【作者】 程兴群； 吴红崑； 宋瑜； 徐欣； 周学东；

【Author】 Xingqun Cheng;Hongkun Wu;Yu Song;Xin Xu;Xuedong Zhou;The State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases,West China Hospital of Stomatology, Sichuan University;The Department of Geriatric Dentistry, West China Hospital of Stomatology,Sichuan University;The Department of Cariology and Endodontics, West China Hospital of Stomatology,Sichuan University;

【机构】 口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学； 四川大学华西口腔医院老年口腔科； 四川大学华西口腔医院牙体牙髓病科；

【摘要】 目的 :牙菌斑生物膜微生态平衡影响龋病发生发展。血链球菌和戈登链球菌等口腔共生菌通过丙酮酸氧化酶SpxB,合成细菌源性过氧化氢（hydrogen peroxide,H₂O₂）,抑制变异链球菌等致龋菌生长;牙菌斑中H₂O₂浓度与致病菌丰度呈负相关,提示H₂O₂是调控牙菌斑生物膜微生态平衡的重要因子。靶向调控细菌H₂O₂合成能力,阻止生理性牙菌斑向病理性牙菌斑进展,维持或一定程度恢复口腔微生态平衡,可能是预防龋病的有用策略。本研究的目的是探讨外源性镁离子能否促进细菌源性H₂O₂合成,提高口腔共生菌生态竞争力,在维持口腔微生态平衡中发挥作用。方法 :检测血链球菌、戈登链球菌和其它口腔链球菌在Mg²⁺存在环境中的生长和H₂O₂产生情况。采用RT-qPCR、Western blot和荧光素酶法检测SpxB蛋白表达水平变化。构建Mg²⁺与SpxB蛋白相互作用的三个必须氨基酸（N 474、D 447和Q 476）突变株,检测细菌H₂O₂产生。通过平板抑菌实验检测Mg²⁺存在环境中产H₂O₂细菌与变异链球菌的竞争。构建双菌种生物膜,通过CFU计数检测细菌分布情况。构建大鼠动物模型,利用生物光子成像技术原位评估大鼠口腔内生物膜细菌组分及定植分布。结果 :在低浓度Mg²⁺环境中,血链球菌和戈登链球菌等口腔共生菌H₂O₂合成能力显著提高,对变异链球菌的抑制作用增强,在双菌种生物膜中丰度和小鼠口腔内定植数量升高。结论 :外源性Mg²⁺可提高血链球菌和戈登链球菌等口腔共生菌H₂O₂合成能力和在生物膜中的竞争能力,提示Mg²⁺补充剂作为一个潜在的益生元,在维持口腔微生态平衡和/或逆转口腔菌群失调中可能发挥重要作用。更多还原

【Abstract】 Objective: The pyruvate oxidase（SpxB） dependent production of H₂O₂ is widely distributed among oral commensal streptococci. Several studies confirmed the ability of H₂O₂ to antagonize susceptible oral bacterial species including caries associated Streptococcus mutans as well as several periodontal pathobionts, establishing H₂O₂ production as a mechanism of molecular commensalism. Oral diseases like caries and periodontal disease are the result of a dysbiotic community. An obvious characteristic of a dysbiotic community is the decline in the abundance of commensal streptococci. Modulating H₂O₂ production under environmental conditions that otherwise support the development of a dysbiotic community could be a useful strategy to prevent oral diseases and maintain a symbiotic dental biofilm. The objective of this study was to explore whether magnesium supplementation was able to positively influence the competitive production of H₂O₂ thus favoring commensal streptococci fitness. Methods: The growth and H₂O₂ production of S. sanguinis, S. gordonii and other streptococci in the presence of Mg²⁺ were measured. Magnesium dependent SpxB expression was quantified with qRT-PCR, Western blot and luciferase assay. Magnesium binding site mutants in S. gordonii and S. sanguinis were constructed by exchanging D 441 with alanine and N 468 with valine to disrupt the interaction of SpxB with Mg²⁺. Inhibition assays between S. sanguinis/S. gordonii and S. mutans were tested on BHI plates with/without Mg²⁺. Dual species biofilm competition was used to verify whether an increased level of Mg²⁺ has an effect on bacterial cell number and final H₂O₂ concentration. A luciferase-based murine biophotonic imaging assay was developed to measure oral bacteria ecological colonization in situ. Results: Magnesium is a cofactor for SpxB catalytic activity and supplementation is increasing the production of H₂O₂ in vitro. Mg²⁺ affects spxB transcription and SpxB abundance in Streptococcus sanguinis and Streptococcus gordonii. The competitiveness of low passage streptococcal clinical isolates is positively influenced in antagonism assays against S. mutans. In growth conditions normally selective for S. mutans, Mg²⁺ supplementation is able to increase the abundance of S. sanguinis in dual species biofilms. Using an in vivo biophotonic imaging platform, we further demonstrate that dietary Mg²⁺ supplementation significantly improves S. sanguinis oral colonization and persistence in mice. Conclusions: In summary, our results support a role for Mg²⁺ supplementation as a potential prebiotic to promote a symbiotic dental microbiota and/or reverse oral dysbiosis.更多还原