中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (25): 4005-4012.doi: 10.12307/2024.196

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

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

赵广建，刘大男，周  博，王  尧   

  1. 贵州医科大学附属医院心内科，贵州医科大学医学科学研究所，贵州省贵阳市   550004
  • 收稿日期:2023-07-24 接受日期:2023-09-07 出版日期:2024-09-08 发布日期:2023-11-23
  • 通讯作者: 刘大男，博士，教授，主任医师，硕士、博士生导师，贵州医科大学附属医院心内科，贵州医科大学医学科学研究所，贵州省贵阳市 550004
  • 作者简介:赵广建，男，1994年生，汉族，贵州医科大学在读硕士，主要从事动脉粥样硬化形成机制研究。
  • 基金资助:
    国家自然科学基金项目(81660083)，项目负责人：刘大男；贵州省科技创新人才团队项目[黔科合平台人才(2020)5014]，项目负责人：刘大男；贵州省“百”层次创新型人才培养计划项目(黔科合人才(2015)4026号)，项目负责人：刘大男；贵州医科大学国家自然科学基金培育项目(TJ20073)，项目负责人：刘大男

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

Zhao Guangjian, Liu Danan, Zhou Bo, Wang Yao   

  1. Department of Cardiology, Affiliated Hospital of Guizhou Medical University, Institute of Medical Science, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Received:2023-07-24 Accepted:2023-09-07 Online:2024-09-08 Published:2023-11-23
  • Contact: Liu Danan, MD, Professor, Chief physician, Master’s/Doctoral supervisor, Department of Cardiology, Affiliated Hospital of Guizhou Medical University, Institute of Medical Science, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Zhao Guangjian, Master candidate, Department of Cardiology, Affiliated Hospital of Guizhou Medical University, Institute of Medical Science, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 81660083 (to LDN); Guizhou Science and Technology Innovation Talent Team Project, No. Guizhou Science and Technology Cooperation Platform Talent (2020)5014 (to LDN); Guizhou Province “One Hundred” Level Innovative Talent Training Program, No. Guizhou Science and Technology Talents (2015)4026 (to LDN); National Natural Science Foundation of Guizhou Medical University, No. TJ20073 (to LDN)

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摘要：


文题释义：

Ⅲ型纤连蛋白结构域包含蛋白5(fibronectin type Ⅲ domain-containing 5，FNDC5)：是一种运动诱导的肌肉因子，主要由心肌、脑组织、骨骼肌和肝脏等组织合成并分泌。FNDC5经蛋白酶水解后形成112个氨基酸水溶性生物活性肽——鸢尾素(Irisin)。FNDC5可以调节糖脂代谢，抗炎、抗氧化应激及改善胰岛素抵抗，抑制动脉粥样硬化、肥胖及糖尿病等糖脂代谢相关疾病发生发展。

细胞焦亡：是炎性程序性细胞死亡，即快速质膜破裂和白细胞介素1β及白细胞介素18等炎症递质释放。焦亡途径主要包括caspase-1依赖的经典途径和caspase-4/5/11参与的非经典途径。在经典途径中，活化的NLRP3等炎症小体可激活caspase-1；caspase-1将白细胞介素1β和白细胞介素18前体变成成熟体，也将GSDMD切割成GSDMD-C端和GSDMD-N端；GSDMD-N端在胞膜上成孔，最终导致细胞肿胀和炎症因子等内容物外渗。


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。

https://orcid.org/0009-0006-9051-9800 (赵广建) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 巨噬细胞, Ⅲ型纤连蛋白结构域包含蛋白5, FNDC5, NF-κB, NLRP3, 细胞焦亡

Abstract: BACKGROUND: Fibronectin type III domain-containing protein 5 (FNDC5) is a muscle factor that can regulate glucose and lipid metabolism, and exert anti-inflammatory, 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-1β in 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-1β and 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.

Key words: macrophage, fibronectin type III domain-containing protein 5, FNDC5, NF-κB, NLRP3, cell pyroptosis 

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