Ⅲ型纤连蛋白结构域包含蛋白5抑制巨噬细胞焦亡的作用机制

Action mechanism by which fibronectin type III domain-containing protein 5 inhibits macrophage pyroptosis

背景:Ⅲ型纤连蛋白结构域包含蛋白5(fibronectin type Ⅲ domain-containing protein 5,FNDC5)是一种肌肉因子,具有调节糖脂代谢、抗炎、抗氧化及改善胰岛素抵抗等功能,并且具有调节多种细胞焦亡的能力。目的:探讨FNDC5对巨噬细胞焦亡的作用及潜在机制。方法:(1)构建FNDC5基因过表达和沉默慢病毒载体,将慢病毒载体转染至THP-1细胞株,通过观察细胞绿色荧光表达、qPCR和Western blot验证转染效果。(2)佛波酯诱导THP-1细胞分化为巨噬细胞,通过氧化性低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)构建巨噬细胞焦亡模型,分组:NC组、ox-LDL组、ox-LDL+MOCK1组、ox-LDL+Ov-FNDC5组、ox-LDL+MOCK2组和ox-LDL+shFNDC5组。(3)采用Hoechst 33342/PI荧光双染和乳酸脱氢酶释放实验评估细胞焦亡程度,采用qPCR和Western blot检测相关分子mRNA和蛋白表达水平,采用ELISA检测细胞上清液中白细胞介素1β和白细胞介素18释放水平。结果与结论:与ox-LDL+MOCK1组相比,过表达FNDC5可显著降低巨噬细胞焦亡率以及乳酸脱氢酶、白细胞介素1β、白细胞介素18释放水平,显著抑制NF-κB p65、NF-κB p50、NLRP3、ASC、Caspase-1及GSDMD的mRNA表达,显著抑制NF-κB p65、NF-κB p50、NLRP3、ASC、Cleaved Caspase-1及GSDMD-N的蛋白表达;与ox-LDL+MOCK2组相比,沉默FNDC5则出现相反结果。结果表明:FDNC5可能通过抑制NF-κB/NLRP3通路缓解巨噬细胞焦亡。

BACKGROUND:Fibronectin type III domain-containing protein 5(FNDC5)is a muscle factor that can regulate glucose and lipid metabolism,and exert antiinflammatory,anti-oxidative effects and improvement in insulin resistance.Moreover,FNDC5 could control various cell pyroptosis.OBJECTIVE:To explore the effect and potential mechanism of FNDC5 on macrophage pyroptosis.METHODS:(1)After completing the construction of the lentivirus virus overexpressing FNDC5 or silencing FNDC5,the THP-1 cells were transfected with the lentivirus vector.The result of transfection was detected by the expression of green fluorescence,qPCR,and western blot assay.(2)Phorbol ester induced THP-1 cells to differentiate into macrophages.Oxidized low-density lipoprotein(ox-LDL)was added to induce the cell pyroptosis model.There were six groups,i.e.,NC group,ox-LDL group,ox-LDL+MOCK1 group,ox-LDL+Ov-FNDC5 group,ox-LDL+MOCK2 group,and ox-LDL+shFNDC5 group.(3)The level of cell pyroptosis was evaluated by Hoechst 33342/propidium iodide fluorescence double staining and lactate dehydrogenase release assay.The expression levels of related molecules in THP-1 cells were analyzed by qPCR and western blot assay.The interleukin-18 and interleukin-1βin cell supernatant were detected by ELISA.RESULTS AND CONCLUSION:Compared with the ox-LDL+MOCK1 group,overexpression of FNDC5 significantly reduced the pyroptosis rate of macrophages and the release levels of lactate dehydrogenase,interleukin-1βand interleukin-18,significantly inhibited the mRNA expression of NF-κB p65,NF-κB p50,NLRP3,ASC,Caspase-1,and GSDMD,and significantly inhibited the protein expression of NF-κB p65,NF-κB p50,NLRP3,ASC,cleaved Caspase-1 and GSDMD-N.Compared with the ox-LDL+MOCK2 group,the silence of FNDC5 showed the opposite result.These findings suggest that FNDC5 attenuates pyroptosis in macrophages by inhibiting the NF-κB/NLRP3 pathway.