基于“胆主窦窍”研究清胆泄热法抑制LuxS/AI-2表达拮抗鼻-鼻窦细菌生物膜形成的分子机制

The forming of bacterial biofilm(BBF), key pathological mechanism of Chronic Rhino sinusitis(CRS) is regulated by LuxS/AI-2, the bacterial quorum sensing (QS) system. “Gallbladder governs sinus orifices” theory is presented in the paper on the basis of the previous researches carried out by the Research Team. Long-term & massive-applied clinical practice has proved the definite curative effect of Clearing Gallbladder and Purging Heat Remedy upon CRS, illustrated by the application of Bi Yuan Shu Decoction, one of the well known traditional Chinese oral Liquid medicine.This research assumes that Clearing Gallbladder and Purging Heat can restrain BBF by inhibiting Luxs/AI-2 expression. The research is to select 50 CRS cases in Chengdu area and another 50 cases inYongchuan area and analyze the difference of microbiome composition on the nasal secretions before and after treatment with Biyuanshu Decoction by 16S rRNA sequential method. Ultrastructures of bioilm in Ostiomeatalex(OMC) is observed under Co focal Laser Scanning Microscope (CLSM). Content and activity of AI-2 ismeasured by fluorescence photometer. Activity changes of LuxS is detected by Elisa. Effect that Bi yuan shu Oral liquid can make on the expression of Luxs/AI-2 QS system’s representative genes is analyzed by quantitative PCR and use Bi yuan shu Decoction to restrain the BBF on OMC mucosa in vitro. In expectation of explaining the molecular mechanisms of clearing gallbladder and purging heat’s antagonism to CRS and reveal the scientific connotation of “Gallbladder governs sinus orfices” and innovate and develop TCM rhinology theory..

细菌生物膜形成是慢性鼻-鼻窦炎（Chronic Rhino Sinusitis，CRS）发生的关键病理环节，其形成受细菌群体感应系统LuxS/AI-2的调节。课题组在前期研究基础上提出了“胆主窦窍”学说。长期、大规模的临床实践证实清胆泄热法（鼻渊舒口服液为其代表方）治疗CRS疗效确切。本课题假设清胆泄热法通过有效抑制Luxs/AI-2表达而阻碍局部细菌生物膜形成，达到临床疗效。拟选取成都及永川地区CRS患者各50例，16S rRNA测序法分析鼻渊舒口服液治疗前后鼻腔菌群结构变化；激光共聚焦法分析窦口鼻道复合体黏膜细菌生物菌膜形成情况；荧光光度计法测定AI-2含量与活性；ELISA法检测LuxS酶活性变化；定量PCR分析Luxs/AI-2系统代表基因（lrgB，luxS,icaA）的表达水平。同时取CRS窦口鼻道复合体黏膜，进行鼻渊舒口服液抑制细菌生物菌膜形成的体外实验，以期阐释清胆泄热法拮抗CRS分子效应机制，揭示“胆主窦窍”的科学内涵，创新发展中医鼻科理论。

细菌生物膜形成是慢性鼻-鼻窦炎（Chronic Rhino Sinusitis，CRS）发生的关键病理环节，其形成受细菌群体感应系统LuxS/AI-2的调节。课题组在前期研究基础上提出了“胆主窦窍”学说，以此指导的清胆泄热法（鼻渊舒口服液为其代表方）治疗CRS疗效被长期、大规模的临床实践证实。本课题通过16S rRNA测序法行菌群分析、激光共聚焦法分析细菌生物菌膜形成情况、荧光光度计法测定AI-2含量与活性、定量PCR分析Luxs/AI-2系统代表基因（luxS,icaA）的表达水平、抑制细菌生物菌膜形成的体外实验等方法对本治法方药进行起效机制探究，结果显示：（1）CRS患者鼻腔优势菌种为表皮葡萄球菌，慢性鼻-鼻窦炎发病与条件致病菌表皮葡萄球菌相关，而表皮葡萄球菌形成生物膜是导致发病的关键。（2）服用鼻渊舒口服液后慢性鼻-鼻窦炎患者鼻腔菌群多样性降低（低于正常人），且优势菌群变为肠杆菌科。鼻渊舒口服液可以调整慢性鼻-鼻窦炎鼻腔菌群结构，减低菌群多样性，改变鼻腔内环境。（3）鼻渊舒口服液通过降低表葡菌生物膜形成率、破坏细菌生物膜结构而达到治疗效果。（4）鼻渊舒口服液可通过抑制LuxS/AI-2 信号系统及相关基因icaA表达影响生物膜形成。（5）据研究结果，我们推测鼻渊舒口服液可能影响其他生物膜相关调控基因信号，并与本系统共同构成对表皮葡萄球菌生物膜调控网络而起到治疗效果。这些结果初步阐明了以鼻渊舒口服液为代表的“清胆泻热法”治疗CRS的起效机制，也揭示了“胆主窦窍”学说的科学内涵，为下一步深入揭示本方法调节BF形成的基因调控网络研究奠定了基础。