TLR4/9介导牙周炎宿主CD25+B细胞表达IL-10、IL-35及TGF-β的效应及机制

The effects and mechanisms of Toll like receptors 4/9 mediating IL-10,IL-35 and TGF-βexpressions by CD25+B cells from periodontitis individuals

目的分析牙周炎宿主CD25^(+)B细胞中IL-10、IL-35及TGF-β的表达特点,评价TLR4/9的表达活化对上述过程的影响。方法SD大鼠随机分为健康组、早期牙周炎组、晚期牙周炎组,采用结扎法建立牙周炎模型。检测各组动物牙龈及外周血CD25^(+)B细胞内IL-10、IL-35和TGF-βmRNA的表达水平,牙龈CD25^(+)B细胞内TLR 2/4/7/9、MyD88、TRAF6的表达活化水平。建立细胞培养体系,分析TLRs/MyD88信号对CD25^(+)B细胞表达分泌IL-10、IL-35和TGF-β的影响。结果早期牙周炎组牙龈CD25^(+)B细胞内IL-10、TGF-βmRNA表达水平高于健康组(P<0.05)。晚期牙周炎组牙龈CD25^(+)B细胞内IL-10、IL-35及TGF-βmRNA表达水平高于健康组(P<0.05),外周血CD25^(+)B细胞内IL-10 mRNA高于健康组(P<0.05),余差异无统计学意义(P>0.05)。相比健康组,晚期牙周炎组牙龈CD25^(+)B细胞内TLR4/9及MyD88的表达磷酸化水平均升高(P<0.01)。细胞实验结果显示,TLR4激动剂上调IL-10、IL-35、TGF-βmRNA表达及IL-10的浓度(P<0.05),TLR9激动剂上调IL-10、TGF-βmRNA表达及IL-10的浓度(P<0.05),TLR4/TLR9激动剂联合应用上调所有检测指标的表达及浓度(P<0.05),MyD88拮抗抑制上述过程(P<0.05)。结论牙周炎晚期,牙龈CD25^(+)B细胞表达IL-10、IL-35和TGF-β水平升高,这一过程可能受TLR4/9-MyD88信号调控。

Objective To analyze the expressions of IL-10,IL-35 and TGF-βin CD25^(+)B cells from periodontitis individuals,and then establish how the activation of TLR4/9 affects the above processes.Methods SD rats were randomly divided into healthy group,primary periodontitis groups and severe periodontitis group;experimental models were performed by ligation.Expression of IL-10,IL-35 and TGF-βmRNA in CD25^(+)B cells from gingiva and peripheral blood,expression and activation of TLR 2/4/7/9,MyD88,TRAF6 in gingival CD25^(+)B cells were detected.The effect of TLRs/MyD88 on IL-10,IL-35 and TGF-βexpressions and production were evaluated by cell culture experiments.Results CD25^(+)B cells from gingiva of primary periodontitis individuals showed improved expression of IL-10 and TGF-βmRNA compared with the healthy ones(P<0.05);cells from peripheral blood did not present the same tendency.CD25^(+)B cells from gingiva of severe periodontitis individuals showed improved expression of IL-10,IL-35 and TGF-βmRNA compared with the healthy ones(P<0.05),cells from peripheral blood showed higher IL-10 mRNA level than the healthy ones(P<0.05).Compared with healthy individuals,the expression and phosphorylation of TLR4/9 and MyD88 in CD25^(+)B cells from gingiva of severe periodontitis individuals were increased(P<0.01).In cell culture experiments,TLR4 agonist promoted IL-10,IL-35 and TGF-βmRNA expression and IL-10 secretion(P<0.05);TLR9 agonist improved IL-10 and TGF-βmRNA expression and IL-10 secretion(P<0.05).The combined use of TLR4/9 agonist could increase the expression and secretion of all the detected indexes(P<0.05);MyD88 antagonism decrease the above effects(P<0.05).Conclusion The expressions of IL-10,IL-35 and TGF-βin gingiva CD25^(+)B cells increase during periodontitis,which may be regulated by TLR4/9-MyD88 pathway.