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)

RichHTML

24

PDF (PC)

679

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。"

1 Kwon T, Lamster IB, Levin L. Current concepts in the management of periodontitis[J]. Int Dent J, 2021, 71(6): 462-476.
2 Fischer RG, Lira Junior R, Retamal-Valdes B, et al. Periodontal disease and its impact on general health in Latin America. Section V: treatment of periodontitis[J]. Braz Oral Res, 2020, 34(supp1 1): e026.
3 Simpson TC, Clarkson JE, Worthington HV, et al. Treatment of periodontitis for glycaemic control in people with diabetes mellitus[J]. Cochrane Database Syst Rev, 2022, 4(4): CD004714.
4 Lin HB, Chen HS, Zhao XT, et al. Advances of exosomes in periodontitis treatment[J]. J Transl Med, 2022, 20(1): 279.
5 GBD Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017[J]. Lancet, 2018, 392(10159): 1789-1858.
6 台保军. 中国居民口腔健康状况及防控策略：第四次全国口腔健康流行病学调查结果解读[C]//第十三次全国老年口腔医学学术年会论文汇编. 武汉, 2018: 16-17.
Tai BJ. Oral health status of Chinese residents and prevention and control strategies: interpretation of the results of the fourth national oral health epide-miological survey[C]//The 13th National Annual Conference of Geriatric Stomatology. Wuhan, 2018: 16-17.
7 Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation[J]. Nat Rev Immunol, 2015, 15(1): 30-44.
8 Hajishengallis G, Chavakis T. Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities[J]. Nat Rev Immunol, 2021, 21(7): 426-440.
9 Genco RJ, Sanz M. Clinical and public health implications of periodontal and systemic diseases: an overview[J]. Periodontol 2000, 2020, 83(1): 7-13.
10 Pisanti S, Malfitano AM, Ciaglia E, et al. Cannabi-diol: state of the art and new challenges for therapeutic applications[J]. Pharmacol Ther, 2017, 175: 133-150.
11 Oláh A, Tóth BI, Borbíró I, et al. Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes[J]. J Clin Invest, 2014, 124(9): 3713-3724.
12 Wroński A, Jarocka-Karpowicz I, Stasiewicz A, et al. Phytocannabinoids in the pharmacotherapy of psoriasis[J]. Molecules, 2023, 28(3): 1192.
13 Ramer R, Hinz B. Cannabinoid compounds as a pharmacotherapeutic option for the treatment of non-cancer skin diseases[J]. Cells, 2022, 11(24): 4102.
14 Casares L, García V, Garrido-Rodríguez M, et al. Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1[J]. Redox Biol, 2020, 28: 101321.
15 Malfait AM, Gallily R, Sumariwalla PF, et al. The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis[J]. Proc Natl Acad Sci U S A, 2000, 97(17): 9561-9566.
16 Qi X, Lin W, Wu Y, et al. CBD promotes oral ulcer healing via inhibiting CMPK2-mediated inflammasome[J]. J Dent Res, 2022, 101(2): 206-215.
17 Singh S, Khanna D, Kalra S. Minocycline and doxycycline: more than antibiotics[J]. Curr Mol Pharmacol, 2021, 14(6): 1046-1065.
18 Asadi A, Abdi M, Kouhsari E, et al. Minocycline, focus on mechanisms of resistance, antibacterial activity, and clinical effectiveness: back to the future[J]. J Glob Antimicrob Resist, 2020, 22: 161-174.
19 Afshari AR, Mollazadeh H, Sahebkar A. Minocycline in treating glioblastoma multiforme: far beyond a conventional antibiotic[J]. J Oncol, 2020, 2020: 8659802.
20 Derfuss T, Mehling M, Papadopoulou A, et al. Advances in oral immunomodulating therapies in relapsing multiple sclerosis[J]. Lancet Neurol, 2020, 19(4): 336-347.
21 Dogaru IA, Puiu MG, Manea M, et al. Current perspectives on pharmacological and non-pharmacolo-gical interventions for the inflammatory mechanism of unipolar depression[J]. Brain Sci, 2022, 12(10): 1403.
22 Morris G, Walker AJ, Berk M, et al. Cell death pathways: a novel therapeutic approach for neuroscientists[J]. Mol Neurobiol, 2018, 55(7): 5767-5786.
23 Shankar C, Nabarro LEB, Anandan S, et al. Minocycline and tigecycline: what is their role in the treatment of carbapenem-resistant gram-negative orga-nisms[J]. Microb Drug Resist, 2017, 23(4): 437-446.
24 Franco V, Gershkovich P, Perucca E, et al. The interplay between liver first-pass effect and lymphatic absorption of cannabidiol and its implications for cannabidiol oral formulations[J]. Clin Pharmacokinet, 2020, 59(12): 1493-1500.
25 Kim EK, Jang SH, Choi YH, et al. Effect of an oral hygienic care program for stroke patients in the intensive care unit[J]. Yonsei Med J, 2014, 55(1): 240-246.
26 de Molon RS, Mascarenhas VI, de Avila ED, et al. Long-term evaluation of oral gavage with periodontopathogens or ligature induction of experimental periodontal disease in mice[J]. Clin Oral Investig, 2016, 20(6): 1203-1216.
27 Polak D, Wilensky A, Shapira L, et al. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response[J]. J Clin Perio-dontol, 2009, 36(5): 406-410.
28 Chen WP, Chang SH, Tang CY, et al. Composition analysis and feature selection of the oral microbiota associated with periodontal disease[J]. Biomed Res Int, 2018, 2018: 3130607.
29 Teles F, Collman RG, Mominkhan D, et al. Viruses, periodontitis, and comorbidities[J]. Periodontol 2000, 2022, 89(1): 190-206.
30 Xu WZ, Zhou W, Wang HZ, et al. Roles of Porphyromonas gingivalis and its virulence factors in pe-riodontitis[J]. Adv Protein Chem Struct Biol, 2020, 120: 45-84.
31 Herrera D, Sanz M, Kebschull M, et al. Treatment of stage Ⅳ periodontitis: the EFP S3 level clinical practice guideline[J]. J Clin Periodontol, 2022, 49(): 4-71.
32 West N, Chapple I, Claydon N, et al. BSP implementation of European S3-level evidence-based treatment guidelines for stage Ⅰ-Ⅲ periodontitis in UK clinical practice[J]. J Dent, 2021, 106: 103562.
33 Oh TJ, Yu SH. Evidence-based clinical practice guideline for treatment of stage IIII periodontitis[J]. J Evid Based Dent Pract, 2021, 21(4): 101638.
34 Stahl V, Vasudevan K. Comparison of efficacy of cannabinoids versus commercial oral care products in reducing bacterial content from dental plaque: a preliminary observation[J]. Cureus, 2020, 12(1): e6809.
35 Fatima T, Khurshid Z, Rehman A, et al. Gingival crevicular fluid (GCF): a diagnostic tool for the detection of periodontal health and diseases[J]. Molecules, 2021, 26(5): 1208.
36 Surlin P, Gheorghe DN, Popescu DM, et al. Interleukin-1α and-1β assessment in the gingival crevicular fluid of periodontal patients with chronic hepatitis C[J]. Exp Ther Med, 2020, 20(3): 2381-2386.
37 Preshaw PM, Taylor JJ, Jaedicke KM, et al. Treatment of periodontitis reduces systemic inflammation in type 2 diabetes[J]. J Clin Periodontol, 2020, 47(6): 737-746.
38 Li C, Yu R, Ding YM. Association between Porphyromonas Gingivalis and systemic diseases: focus on T cells-mediated adaptive immunity[J]. Front Cell Infect Microbiol, 2022, 12: 1026457.
39 Yang RQ, Guo SJ, Xiao SM, et al. Enhanced wound healing and osteogenic potential of photodynamic therapy on human gingival fibroblasts[J]. Photodiagnosis Photodyn Ther, 2020, 32: 101967.
40 Amberg R, Elad A, Rothamel D, et al. Design of a migration assay for human gingival fibroblasts on biodegradable magnesium surfaces[J]. Acta Biomater, 2018, 79: 158-167.
[1] 杨再目,曹沛,刘振华,栾庆先. 血浆无细胞线粒体外线粒体DNA与牙周炎临床指标的相关性研究[J]. 国际口腔医学杂志, 2024, 51(3): 288-295.
[2] 马瑜鸿,赵蕾. 微创非手术牙周治疗技术的临床研究进展[J]. 国际口腔医学杂志, 2024, 51(2): 227-232.
[3] 傅豫, 何薇, 黄兰. 铁死亡在口腔疾病中的研究进展[J]. 国际口腔医学杂志, 2024, 51(1): 36-44.
[4] 罗晓洁,王德续,陈晓涛. 基于生物信息学分析铁死亡调控基因与牙周炎的关系[J]. 国际口腔医学杂志, 2023, 50(6): 661-668.
[5] 黄元鸿,彭显,周学东. 骨碎补在治疗口腔骨相关疾病的研究进展[J]. 国际口腔医学杂志, 2023, 50(6): 679-685.
[6] 王仁义,赵呈智,潘剑. 拔牙围手术期预防性使用抗生素对术后并发症影响的研究进展[J]. 国际口腔医学杂志, 2023, 50(5): 558-565.
[7] 龚美灵,程兴群,吴红崑. 牙周炎与帕金森病相关性的研究进展[J]. 国际口腔医学杂志, 2023, 50(5): 587-593.
[8] 孙佳,韩烨,侯建霞. 白细胞介素-6-铁调素信号轴调控牙周炎相关性贫血致病机制的研究进展[J]. 国际口腔医学杂志, 2023, 50(3): 329-334.
[9] 刘体倩,梁星,刘蔚晴,李晓虹,朱睿. 咬合创伤在牙周炎发生发展中的作用及机制的研究进展[J]. 国际口腔医学杂志, 2023, 50(1): 19-24.
[10] 李琼,于维先. 白藜芦醇治疗牙周炎及其生物利用度的研究进展[J]. 国际口腔医学杂志, 2023, 50(1): 25-31.
[11] 黄伟琨,徐秋艳,周婷. 黄芩苷抑制脂多糖促巨噬细胞氧化应激损伤作用的研究[J]. 国际口腔医学杂志, 2022, 49(5): 521-528.
[12] 周剑鹏,谢旭东,赵蕾,王骏. 辅助性T细胞17及白细胞介素17在牙周炎中的作用及机制的研究进展[J]. 国际口腔医学杂志, 2022, 49(5): 586-592.
[13] 陈荟宇,白明茹,叶玲. 信号素3A与口腔常见病关系的研究进展[J]. 国际口腔医学杂志, 2022, 49(5): 593-599.
[14] 周佳佳,赵蕾,徐欣. 牙周炎相关基因多态性的研究进展[J]. 国际口腔医学杂志, 2022, 49(4): 432-440.
[15] 马玉,左玉,张鑫. 光动力疗法辅助治疗牙周炎治疗效果的Meta分析[J]. 国际口腔医学杂志, 2022, 49(3): 305-316.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!