检测不同浓度芦丁(12.5、25、50μmol·L^(-1))对脂肪细胞产热相关蛋白解偶联蛋白1(UCP1)、PR结构域蛋白16(PRDM16)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(PGC-1α)表达的影响。确定芦丁最佳浓度后,油红O染色观察芦丁对脂肪细胞中脂滴生成的影响,Western blot检测线粒体生物合成标志性蛋白核呼吸因子1(NRF1)、核呼吸因子2(NRF2)和线粒体转录因子A(TFAM)的表达。结果:与空白组比较,200μmol·L^(-1)芦丁显著抑制3T3-L1细胞活性(P<0.01);在12.5、25、50μmol·L^(-1)浓度下,50μmol·L^(-1)芦丁显著促进产热蛋白(UCP1、PRDM16、PGC-1α)表达(P<0.01),确定为最佳浓度。与空白组比较,芦丁50μmol·L^(-1)显著增加3T3-L1细胞线粒体UCP1蛋白的免疫荧光强度(P<0.01)及线粒体生物合成标志性蛋白NRF1、NRF2和TFAM的表达(P<0.01),显著抑制3T3-L1脂肪细胞脂滴生成(P<0.01)。结论:芦丁可抑制3T3-L1脂肪细胞脂滴沉积,增加产热相关蛋白(UCP1、PRDM16和PGC-1α)及线粒体生物合成标志性蛋白(NRF1、NRF2和TFAM)的表达,从而诱导3T3-L1脂肪细胞棕色化,为开发安全调节白色细胞棕色化的药物提供实验室基础。

Objective:To investigate the effect of rutin on the browning of 3T3-L1 preadipocytes and the mechanism. Method:Cell counting kit-8(CCK-8) assay was used to detect the effect of different concentration of rutin(3.125, 6.25, 12.5, 25, 50, 100, 200 μmol·L^(-1)) on 3T3-L1 cell activity, and Western blot to examine the effect of rutin(12.5, 25, 50 μmol·L^(-1)) on the expression of thermogenesis-associated proteins uncoupling protein 1(UCP1), PR domain containing 16(PRDM16) and peroxisome proliferatoractivated receptor γ coactivator-1α(PGC-1α) in adipocytes. After the optimal concentration of rutin was determined, the effect of rutin on lipid droplet formation in adipocytes was observed based on oil red O staining, and the expression of nuclear respiratory factor 1(NRF1), nuclear respiratory factor 2(NRF2) and mitochondrial transcription factor A(TFAM), which were the landmark proteins of mitochondrial biosynthesis, was detected by Western blot. Result:Compared with the blank group, 200 μmol·L^(-1)rutin inhibited 3T3-L1cell activity(P<0.01). Compared with the blank group, at the concentration of 12.5, 25, 50 μmol·L^(-1)rutin significantly promoted the expression of thermogenesis-associated proteins(UCP1, PRDM16, and PGC-1α)(P<0.01), which was determined as the optimal concentration. Compared with the blank group, 50 μmol·L^(-1)rutin significantly increased the immunofluorescence intensity of mitochondrial UCP1 protein in 3T3-L1 cells(P<0.01) and the expression of the markers of mitochondrial biosynthesis(NRF1, NRF2, and TFAM)(P<0.01). In addition, 50 μmol·L^(-1)rutin significantly inhibited lipid droplet formation of 3T3-L1 adipocytes(P<0.01). Conclusion:Rutin inhibited lipid droplet deposition in 3T3-L1 adipocytes and increased the expression of thermogenesis-related proteins(UCP1, PRDM16, and PGC-1α) and markers of mitochondrial biosynthesis(NRF1, NRF2, and TFAM), thereby inducing the browning of 3T3-L1 adipocytes. This lays a basis for the development of drugs that safely regulate the browning of white cells.