当归有效组分调经止痛的机制及效应小分子研究

A number of primary dysmenorrhea can be found in adolescent girls after menarche, their learning and working will be delayed while dysmenorrhea happens severely. At present, main treatment for Dysmenorrhea with nsaids, COX-2 inhibitors, analgesics, traditional Chinese medicine, angelica sinensis is an important medicine for Treating Dysmenorrhea in Clinical Chinese medicine. Our previous studies have shown that the low polar components from Radix of angelica sinensis are really effective components for relieving spasms of the smooth and treating dysmenorrheal, but the effect mechanism and composition of effective components has not been revealed yet. Current study shows prostaglandin secretion in primary dysmenorrhea is an important factor, prostaglandin PGF2 Alpha, and its transformation products can cause contraction of uterine smooth muscle and vascular smooth muscle, TXA2 promoting generation of small vessel thrombosis and platelet aggregation which inducing ischemia and dysmenorrheal. Prostaglandin concentrations are regulated by endometrial progesterone, estrogen, vasopressin regulation, PGE2 and PGI2 can relax uterine smooth muscle, causing vasodilatation, preventing thrombosis generation, improving microcirculation then ease the pain. Base on above all, We think that Generation of PGF2 Alpha, PGE2, and PGI2 and metabolism of prostaglandin are regulated by Angelica sinensis in uterine Smooth Muscle tissue, that may be one of the molecular biological mechanism for Angelica sinensis preventing and treating Dysmenorrhea. Above on this reason, from view of the regulation of prostaglandin secretion on dysmenorrhea model rats, this project aims to analyze effective molecules and possible mechanism of effective components from Angelica sinensis for treating dysmenorrhea by using flow cytometry and molecular biology and liquid chromatography-mass spectrometry technology.

原发性痛经严重时误工误学，目前治疗以非甾体抗炎药、COX-2抑制药、镇痛药、中药为主，当归为中医妇科调经止痛要药，项目组前期研究显示：当归中低极性组分是缓解平滑肌痉挛与治疗痛经的高效组分，但尚未揭示其效应机制与效应组分组成。研究证实前列腺素分泌异常是原发性痛经的重要因素，PGF2α、前列腺素的转化产物均可引起子宫平滑肌和血管平滑肌收缩，TXA2促进微小血管收缩和血小板聚集栓塞而致缺血引起痛经，前列腺素在子宫内膜的浓度受到孕激素、雌激素、加压素的调节， PGE2和PGI2则引起子宫平滑肌松弛、血管扩张、防止血栓生成改善微循环，缓解痛经。据此。我们认为调控子宫平滑肌组织前列腺素PGF2α、PGE2和PGI2的生成与代谢可能是当归防治痛经的分子生物学机制之一。基于此，本项目拟采用流式细胞术、分子生物学与液-质联用技术从调控痛经模型大鼠前列腺素的分泌角度，解析当归调经止痛的效应分子与可能机制。

为探讨当归有效组分调经止痛的机制及效应小分子研究，本实验对当归治疗原发性痛经的效应组分进行提取分离，采用GC-MS方法对该组分进行成分分析，运用网络药理学方法对该组分治疗原发性痛经的药效物质和潜在作用机制进行分析，通过当归效应组分对痛经小鼠模型、大鼠模型、细胞模型的干预作用研究，探讨当归效应组分对痛经细胞模型的干预作用。研究发现，组分1为抑制原发性痛经离体子宫收缩作用最显著部位；GC-MS结果显示当归治疗原发性痛经主要效应组分经鉴定有3-丁烯基苯酞、藁本内酯、β-金合欢烯、β-花柏烯、β-红没药烯、β-柏木烯、β-雪松烯、桉油烯醇等44个化学成分；网络药理学研究显示显示，3-丁烯基苯酞、藁本内酯、β-金合欢烯、β-花柏烯、β-红没药烯、β-柏木烯、β-雪松烯、桉油烯醇、十五烷酸、十五酸乙酯可能是当归治疗原发性痛经的主要药效物质基础，当归治疗原发性痛经效应组分可能作用于PTGS2、PTGS1、PPARG、UGT2B4、CA2、ALOX5、UGT2B7、PPARA、FABP2、CA3等10个靶点进而参与调控花生四烯酸代谢、卵巢类固醇生成、类固醇生物激素合成等生物过程而发挥原发性痛经的作用，预测结果中核心成分藁本内脂对原发性痛经具有缓解作用，可降低小鼠血清及子宫组织中PGF2α水平及PGF2α/PGE2比值，升高PGE2水平，提示藁本内酯通过与PTGS靶点结合，调节花生四烯酸代谢是其治疗原发性痛经的机制之一；痛经小鼠模型研究结果显示，己烯雌酚或戊酸雌二醇作为造模药物均可，0.29g/kg为有效作用剂量，当归效应组份可改善痛经模型小鼠的疼痛症状，降低痛经模型小鼠血清及子宫AVP、PGE2、PGF2a含量；当归效应组分对PD模型大鼠干预作用研究结果显示，当归效应组分对PD具有显著的镇痛作用，降低模型大鼠血清及子宫组织中PGE2、AVP、PGF2α、ET-1含量而升高P、NO含量，抑制PD大鼠子宫组织中PGES、PGFS 、OTR及VPRmRNA的表达，改善痛经模型大鼠病理学状，减轻PD模型大鼠子宫组织中ET-1蛋白的表达；当归效应组分对PD细胞模型干预作用研究显示，在PGE2、P、AVP药物终浓度在10-9 mol/L，培养时间为24h时，细胞中的PGES和PGFS mRNA表达水平最高，在3.0 mg/ml、2.5 mg/ml、2.0 mg/ml、1.5mg/ml浓度下，P