中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (7): 1328-1335.doi: 10.12307/2025.014

• 牙髓及牙周膜干细胞 Dental pulp and periodontal ligament stem cells • 上一篇    下一篇

淫羊藿苷预处理增强人牙周膜干细胞对M1型巨噬细胞的影响

喻  婷1，2，3，吕冬梅1，2，3，邓  浩1，2，4，孙  涛1，2，4，程  钎1，2，3   

  1. 西南医科大学，1口颌面修复重建和再生泸州市重点实验室，2口腔医学研究所，4口腔医学院，四川省泸州市   646000；3西南医科大学附属口腔医院正畸科，四川省泸州市   646000
  • 收稿日期:2023-11-10 接受日期:2024-01-17 出版日期:2025-03-08 发布日期:2024-06-27
  • 通讯作者: 程钎，硕士，主治医师，西南医科大学，口颌面修复重建和再生泸州市重点实验室，口腔医学研究所，四川省泸州市 646000；西南医科大学附属口腔医院正畸科，四川省泸州市 646000
  • 作者简介:喻婷，女，1997年生，重庆市人，汉族，西南医科大学在读硕士，医师，主要从事口腔正畸学的研究。
  • 基金资助:
    泸州市科技计划项目(2023JYJ055)，项目负责人：程钎；四川省医学青年创新科研课题计划(Q21053)，项目负责人：程钎；西南医科大学附属口腔医学院导师组能力提升资助项目(2022DS14)，项目负责人：程钎；西南医科大学大学生创新创业训练项目(S202210632302)，项目负责人：邓浩

Icariin pretreatment enhances effect of human periodontal stem cells on M1-type macrophages

Yu Ting1, 2, 3, Lyu Dongmei1, 2, 3, Deng Hao1, 2, 4, Sun Tao1, 2, 4, Cheng Qian1, 2, 3   

  1. 1Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 3Department of Orthodontics, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 4School of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2023-11-10 Accepted:2024-01-17 Online:2025-03-08 Published:2024-06-27
  • Contact: Cheng Qian, Master, Attending physician, Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Orthodontics, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Yu Ting, Master candidate, Physician, Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Orthodontics, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    Luzhou Science and Technology Program, No. 2023JYJ055 (to CQ); Sichuan Medical Youth Innovative Scientific Research Project Program, No. Q21053 (to CQ); Mentor Group Capacity Enhancement Funding Project of Affiliated School of Stomatology, Southwest Medical University, No. 2022DS14 (to CQ); Innovation and Entrepreneurship Training Project for Undergraduates of Southwest Medical University, No. S202210632302 (to DH)

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摘要：

文题释义：

淫羊藿苷：是一种黄酮苷类化合物，具有广泛的药理活性，能促进成骨、抑制破骨，也能进行免疫调节，具有抗炎等功能。
M1型巨噬细胞：表面标志物为CD86，是在炎症早期富集于炎症部位的免疫细胞之一，能被脂多糖及肿瘤坏死因子α等激活，通过分泌炎症因子的方式促进炎症的发生发展，核转录因子κB通路是其发挥促炎作用的关键信号途径。



背景：人牙周膜干细胞对M1型巨噬细胞促炎功能有一定抑制作用，具有抗炎等药理活性的淫羊藿苷是否能增强人牙周膜干细胞对M1型巨噬细胞的抑制作用尚不明确。

目的：探究淫羊藿苷预处理人牙周膜干细胞后对M1型巨噬细胞的影响。
方法：分离培养原代人牙周膜干细胞，并进行鉴定。对THP-1细胞进行诱导，采用免疫荧光染色及PCR进行M1型巨噬细胞鉴定。用含浓度10-7，10-6，10-5，10-4 mol/L淫羊藿苷的α-MEM完全培养基培养人牙周膜干细胞1，3，5，7 d，采用CCK-8法筛选合适的淫羊藿苷浓度进行实验。将α-MEM完全培养基、未处理的人牙周膜干细胞α-MEM条件培养基及淫羊藿苷预处理24 h的人牙周膜干细胞α-MEM条件培养基与RPMI-1640完全培养基以1∶1的比例对M1型巨噬细胞进行条件培养，分别为对照组、未处理组及预处理组，24 h后RT-PCR法检测M1型巨噬细胞炎症因子的mRNA表达情况；ELISA法检测M1型巨噬细胞炎症因子的蛋白表达情况；Western blot检测M1/M2型巨噬细胞表面标记物及核转录因子κB通路相关蛋白的表达。
结果与结论：①CCK-8检测结果显示，10-7，10-6，10-5，10-4 mol/L淫羊藿苷对人牙周膜干细胞均无细胞毒性，且从第5天起，各浓度都提高了细胞活力，促进细胞增殖，选择10-4 mol/L淫羊藿苷进行后续实验。②RT-PCR法及ELISA检测结果显示，与对照组相比，未处理组及预处理组均降低了M1型巨噬细胞白细胞介素1β、白细胞介素6及肿瘤坏死因子α的表达与分泌(P < 0.05)，且预处理组低于未处理组(P < 0.05)。③Western blot检测结果显示，与未处理组相比，预处理组CD86的表达明显降低(P < 0.05)；与对照组相比，未处理组和预处理组M2型巨噬细胞表面标志物CD206的表达均升高(P < 0.01)，且预处理组明显高于未处理组(P < 0.01)；M1型巨噬细胞经24 h条件培养后，与对照组相比，核转录因子κB/P65的表达在未处理组和预处理组均有降低(P < 0.01)，p-IκBα的表达仅在预处理组降低(P < 0.01)；与未处理组相比，预处理组核转录因子κB/P65和p-IκBα的表达均显著降低(P < 0.05)，而IκBα在3组中的表达差异无显著性意义。④上述结果证实，淫羊藿苷增强了人牙周膜干细胞对M1型巨噬细胞的抑制作用，此作用可能与抑制了巨噬细胞的核转录因子κB信号通路相关。

https://orcid.org/0009-0000-6270-7544 (喻婷) ；https://orcid.org/0000-0003-3833-1545 (程钎)


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: 人牙周膜干细胞, 淫羊藿苷, 巨噬细胞, 条件培养基共培养, 表面标志物, 炎症因子, 抗炎, 核转录因子κB信号通路

Abstract: BACKGROUND: Human periodontal stem cells have a certain inhibitory effect on the pro-inflammatory function of M1-type macrophages, and it is not clear whether icariin, which has anti-inflammatory and other pharmacological activities, can enhance the inhibitory effect of human periodontal stem cells on M1-type macrophages.
OBJECTIVE: To investigate the effect of icariin on M1 macrophages after pretreatment of human periodontal stem cells. 
METHODS: Primary human periodontal stem cells were isolated, cultured and characterized. THP-1 was induced and M1-type macrophages were identified by immunofluorescence staining and PCR. Human periodontal stem cells were cultured with α-MEM complete medium containing concentrations of 10-7, 10-6, 10-5, and 10-4 mol/L icariin, and the cytotoxicity of Icariin on human periodontal stem cells was detected by the CCK-8 assay at 1, 3, 5, and 7 days, respectively. α-MEM complete medium, untreated α-MEM conditioned medium for human periodontal stem cells and α-MEM conditioned medium for human periodontal stem cells pretreated with icariin for 24 hours were conditioned with RPMI-1640 complete medium in a 1:1 ratio for M1-type macrophages in the control, untreated, and pretreated groups, and 24 hours later, the mRNA expression of inflammatory factors in M1 macrophages was detected by RT-PCR. The protein expression of inflammatory factors in M1 macrophages was detected by ELISA. The expression of surface markers and nuclear factor-κB pathway-related proteins in M1/M2 macrophages was detected by western blot assay. 
RESULTS AND CONCLUSION: (1) CCK-8 assay results showed that 10-7, 10-6, 10-5, 10-4 mol/L icariin was not cytotoxic to the human periodontal stem cells, and from day 5 onwards, all the concentrations increased the cell viability, and promoted the cell proliferation. 10-4 mol/L icariin was selected for follow-up experiment. (2) RT-PCR and ELISA results showed that compared with the control group , the untreated group and the pretreated group both decreased the expression and secretion of interleukin-1β, interleukin-6, and tumor necrosis factor-α of M1-type macrophages (P < 0.05), and the pretreated group was lower than the untreated group (P < 0.05). (3) Western blot assay results showed that compared with the untreated group, the expression of CD86 was significantly lower in the pretreated group (P < 0.05); compared with the control group, the expression of CD206, a surface marker of M2-type macrophages, was elevated in both the untreated and pretreated groups (P < 0.01), and it was significantly higher in the pretreated group than in the untreated group (P < 0.01). In M1-type macrophages after 24 hours of conditioned culture, compared with the control group, the expression of nuclear factor-κB/P65 was decreased in the untreated group and the pretreated group (P < 0.01), and the expression of p-IκBα was decreased only in the pretreated group (P < 0.01); the expression of both nuclear factor-κB/P65 and p-IκBα was significantly reduced in the pretreated group compared with the untreated group (P < 0.05), while the difference of IκBα in the three groups was not statistically significant. (4) These results indicated that icariin enhanced the inhibitory effect of human periodontal stem cells on M1-type macrophages, and this effect may be related to the inhibition of the nuclear factor-κB signaling pathway of macrophages.

Key words: human periodontal ligament stem cell, icariin, macrophage, conditioned medium co-culture, surface marker, inflammatory factor, anti-inflammatory, nuclear factor-κB signaling pathway

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