牙周炎患者唾液菌群对小鼠色氨酸-芳香烃受体信号轴的影响

Effects of salivary microbiota on tryptophan-aryl hydrocarbon receptor signaling axis in mice with periodontitis

目的研究牙周炎患者唾液菌群对牙周炎小鼠色氨酸-芳香烃受体(aryl hydrocarbon receptor,AhR)信号轴的影响,为牙周炎对全身代谢的影响提供理论依据和新思路。方法于2020年6至12月在南京大学医学院附属口腔医院·南京市口腔医院收集12名牙周健康者及14例牙周炎患者唾液。根据随机数字表法将24只8周龄雄性C57/J小鼠随机分为3组:Sham组(对照组,给予无菌磷酸盐缓冲液灌胃)、P组(牙周炎患者唾液菌群灌胃组)及H组(牙周健康者唾液菌群灌胃组),每组8只。所有小鼠均用丝线结扎法诱导牙周炎,将磷酸盐缓冲液、牙周健康者和牙周炎患者唾液菌群分别对牙周炎小鼠灌胃2周。通过酶联免疫吸附测定检测小鼠血清中炎症因子水平,通过液相色谱-质谱法检测肠道和血清中的色氨酸及吲哚类代谢物的含量,使用免疫组化、荧光定量PCR检测小鼠肠道中AhR的表达,利用16S rRNA基因测序检测小鼠肠道菌群的组成。剩余的唾液样本用于检测人唾液中色氨酸及吲哚类代谢物的含量。结果P组小鼠血清中白细胞介素-1β[(162.38±39.46)pg/ml]显著高于H组[(82.83±20.01)pg/ml](t=4.40,P=0.001),TNF-α[(361.16±123.90)pg/ml]也显著高于H组[(191.66±106.87)pg/ml](t=2.54,P=0.030),P组小鼠结肠(1.18±0.05)和回肠中AhR的表达(0.80±0.13)均显著低于H组小鼠结肠(1.83±0.47)(t=3.09,P=0.015)和回肠AhR的表达(0.80±0.13)(t=4.93,P=0.001)。P组小鼠血清色氨酸[(18.1±3.8)×10^(7)]和吲哚乳酸含量[(1.9±0.7)×10^(7)]均显著低于H组血清色氨酸[(26.6±6.6)×10^(7)](t=2.49,P=0.037)和吲哚乳酸[(3.7±0.6)×10^(7)](t=4.49,P=0.002)。牙周炎患者唾液样本中色氨酸及吲哚类代谢物含量均高于牙周健康者,其中P组唾液样本吲哚丙酸检测值[(1239.39±818.72)nmol/L]显著高于H组[(56.96±38.33)nmol/L](t=2.83,P=0.022)。吲哚乳酸代谢在唾液、血清及肠道中的表达一致,牙周炎患者唾液菌群导致小鼠肠道中吲哚乳酸产生菌相对丰度下降。结论牙周炎患者唾液菌群可能通过下调肠道色氨酸-AhR信号轴的表达影响全身炎症状态。

Objective To study the effects of salivary microbiota in patients with periodontitis on the tryptophan-aryl hydrocarbon receptor(AhR)signaling axis in mice with periodontitis and to provide theoretical basis as well as new ideas for the influences of periodontitis on systemic metabolism.Methods Salivary microbiota of 12 healthy individuals and 14 patients with periodontitis were collected in Nanjing Stomatological Hospital,Medical School of Nanjing University from June to December of 2020.According to the random number table method,twenty-four mice were randomly divided into three groups:Sham group(control group),P group(periodontitis patients′salivary microbiota group)and H group(periodontal healthy individuals′salivary microbiota group).The maxillary second molars of all mice were treated with silk thread ligation to induce periodontitis.Phosphate buffer as well as salivary microbiota of periodontal healthy individuals and periodontitis patients were gavaged into periodontitis mice for 2 weeks.The expression of inflammatory factors in mice serum were detected by enzyme linked immunosorbent assay,and the expression of tryptophan and indole metabolites in intestinal tract and serum were detected by liquid chromatography-mass spectrometry.The expression of AhR in intestinal tract of mice was detected by immunohistochemistry and quantitative real time-PCR while gut microbiota constitution was detected by 16S rRNA gene sequencing.The remaining saliva samples of periodontitis patients and periodontal healthy individuals were applied to detect the expression of tryptophan and indole metabolites themselves.Results The salivary microbiota of periodontitis patients could induce the expression of interleukin-1β[P group:(162.38±39.46)pg/ml,H group:(82.83±20.01)pg/ml;t=4.40,P=0.001]and tumor necrosis factor-α[P group:(361.16±123.90)pg/ml,H group:(191.66±106.87)pg/ml;t=2.54,P=0.030]in serum of periodontitis mice,and reduce the expression of AhR in colon(P group:1.18±0.05,H group:1.83±0.47;t=3.09,P=0.015)and ileum(P group:0.80±0.13,H group:1.18±0.11;t=4.93,P=0.001).After gavage of salivary microbiota of periodontitis patients to the mice,tryptophan[P group:(18.1±3.8)×10^(7),H group:(26.6±6.6)×10^(7);t=2.49,P=0.037]and indole lactic acid[P group:(1.9±0.7)×10^(7),H group:(3.7±0.6)×10^(7);t=4.49,P=0.002]in serum of periodontitis mice were significantly decreased,but was relatively disorder in intestinal tract.However,the expressions of tryptophan and indole metabolites in saliva of periodontitis patients were higher than those of periodontal healthy individuals.There were significant differences in indole propionic acid[P group:(1239.39±818.72)nmol/L,H group:(56.96±38.33)nmol/L;t=2.83,P=0.022].What we find noteworthy was that the expressions of indolelactic acid metabolism in saliva,serum and intestinal were consistent,and salivary microbiota of periodontitis patients could reduce the relative abundance of indolelactic acid-producing bacteria in the gut,suggesting that the salivary microbiota of periodontitis patients might affect the expression of AhR through gut microbiota disorder and indolelactic acid downregulation.Conclusions Salivary microbiota in patients with periodontitis may affect the systemic inflammatory state through down-regulating the expression of tryptophan-AhR signal axis.