葛根对选择性毒蕈碱M3受体拮抗剂治疗膀胱过度活动症的增效减毒机制

Selective muscarinic acetylcholine receptor-3 (M3 receptor) antagonists were used for the treatment of urinary incontinence caused by the overactive bladder (OAB). However, when their dosing increases, their side effects (e.g. dry mouth) are more serious. Puerariae Lobatae Radix (Gegen in Chinese), as a Chinese herbal medicine traditionally used for promoting salivary secretion, is potentially good for dry mouth induced by taking M3 receptor antagonists. Our previous in vivo study has preliminarily shown that after 3-week treatment in rats, Gegen water extract reduced muscarinic agonist-induced tonic contraction and phasic frequency in detrusors as well as electrical field stimulation (EFS)-induced tonic contraction; Meanwhile, synergism between Gegen water extract and M3 receptor antagonist has been found on EFS-induced tonic contraction. Gegen water extract also reduced the water intake in rats treated with high dose of darifenacin. However, the pharmacological substances in Gegen water extract responsible for M3 receptor inhibition and further pharmacological mechanisms have not been elucidated. The current study will investigate the beneficial effects of Gegen water extract combined M3 receptor antagonist for treating OAB, according to their pharmacological herb-drug interactions. This study will firstly investigate the pharmacological substances in Gegen water extract responsible for M3 receptor inhibition by using preparative chromatography, ex vivo detrusor contraction, LC-Q-TOF-MS and NMR technics. The possible signaling pathways of M3 receptor involved in Gegen-induced relaxation will be investigated by transcriptomics, quantitative real-time PCR and Western blotting. Furthermore, the promoting effect of Gegen on salivary secretion against M3 receptor antagonist and the underlying mechanisms will be studied. After these investigations, this study will provide information on the pharmacological interactions for treating OAB between Gegen and M3 receptor inhibitors, which will be the evidence for their combinational uses in clinics.

选择性毒蕈碱M3受体拮抗剂能缓解膀胱过度活动症(overactive bladder，OAB)导致的尿频、尿失禁，但能引起口干等副作用。葛根生津，能促进唾液分泌，理论上能缓解M3受体拮抗剂引起的口干。前期研究发现，大鼠灌胃给药三周，葛根水提物能抑制M受体激动剂引起的逼尿肌收缩，且协同M3受体拮抗剂抑制电流脉冲引起的逼尿肌收缩；而葛根还减少了M3受体拮抗剂高剂量组大鼠的饮水量。但所涉及的药效物质和分子药理机制尚不明确。基于前期研究，本课题拟研究葛根加强M3受体拮抗剂治疗OAB的增效机制及缓解口干的减毒机制。本课题拟通过制备液相、LC-Q-TOF-MS和NMR等技术确定葛根抑制M3受体的药效物质基础，并通过转录组学、实时定量PCR、免疫印迹等分子生物学技术考察葛根对M3受体通路的影响，及其缓解M3受体拮抗剂引起口干的分子机制。本研究将会为葛根联合M3受体拮抗剂治疗OAB的临床应用提供理论依据。

选择性毒蕈碱M3受体拮抗剂（如达非那新）能缓解膀胱过度活动症(overactive bladder，OAB)导致的尿频、尿失禁，但能引起口干等副作用。葛根生津，能促进唾液分泌，理论上能缓解M3受体拮抗剂引起的口干。前期研究发现了葛根对达非那新有增效作用，且能减少大鼠饮水量。为了明确葛根对选择性M3受体拮抗剂的增效减毒作用，本研究研究了葛根抑制逼尿肌的药效物质基础和分子机制，以及缓解该类拮抗剂导致口干的减毒机制。本研究首先基于自发性高血压大鼠（膀胱缺血OAB）模型，采用尿流动力学技术明确了口服葛根能够显著改善膀胱顺应性、排尿间隔等储尿期参数。基于制备液相分离技术和离体逼尿肌收缩实验的活性追踪实验，明确了葛根水提物主要成分葛根及其糖苷衍生物作为前体药物，分别经体内代谢为大豆苷元后于体内抑制膀胱M3受体的活性，并以分子对接技术佐证。基于转录组学和代谢组学整合分析技术以及分子生物学技术验证，葛根还通过花生四烯酸代谢介导的PTGFR/PLCβ1信号通路、NTSR1/PLCβ1信号通路和p-SMAD/MMP13/Type III collagen信号通路对逼尿肌过度活跃进行调控。此外，在明确葛根促进唾液分泌、缓解达非那新所致口干的药效基础上，基于代谢组学和网络药理学技术阐明了葛根缓解口干的分子药理机制，主要是通过改变唾液腺组织内的代谢通路如苯丙氨酸、酪氨酸和色氨酸的生物合成等，进而调控花生四烯酸、钙离子释放和转运体能力等分子功能。本研究的成果为葛根治疗OAB的临床应用提供了理论依据和科学证据。