国际口腔医学杂志

国际口腔医学杂志 ›› 2024, Vol. 51 ›› Issue (4): 392-400.doi: 10.7518/gjkq.2024078

• 牙周病学专栏 • 上一篇    

大麻二酚联合米诺环素对牙周炎治疗作用的实验研究

漆美瑶(),祁星颖,周欣奕,谭震,袁泉()   

  1. 口腔疾病防治全国重点实验室 国家口腔医学中心 国家口腔疾病临床医学研究中心四川大学华西口腔医院种植科 成都 610041
  • 收稿日期:2023-03-25 修回日期:2023-06-29 出版日期:2024-07-01 发布日期:2024-06-24
  • 通讯作者: 袁泉
  • 作者简介:漆美瑶，医师，博士，Email：727198073@qq.com
  • 基金资助:
    四川大学华西口腔医院交叉学科创新项目(RD-03-202010);四川省国际科技合作项目(2021YFH0015)

Therapeutic effect of cannabidiol combined with minocycline on periodontitis

Meiyao Qi(),Xingying Qi,Xinyi Zhou,Zhen Tan,Quan Yuan()   

  1. State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2023-03-25 Revised:2023-06-29 Online:2024-07-01 Published:2024-06-24
  • Contact: Quan Yuan
  • Supported by:
    West China Hospital of Stomatology Sichuan University Interdisciplinary Innovation Project(RD-03-202010);Sichuan Province International Science and Technology Cooperation Project(2021YFH0015)

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摘要：

目的 通过体内外实验探索大麻二酚（CBD）联合米诺环素（MINO）对小鼠实验性牙周炎的治疗作用。 方法 丝线结扎法建立小鼠牙周炎模型，口腔局部分别给予CBD、MINO及二者联用， 通过定量反转录聚合酶链反应（qRT-PCR）检测牙周炎小鼠用药后外周血炎症因子变化，Micro CT、免疫印迹（WB）及苏木精-伊红（HE）染色分析局部位点炎症改善情况；牙龈卟啉单胞菌抑菌实验检测联用药物体外抗菌性能；体外诱导巨噬细胞及牙龈成纤维细胞炎症，通过qRT-PCR检测炎症相关mRNA表达，划痕实验探索药物对细胞迁移的影响，并通过酶联免疫吸附（ELISA）测定探究药物对于胶原生成的作用。 结果 联用CBD与MINO后，与单独用药相比，小鼠全身炎症指标下降，局部附着丧失减轻、组织炎症水平降低、牙周软硬组织破坏得到改善；体外抑菌实验结果表明联用药物表现出良好的抗菌性能；体外细胞实验证明联用药物降低巨噬细胞多种炎症因子的表达水平，促进炎症状态下人牙龈成纤维细胞增殖、迁移，提升胶原形成功能。 结论 相较单一用药方式法，局部应用CBD联合MINO对小鼠实验性牙周炎有着显著疗效。

关键词: 牙周炎, 大麻二酚, 米诺环素, 抗生素

Abstract:

Objective The therapeutic effect of cannabidiol combined with minocycline on experimental periodontitis in mice was studied in vitro and in vivo. Methods The mouse periodontitis model was established by thread ligation. The oral area was treated with cannabidiol, minocycline, and both. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the changes in inflammatory factors in peripheral blood of mice with periodontitis. Micro CT, western blot (WB), and hematoxylin-eosin (HE) staining were used to analyze the improvement in local inflammation. Porphyromonas gingivalis (P. gingivalis) bacteriostatic assay was used to detect the antibacterial properties of the combined drugs in vitro. Inflammation of macrophages and gingival fibroblasts was induced in vitro, and mRNA expression related to inflammation was detected by qRT-PCR. The effect of drugs on cell migration was explored by wound-healing assay, and the effect of drugs on collagen production was explored by enzyme-linked immunosorbent assay. Results After combined administration of cannabidiol and minocycline, the systemic inflammation index of mice decreased, the local attachment loss was alleviated, the tissue inflammation level was reduced, and the destruction of periodontal soft and hard tissues improved compared with single administration. The results of the antibacterial experiment in vitro showed that the combined drugs had good antibacterial properties. In vitro cell experiments proved that the combination of drugs reduced the expression levels of various inflammatory factors in macrophages, promoted the proliferation and migration of human gingival fibroblasts in inflammatory states, and improved the collagen formation function. Conclusion Compared with the single medication method, the local application of cannabidiol combined with minocycline has a significant effect on experimental periodontitis in mice.

Key words: periodontitis, cannabidiol, minocycline, antibiotic

中图分类号: 

  • R781.4+2

图 1

给药后的小鼠颌骨Micro CT三维重建与分析A：小鼠颌骨三维重建图及横断面展示（舌侧观），所示依次为5组小鼠上颌第一、二磨牙牙槽骨丧失高度标记及横断面骨量丧失情况，红色箭头所示为CEJ-ABC距离，标尺为500 μm；B：小鼠上颌第一磨牙远中CEJ-ABC的垂直高度；C：小鼠上颌第一、二磨牙牙根近远中轴面所围成的横截面积；D：小鼠上颌第一、二磨牙间牙槽骨BV/TV值。*示P≤0.05，**示P≤0.01。"

图 2

给药后小鼠牙周局部及全身炎症水平变化A：小鼠牙周组织石蜡切片的HE染色结果，所框选截面为放大倍率扫描结果，G是牙龈乳头，B是ABC，1是小鼠上颌第一磨牙，2是小鼠上颌第二磨牙，标尺为50 μm；B：小鼠牙龈组织蛋白的免疫印迹检测；C：小鼠外周血中全身炎症相关蛋白转录水平检测。*示P≤0.05。"

图 3

CBD+MINO体外抑菌作用探索A：有效抑制P. gingivalis的组合药物浓度探索，红色三角示最低抗菌组合浓度；B：不同浓度组合药物对P. gingivalis生长曲线的影响，用光密度值（optical density，OD）表征。"

图 4

CBD+MINO处理影响炎症下RAW264.7细胞炎症因子分泌A：qRT-PCR检测不同药物处理的炎症RAW264.7细胞，pro-IL-1β、IL-1β、TNF-α、Irf-1 mRNA水平，*示P≤0.05，***示P≤0.001；B：WB检测不同药物处理的炎症RAW264.7细胞中炎症因子的表达水平。"

图 5

HGF细胞划痕实验结果A：划痕实验结果，标尺为200 μm；B：48 h划痕实验定量结果，***示P≤0.001。"

图 6

联用药物提升炎症下HGF细胞胶原生成A：qRT-PCR实验结果；B：ELISA实验结果。***示P≤0.001。"

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