补肾活血方靶向Nrf2介导的铁死亡调控3T3-L1细胞成脂分化功能障碍的研究

Study of Bushen Huoxue Formula modulate iron overload induced adipogenic differentiation dysfunction of 3T3-L1 cells by targeting Nrf2 mediated ferroptosis

目的 探讨补肾活血方对铁过载小鼠前脂肪细胞株(3T3-L1)成脂分化功能的影响及作用机制。方法 连续7 d给45只SD大鼠灌胃补肾活血方(15 g/kg)以制备补肾活血方含药血清。将3T3-L1细胞分为正常对照组、空载体对照组、模型组、铁过载组、Nrf2基因沉默组。采用3T3-L1细胞,使用慢病毒颗粒转染构建核因子E2相关因子-2(Nrf2)基因沉默3T3-L1细胞(3T3-L1 shNrf2)与空载体对照3T3-L1细胞(3T3-L1 shCtrl)。使用柠檬酸铁铵(FAC)构建3T3-L1铁过载细胞模型,并筛选出最优浓度。正常对照组与空载体对照组不予干预,模型组给予FAC,铁过载组和Nrf2基因沉默组给予FAC+补肾活血方含药血清。采用CCK-8法、油红O染色法分别检测各组细胞的存活率及脂质沉积情况。采用实时荧光PCR法检测过氧化物酶体增殖物激活受体(PPARγ)、激素敏感脂肪酶(HSL)、脂肪甘油三酯脂肪酶(ATGL)mRNA的表达量。采用酶联免疫吸附剂测定(ELISA)法检测细胞上清液中还原型谷胱甘肽(GSH)和脂质过氧化物(LPO)的含量。采用蛋白质印迹法检测转铁蛋白受体(TFR)、铁蛋白轻链(FTL)、铁蛋白重链(FTH)、铁调节蛋白(IRP)、谷胱甘肽过氧化物酶4(GPX4)、胱氨酸/谷氨酸反向转运体(XCT)、血红素氧合酶1(HO-1)、Nrf2的蛋白表达量。结果 与空载体对照组比较,Nrf2基因沉默组中3T3-L1细胞的Nrf2的表达水平降低(P<0.01),表明3T3-L1 shNrf2构建成功。并确定15μmol/L为FAC的最优浓度。与空载体对照组比较,模型组的细胞存活率降低(P<0.01),甘油三酯相对含量增加(P<0.01)、细胞内脂质沉积增加,PPARγ、HSL和ATGL mRNA的表达量均降低(P<0.01),GSH和LPO含量均降低(P<0.01),Nrf2、IRP的蛋白表达量均增加(P<0.05,P<0.01),TFR、FTL、GPX4、XCT、FTH、HO-1的蛋白表达量均降低(P<0.01)。与模型组比较,铁过载组的细胞存活率增加(P<0.05),甘油三酯相对含量降低(P<0.01)、细胞内脂质沉积降低,PPARγ、HSL和ATGL mRNA的表达量均增加(P<0.05),GSH和LPO含量增加(P<0.05),IRP蛋白表达量降低(P<0.05),Nrf2、TFR、FTL、GPX4、XCT、FTH、HO-1蛋白表达量均增加(P<0.05,P<0.01)。与铁过载组比较,Nrf2基因沉默组的细胞存活率降低(P<0.05),甘油三酯相对含量增加(P<0.05)、细胞内脂质沉积增加,PPARγ、HSL和ATGL mRNA的表达量均降低(P<0.05),GSH和LPO含量均降低(P<0.05),IRP蛋白表达量增加(P<0.05),Nrf2、TFR、FTL、GPX4、XCT、FTH、HO-1蛋白表达量均降低(P<0.05,P<0.01)。结论 补肾活血方可以有效保护3T3-L1细胞免受铁过载诱导的氧自由基损伤与细胞铁死亡,有利于脂肪细胞的增殖,并且可以抑制其终末成熟。

Objective We aimed to investigate the effects and the underlying mechanism of Bushen Huoxue Formula(BSHXF)on the adipogenic differentiation function of an iron overload(IO)mouse preadipocyte strain(3T3-L1 cells)and the underlying mechanism.Methods In total,45 Sprague-Dawley rats were given BSHXF(15 g/kg)by gavage for 7 days to prepare the medicated serum of BSHXF.3T3-L1 cells were divided into the normal group,the shCtrl group,the model group,the BSHXF+IO shCtrl group and the BSHXF+IO shNrf2 group.3T3-L1 cells were transfected with lentivirus particles to construct nuclear factor E2 related factor-2(Nrf2)silenced 3T3-L1 cells(3T3-L1 shNrf2)and empty vector control 3T3-L1 cells(3T3-L1 shCtrl).Ferric ammonium citrate(FAC)was used to establish the 3T3-L1 IO cell model,and the optimal FAC concentration was determined.The normal group and the shCtrl group were not treated.The model group was given FAC,the BSHXF+IO shCtrl group and the BSHXF+IO shNrf2 group were given FAC+BSHXF medicated serum.The cell survival rate and lipid deposition of each group were detected by the CCK-8 assay and Oil red O staining.The mRNA expression levels of peroxisome proliferator-activated receptor gamma(PPARγ),hormonesensitive triglyceride lipase(HSL)and adipose triglyceride lipase(ATGL)were detected by real-time PCR.The levels of glutathione reduced(GSH)and lipid peroxidation(LPO)in cell supernatant were detected by ELISA.The protein expression levels of transferrin receptor(TFR),ferritin light chain(FTL),ferritin heavy chain(FTH),iron regulatory protein(IRP),glutathione peroxidase 4(GPX4),cystine/glutamate antiporter system(XCT),heme oxygenase 1(HO-1)and Nrf2 were detected by Western blotting.Results Compared with the shCtrl group,the expression level of Nrf2 in the 3T3-L1 cells in the shNrf2 group was decreased(P<0.01),indicating that the establishment was successful.The optimal concentration of FAC was determined to be 15μmol/L.Compared with the shCtrl group,the cell survival rate of the model group was decreased(P<0.01),the relative content of triglyceride was increased(P<0.01),intracellular lipid deposition was increased,the mRNA expression levels of PPARγ,HSL and ATGL were decreased(P<0.01),the levels of GSH and LPO were decreased(P<0.01),the protein expression levels of Nrf2 and IRP were increased(P<0.05,P<0.01),and the protein expression levels of TFR,FTL,GPX4,XCT,FTH and HO-1 were decreased(P<0.01).Compared with the model group,the cell survival rate of the BSHXF+IO shCtrl group was increased(P<0.05),the relative content of triglyceride was decreased(P<0.01),intracellular lipid deposition was decreased,the mRNA expression levels of PPARγ,HSL and ATGL were increased(P<0.05),the levels of GSH and LPO were increased(P<0.05),the protein expression level of IRP was decreased(P<0.05)and the protein expression levels of Nrf2,TFR,FTL,GPX4,XCT,FTH and HO-1 were increased(P<0.05,P<0.01).Compared with the BSHXF+IO shCtrl group,the cell survival rate of the BSHXF+IO shNrf2 group was decreased (P<0. 05), the relative content of triglyceride was increased(P<0. 05), intracellular lipid deposition was increased, the mRNA expression levels of PPARγ, HSLand ATGL were decreased (P <0. 05), the levels of GSH and LPO were decreased (P <0. 05), theprotein expression level of IRP was increased (P <0. 05), and the protein expression levels of Nrf2,TFR, FTL, GPX4, XCT, FTH and HO-1 were decreased (P<0. 05, P<0. 01). Conclusion BSHXFcan effectively protect 3T3-L1 cells from oxygen free radical damage and ferroptosis induced by IO, whichbenefits the proliferation of adipocytes, and can inhibit terminal maturation.