盐酸小檗碱调节牙周炎TGF-β诱导的Treg活化机制

Periodontitis represents a specific inflammatory response to microbial residents of the subgingival biofilm. Accumulated evidence demonstrates that PD involves bacterially derived factors and antigens that stimulate a local inflammatory reaction and activation of the innate immune system. Proinflammatory molecules and cytokine networks play essential roles in this process. Treg (regulatory T cell) have been described as a protective T-cell subset concerning tissue damage in the periodontal environment. Treg characteristically express as phenotypic markers the transcription factor forkhead box P3 (FOXp3), CD103, the glucocorticoid-inducible TNF receptor (GITR) and the inhibitory molecule cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4). Transforming growth factor-β (TGF-β) induces naive peripheral human T cells to become functional Tregs. IL-2 was also necessary for TGF-β -induced Foxp3 expression and Treg development via STAT5. IL-6 fails to regulate TGF-β-induced Foxp3 expression through Stat3 in mice. Periodontitis are treated by antibiotics in local tissue with patients, but it is the unsatisfactory therapeutic effect is unexpect. Traditional Chinese Medicine considers that intense stomach fire, yangming fire floating and damp-heat fumigating upward contribute to periodontitis. The exact of coptis in water, berberine hydrochloride, inhibits pro-inflammatory cytokines and reduces inflammatory cells infiltration. However, it is not clear that berberine hydrochloride regulates the activation of Treg. We hypothesize that berberine hydrochloride improves TGF-β-induced Treg activation via regulation of IL-2 and IL-6, and their downstream signals in periodontic tissue. In this project, the periodontitis model in rats and Treg activation in vitro were established. Using flow cytometry, western blot, realtime-PCR and so on, phenotype, receptors on membrance (IL-2R, IL-6R, TGF-βR), cytokines (IL-2, IL-6, TGF-β, IL-10) and transcript factors (Smad3, STAT5, STAT3) of Treg in periodontic tissue and circulating blood are examined to disclose the mechaniam of berberine hydrochloride ACh that regulate TGF-β-induced Treg activation in periodontitis, offering new strategy and promising approach for therapy of periodontitis.

牙周炎是宿主对牙龈下微生物的特异性炎症反应。调节性T细胞（Treg）可对抗过度炎症反应引起的牙周损伤；转化生长因子-β（TGF-β）可诱导Treg的发育，受IL-2和IL-6调节。临床以局部给予抗生素治疗为主，但疗效不佳。中医认为牙周炎是胃腑积热，阳明火炽，湿热熏蒸于上而形成牙宣。研究发现黄连水提取物盐酸小檗碱可降低牙周炎致炎因子并减少炎性细胞的浸润，对Treg的调节作用尚不清楚。本项目建立大鼠牙周炎模型和体外Treg培养模型，给予盐酸小檗碱，运用流式细胞仪、western blot和realtime-PCR等方法，检测牙周组织和循环血中IL-2、IL-6、TGF-β、IL-10，Treg表面受体（IL-2R, IL-6R, TGF-βR）和表型，转录因子Smad3、STAT5、STAT3活性；阐明盐酸小檗碱调节TGF-β诱导Treg活化的信号通路，为黄连治疗牙周炎提供新的理论依据。

牙周炎主要是由细菌感染引起牙周组织的慢性炎性疾病，造成进行性牙周附着丧失和牙槽骨吸收，形成牙周袋，最终可导致牙齿脱落，是成人失牙的主要原因。研究发现宿主的免疫反应在牙周炎的发生发展中起到了关键性的作用。本课题主要研究了盐酸小檗碱（berberine）和黄芩苷（baicalin）对大鼠牙周炎治疗机制，发现（1）盐酸小檗碱①可呈浓度依赖性改善大鼠牙周情况；②减轻牙槽骨的丧失；③降低龈沟液肿瘤坏死因子-α（TNF-α）含量、增加龈沟液转化生长因子-β（TGF-β）和白介素-10（IL-10）的含量；④减轻牙龈组织TNF-α、IL-1β含量和髓过氧化酶含量；⑤诱导调节性T淋巴细胞（Treg）分化增加；⑥上调牙龈组织TGF-β下游信号分子Smad3的磷酸化水平；⑦抑制炎症通路p38 MAPK/NF-κB的活化；⑧减少牙龈组织MMP-2/9表达并增加TIMP-1表达；最终发挥对牙周炎的抗炎治疗作用。（2）不同浓度黄芩苷（baicalin；50、100、200 mg/kg/d）干预，发现黄芩苷①可呈浓度依赖性改善大鼠牙周情况；②减轻牙槽骨的丧失；③减少牙周炎性细胞浸润；④减轻牙龈组织TNF-α、IL-1β含量和髓过氧化酶含量；⑤抑制牙龈组织TLR-4m RNA和蛋白表达；⑥降低p38 MAPK/NF-κB磷酸化；最终发挥对牙周炎的抗炎治疗作用。综上所述，盐酸小檗碱（berberine）和黄芩苷（baicalin）均可以通过调节宿主的免疫反应减轻大鼠牙周炎牙槽骨的吸收和牙龈的炎症反应，从而发挥对牙周炎的治疗作用，为临床上使用这两种中药单体治疗牙周炎提供了一定的理论依据。