中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (19): 4972-4982.doi: 10.12307/2026.793

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

芦丁促进MC3T3-E1细胞成骨分化：调控中性粒细胞胞外陷阱形成

李  杰1，刘  洋2，王大禹3，万  强3，朱家毅3，冯文俊1，陈锦伦1，揭  珂4，黄艺伟5，辛鹏飞6，曾建春1，曾意荣1，张海涛1   

  1. 1广州中医药大学第一附属医院，广东省广州市  510405；2复旦大学，上海市  200433；3广州中医药大学，广东省广州市  510405；4佛山市中医院，广东省佛山市  528000；5中山市中医院，广东省中山市  528400；6上海中医药大学附属光华医院，上海市  200052
  • 收稿日期:2025-08-09 接受日期:2025-12-24 出版日期:2026-07-08 发布日期:2026-02-24
  • 通讯作者: 张海涛，博士，医师，广州中医药大学第一附属医院，广东省广州市 510405； 共同通讯作者：曾意荣，博士，主任医师，博士生导师，广州中医药大学第一附属医院，广东省广州市 510405
  • 作者简介:李杰，男，1985年生，博士，副主任医师，硕士生导师，主要从事中医药治疗骨与关节疾病方面的研究。 共同第一作者：刘洋，男，1996年生，江西省南昌市人，汉族，复旦大学药学院在读博士，主要从事中医药活性成分疗效及机制方面的研究。
  • 基金资助:
    国家自然科学基金青年项目(82104882)，项目负责人：冯文俊；国家自然科学基金面上项目(82374484)，项目负责人：曾意荣；广州中医药大学第一附属医院国家中医药传承创新科研专项(2022QN16)，项目负责人：李杰；广州中医药大学校院联合科技创新基金(GZYZS2024G42)，项目负责人：黄艺伟；广州中医药大学校院联合科技创新基金(GZYFS2024Y08)，项目负责人：揭珂；广东省中医药局科研项目(20254038)，项目负责人：陈锦伦；广州市科技计划项目(2025A04J2851)，项目负责人：陈锦伦

Rutin promotes osteogenic differentiation of MC3T3-E1 cells: regulating the formation of neutrophil extracellular traps

Li Jie1, Liu Yang2, Wang Dayu3, Wan Qiang3, Zhu Jiayi3, Feng Wenjun1, Chen Jinlun1, Jie Ke4, Huang Yiwei5, Xin Pengfei6, Zeng Jianchun1, Zeng Yirong1, Zhang Haitao1   

  1. 1The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2Fudan University, Shanghai 200433, China; 3Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 4Foshan Hospital of Traditional Chinese Medicine, Foshan 528000, Guangdong Province, China; 5Zhongshan Hospital of Traditional Chinese Medicine, Zhongshan 528400, Guangdong Province, China; 6Guanghua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200052, China
  • Received:2025-08-09 Accepted:2025-12-24 Online:2026-07-08 Published:2026-02-24
  • Contact: Zhang Haitao, MD, Physician, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Co-corresponding author: Zeng Yirong, MD, Chief physician, Doctoral supervisor, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • About author:Li Jie, MD, Associate chief physician, Master’s supervisor, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Liu Yang, Doctoral candidate, Fudan University, Shanghai 200433, China; Li Jie and Liu Yang contributed equally to this article.
  • Supported by:
    National Natural Science Foundation of China - Youth Project, No. 82104882 (to FWJ); National Natural Science Foundation of China - General Project, No. 82374484 (to ZYR); National Research Project for the Transmission and Innovation of Traditional Chinese Medicine at Guangzhou University of Chinese Medicine, No. 2022QN16 (to LJ); Guangzhou University of Chinese Medicine School-University Joint Innovation Science and Technology Fund, No. GZYZS2024G42 (to HYW); Guangzhou University of Chinese Medicine School-University Joint Innovation Science and Technology Fund, No. GZYFS2024Y08 (to JK); Guangdong Provincial Department of Traditional Chinese Medicine Research Project, No. 20254038 (to CJL); Guangzhou Science and Technology Plan Project, No. 2025A04J2851 (to CJL)

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摘要：

文题释义：

芦丁：是一种来源于中药杜仲和枸杞的活性成分，具有抗骨质疏松症的作用。
中性粒细胞胞外陷阱：是由组蛋白-DNA复合物协同髓过氧化物酶、中性粒细胞弹性蛋白酶等抗菌蛋白共同构建的三维防御结构，其空间延展度可达中性粒细胞体积的10-15倍，也被称之为“神奇的网”。

摘要
背景：芦丁能够有效抗骨质疏松症，但作用机制尚不明确。
目的：探讨芦丁在中性粒细胞胞外陷阱作用下对MC3T3-E1细胞成骨的影响。
方法：①采用佛波酯刺激人源髓细胞白血病dHL60细胞，体外诱导中性粒细胞胞外陷阱形成。将dHL60细胞分4组进行培养，对照组加入Hank’s平衡盐溶液，其余3组均加入50 nmol/L佛波酯；后2组再加入250 μmol/L芦丁或250 μmol/L芦丁和5 U/mL DNaseⅠ。通过流式细胞术检测dHL60细胞凋亡情况，RT-qPCR和Western blot检测中性粒细胞胞外陷阱形成标志性基因和蛋白的表达。②将MC3T3-E1细胞分为6组进行培养：对照组加入Hank’s平衡盐溶液，其余5组分别加入50 nmol/L佛波酯；dHL60细胞和50 nmol/L佛波酯；100 μmol/L芦丁；dHL60细胞、50 nmol/L佛波酯和250 μmol/L芦丁；dHL60细胞、50 nmol/L佛波酯、250 μmol/L芦丁和5 U/mL DNaseⅠ。通过流式细胞仪检测在中性粒细胞胞外陷阱作用下MC3T3-E1细胞凋亡情况，碱性磷酸酶染色、茜素红染色明确在中性粒细胞胞外陷阱作用下MC3T3-E1细胞的成骨及矿化能力，RT-qPCR和Western blot检测在中性粒细胞胞外陷阱作用下MC3T3-E1细胞的成骨相关基因和蛋白表达。
结果与结论：①与空白对照组比较，芦丁能够显著抑制dHL60细胞中蛋白精氨酸脱亚胺酶4、髓过氧化物酶和中性粒细胞弹性蛋白酶的mRNA和蛋白表达(P < 0.000 1)；与芦丁组比较，芦丁与DNaseⅠ联合干预对蛋白精氨酸脱亚胺酶4、髓过氧化物酶和中性粒细胞弹性蛋白酶的下调作用更加明显(P < 0.05)，说明芦丁能够显著抑制中性粒细胞胞外陷阱的形成；②将dHL60诱导出中性粒细胞胞外陷阱后与MC3T3-E1共培养，MC3T3-E1细胞的Runt相关转录因子2、β-连环蛋白和骨形态发生蛋白2的mRNA和蛋白表达显著下调(P < 0.000 1)，凋亡率显著上升(P < 0.000 1)，说明中性粒细胞胞外陷阱在体外能显著抑制MC3T3-E1细胞的成骨能力，并促进细胞凋亡；在经芦丁单独干预或芦丁与DNaseⅠ联合干预后，中性粒细胞胞外陷阱作用下的MC3T3-E1细胞的凋亡和成骨能力显著改善，且芦丁与DNaseⅠ联合干预的作用较芦丁单独干预更为显著，表明芦丁可能抑制中性粒细胞胞外陷阱的形成，从而改善MC3T3-E1细胞的成骨能力；③分子对接和分子动力学模拟显示，芦丁与蛋白精氨酸脱亚胺酶4、髓过氧化物酶和中性粒细胞弹性蛋白酶靶蛋白的结合作用良好，说明芦丁能够靶向抑制中性粒细胞胞外陷阱的形成。

关键词: 芦丁, DNaseⅠ, 骨质疏松症, 成骨前体细胞, 中性粒细胞胞外陷阱, 成骨, 靶向

Abstract: BACKGROUND: Rutin can effectively prevent osteoporosis, but its mechanism of action remains unclear.
OBJECTIVE: To investigate the effect of rutin on osteogenesis of MC3T3-E1 cells under the action of neutrophil extracellular traps.
METHODS: (1) Human myeloid leukemia dHL60 cells were stimulated with phorbol 12-myristate 13-acetate to induce neutrophil extracellular trap formation in vitro. dHL60 cells were divided into four groups and cultured. The control group received Hank's balanced salt solution, while the other three groups received 50 nmol/L phorbol esters. The latter two groups were then treated with either 250 μmol/L rutin or 250 μmol/L rutin and 5 U/mL DNase I. dHL60 cell apoptosis was assessed by flow cytometry, and the expression of marker genes and proteins associated with neutrophil extracellular trap formation was determined by RT-qPCR and western blot assay. (2) MC3T3-E1 cells were divided into six groups for culture: the control group was treated with Hank's balanced salt solution, and the other five groups were treated with 50 nmol/L phorbol esters; dHL60 cells and 50 nmol/L phorbol esters; 100 μmol/L rutin; dHL60 cells, 50 nmol/L phorbol esters, and 250 μmol/L rutin; and dHL60 cells, 50 nmol/L phorbol esters, 250 μmol/L rutin, and 5 U/mL DNase I. Flow cytometry was used to assess apoptosis in MC3T3-E1 cells treated with neutrophil extracellular traps. Alkaline phosphatase staining and Alizarin red staining were used to determine the osteogenic and mineralization abilities of MC3T3-E1 cells treated with neutrophil extracellular traps. RT-qPCR and western blot assay were used to examine the expression of osteogenesis-related genes and proteins in MC3T3-E1 cells treated with neutrophil extracellular traps.
RESULTS AND CONCLUSION: (1) Compared with the blank control group, rutin significantly inhibited the mRNA and protein expressions of protein arginine deiminase 4, myeloperoxidase, and neutrophil elastase in dHL60 cells (P < 0.000 1). Compared with the rutin group alone, the combined intervention of rutin and DNase I had a more significant downregulation effect on protein arginine deiminase 4, myeloperoxidase, and neutrophil elastase (P < 0.05), indicating that rutin could significantly inhibit the formation of neutrophil extracellular traps. (2) After dHL60 cells induced neutrophil extracellular traps and then co-cultured with MC3T3-E1 cells, the mRNA and protein expressions of Runt-related transcription factor 2, β-catenin, and bone morphogenetic protein 2 in MC3T3-E1 cells were significantly downregulated (P < 0.000 1), and the apoptosis rate was significantly increased (P < 0.000 1), which indicate that neutrophil extracellular traps significantly inhibit the osteogenic capacity of MC3T3-E1 cells in vitro and promote their apoptosis. Rutin alone or combined with DNase I significantly improved the apoptosis and osteogenic capacity of MC3T3-E1 cells exposed to neutrophil extracellular traps, and the combined effect of rutin and DNase I was more pronounced than that of rutin alone, suggesting that rutin may inhibit the formation of neutrophil extracellular traps, thereby improving the osteogenic capacity of MC3T3-E1 cells. (3) Molecular docking and molecular dynamics simulations revealed that rutin binds well to the target proteins of protein arginine deiminase 4, myeloperoxidase, and neutrophil elastase, indicating that rutin can specifically inhibit the formation of neutrophil extracellular traps.

Key words: rutin, DNase I, osteoporosis, osteoblast precursor cells, neutrophil extracellular traps, osteogenesis, targeting

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