SFN通过NFE2L2信号通路缓解FFA诱导的肉牛单核巨噬细胞氧化损伤及凋亡

SFN alleviates FFA-induced oxidative damage and apoptosis in beef cattle mononuclear macrophages through NFE2L2 signaling pathway

旨在探讨游离脂肪酸(free fatty acid,FFA)是否通过核转录因子E2相关因子2(nuclear factor E2-reated factor 2,NFE2L2)信号通路诱导肉牛单核巨噬细胞的氧化应激。采牛颈静脉血,体外分离培养肉牛单核巨噬细胞,用不同浓度的FFA(0、0.3、0.6和1.2 mmol/L)处理肉牛单核巨噬细胞24 h;用10μmol/L的萝卜硫素(sulforaphane,SFN)预处理细胞24 h后加入1.2 mmol/L FFA再处理24 h。使用各类试剂盒分别检测活性氧(reactive oxygen species,ROS)和脂质过氧化物丙二醛(malondialdehyde,MDA)的含量及超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的活性;通过q-PCR对Caspase-3、Caspase-9、NFE2L2及其下游血红素加氧酶1(heme oxyenase-1,HMOX1)和醌氧化还原酶(NAD(P)H quinone oxidoreductase 1,NQO1)的基因表达水平进行定量分析;使用流式细胞仪测定细胞凋亡率。结果显示,与对照组相比,不同浓度FFA处理细胞后,ROS和MDA的含量呈浓度依赖性升高(P<0.05),SOD和GSH-Px的活性逐渐下降(P<0.05);Caspase-3和Caspase-9基因相对表达量增加,细胞凋亡率显著增加;NFE2L2、HMOX1和NQO1的mRNA水平显著降低。与FFA+DMSO组相比,FFA+SFN组的NFE2L2 mRNA表达量显著升高(P<0.05),HMOX1、NQO1的mRNA表达水平显著升高(P<0.05);ROS和MDA的含量显著降低(P<0.05),SOD和GSH-Px活性显著升高(P<0.05)。与FFA+DMSO组相比,FFA+SFN组细胞凋亡率显著降低。结果表明,SFN通过激活NFE2L2信号通路缓解FFA诱导的肉牛单核巨噬细胞氧化应激和凋亡。本研究为探明高FFA导致的肉牛单核巨噬细胞氧化损伤和凋亡的治疗靶点提供了理论依据。

To investigate whether free fatty acid(FFA)induced oxidative stress of mononuclear macrophages in beef cattle through nuclear factor E2-related factor 2(NFE2L2)signaling pathway.Beef cattle mononuclear macrophages were isolated from blood and treated with different concentrations of FFA(0,0.3,0.6 or 1.2 mmol/L)for 24 h.Cells were pretreated with 10μmol/L sulforaphane(SFN)for 24 h and then treated with 1.2 mmol/L FFA for another 24 h.The contents of reactive oxygen species(ROS)and lipid peroxide malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by various kits.The gene expression level of Caspase-3,Caspase-9,NFE2L2,Heme oxyase-1(HMOX1)and NAD(P)H quinone oxidoreductase 1(NQO1)was quantitatively analyzed by q-PCR.The apoptosis rate was determined by flow cytometry.The results showed that compared with the control group,with the increase of FFA concentration,the contents of ROS and MDA increased in a concentration-dependent manner(P<0.05),while the activities of SOD and GSH-Px decreased gradually(P<0.05).Compared with the control group,after FFA treatment,the relative expressions of Caspase-3and Caspase-9 were increased,the apoptosis rate was significantly increased,the mRNA levels of NFE2L2 and its downstream genes HMOX1 and NQO1 were significantly decreased.Compared with FFA+DMSO group,the mRNA expression level of NFE2L2 in FFA+SFN group was significantly increased(P<0.05),and the mRNA expression levels of HMOX1 and NQO1 were significantly increased(P<0.05).Compared with the FFA+DMSO group,the contents of ROS and MDA in FFA+SFN group were significantly decreased(P<0.05),while the activities of SOD and GSH-Px were significantly increased(P<0.05).Compared with the FFA+DMSO group,the apoptosis rate in FFA+SFN group was significantly decreased.The results showed that SFN protected beef cattle mononuclear macrophages from FFA-induced oxidative stress and apoptosis by activating NFE2L2 signaling pathway.This study provides a theoretical basis for exploring therapeutic targets of oxidative damage and apoptosis of mononuclear macrophages in beef cattle induced by high FFA.