Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (7): 1328-1335.doi: 10.12307/2025.014

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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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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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