黄酮类NO供体纳米颗粒通过调控巨噬细胞极化促进PDLSCs成骨分化的体外研究

doi:10.13591/j.cnki.kqyx.2024.11.002

摘要/Abstract

摘要：

目的一氧化氮(nitric oxide,NO)作为一种信号分子,调节关键生理过程,与牙周炎关系密切。本研究拟探讨负载黄酮类NO供体药物的复合纳米颗粒(G10@HAP/MSN@ZnO@COS)通过调控巨噬细胞极化对牙周膜干细胞(periodontal ligament stem cells, PDLSCs)成骨分化的影响。方法将新型NO供体药物G10负载于羟基磷灰石掺杂的介孔二氧化硅颗粒(hydroxyapatite/mesoporous silica nanoparticles, HAP/MSN)上,并用氧化锌(zinc oxide, ZnO)填充,再通过壳聚糖(chitosan, COS)包裹,制备复合纳米颗粒(G10@HAP/MSN@ZnO@COS)。CCK-8细胞实验筛选G10@HAP/MSN@ZnO@COS促进细胞增殖的最佳浓度。通过脂多糖刺激小鼠单核巨噬细胞建立细胞炎症模型后,将其分为Control组、G10组、HAP/MSN@ZnO@COS组和G10@HAP/MSN@ZnO@COS组,各组分别加入新鲜培养基、5 μg/mL G10、5 μg/mL HAP/MSN@ZnO@COS和5 μg/mL G10@HAP/MSN@ZnO@COS,培养72 h,采用ELISA和RT-qPCR检测各组细胞因子(TNF-α、IL-6、IL-1β、iNOS、IL-10)的表达水平,评估其M1/M2表型变化。用各组培养基上清液作为条件培养基培养PDLSCs,并通过碱性磷酸酶活性检测和茜素红染色评估其成骨矿化能力。结果 CCK-8实验显示,5 μg/mL G10@HAP/MSN@ZnO@COS能显著促进PDLSCs的增殖。ELISA结果表明,与Control组相比,G10@HAP/MSN@ZnO@COS组中M1型标志物IL-1β、IL-6、TNF-α和iNOS的表达显著降低(P<0.000 1),而M2型标志物IL-10表达显著升高(P<0.000 1)。RT-qPCR结果与ELISA结果一致,显示G10@HAP/MSN@ZnO@COS组中M1相关基因的表达显著下降(P<0.01)。在G10@HAP/MSN@ZnO@COS调控巨噬细胞的环境下,茜素红染色和碱性磷酸酶活性检测结果表明,G10@HAP/MSN@ZnO@COS-CM作为条件培养基,PDLSCs的矿化结节数量和碱性磷酸酶活性均显著高于其他组(P<0.000 1)。结论复合纳米颗粒(G10@HAP/MSN@ZnO@COS)能有效抑制巨噬细胞向M1表型极化,并促进其向M2表型极化,G10@HAP/MSN@ZnO@COS调控的抗炎微环境能增强PDLSCs的成骨分化能力。

关键词: 一氧化氮供体药物, 巨噬细胞极化, 牙周膜干细胞, 条件培养基, 成骨分化

Abstract:

Objective As a signaling molecule, NO regulates key physiological processes and is closely related to periodontitis. To investigate the effect of flavonoid NO donor composite nanoparticles (G10@HAP/MSN@ZnO@COS) on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) by regulating macrophage polarization. Methods The novel NO donor drug G10 was loaded on hydroxyapatite/mesoporous silicanant particles (HAP/MSN), filled with zinc oxide (ZnO), and then coated with chitosan (COS) to prepare composite nanoparticles (G10@HAP/MSN@ZnO@COS). The best concentration of G10@HAP/MSN@ZnO@COS was screened to promote cell proliferation by CCK-8 cell experiment. After the mouse mononuclear macrophages were stimulated by lipopolysaccharide, the mice were divided into four groups: Control group, G10 group, HAP/MSN@ZnO@COS group and G10@HAP/MSN@ZnO@COS group. Each group was cultured with fresh medium, 5 μg/mL G10, 5 μg/ mL HAP/MSN@ZnO@COS and 5 μg/mL G10@HAP/MSN@ZnO@COS for 72 h respectively. ELISA and RT-qPCR were used to detect the expression of cytokines (TNF-α, IL-6, IL-1β, iNOS, IL-10) and mRNA expression in each group, and the phenotypic changes of M1/M2 were evaluated. The supernatant of each culture medium was used as conditioned medium to culture PDLSCs, and the osteogenic ability and cell mineralization were evaluated by alkaline phosphatase activity test and alizarin red staining. Results CCK-8 experiment showed that G10@HAP/MSN@ZnO@COS of 5 μg/mL could significantly promote the proliferation of PDLSCs. The results of ELISA showed that compared with Control group, the expression of M1 type marker IL-1β, IL-6, TNF-α and iNOS in G10@HAP/MSN@ZnO@COS group was significantly decreased (P<0.000 1), while the expression of M2 type marker IL-10 was significantly increased (P<0.000 1). The results of RT-qPCR were consistent with those of ELISA, which showed that the expression of M1-related genes in G10@HAP/MSN@ZnO@COS group decreased significantly (P<0.01). The results of alizarin red staining and alkaline phosphatase activity test showed that the number of mineralized nodules and alkaline phosphatase activity in G10@HAP/MSN@ZnO@COS-CM group were significantly higher than those in other groups (P<0.000 1). Conclusion Composite nanoparticles (G10@HAP/MSN@ZnO@COS) can effectively inhibit the polarization of macrophages to M1 phenotype and promote it to M2 phenotypic polarization. The anti-inflammatory microenvironment regulated by G10@HAP/MSN@ZnO@COS can enhance the osteogenic differentiation of PDLSCs.

Key words: nitric oxide donor drugs, macrophage polarization, periodontal ligament stem cells, conditioned medium, osteogenic differentiation

中图分类号:

R783.1

丁珂欣, 杨金鑫, 牟杰, 孙哲, 崔雅雯, 刘宗响. 黄酮类NO供体纳米颗粒通过调控巨噬细胞极化促进PDLSCs成骨分化的体外研究[J]. 口腔医学, 2024, 44(11): 806-814.

DING Kexin, YANG Jinxin, MOU Jie, SUN Zhe, CUI Yawen, LIU Zongxiang. In vitro study on flavonoid NO donor nanoparticles promoting osteogenic differentiation of PDLSCs by regulating macrophage polarization[J]. Stomatology, 2024, 44(11): 806-814.

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基因名称	序列(5'—3')
TNF-α	F:CCCCAAAGGGATGAGAAGTT
	R:CACTTGGTGGTTTGCTACGA
IL-6	F:CCAATGCTCTCCTAACAGAT
	R:TGTCCACAAACTGATATGCT
iNOS	F:CTCACCTACTTCTGGACATTAC
	R:GCCTCCAATCTCTGCCTATC
IL-1β	F:AGGACATGAGCACCTTCTTTTCC
	R:ACGTCACACACCAGCAGGTTAT
GAPDH	F:AGGTCGGTGTGAACGGATTTG
	R:TGTAGACCATGTAGTTGAGGTCA