中药橘核治疗自身免疫性睾丸炎的药效物质基础及作用机制研究

Autoimmune orchitis (AO) is one of the important factors that cause male infertility, there is still no ideal drug to cure AO. Semen citri reticulatae, the traditional Chinese medicine, has long been used for the treatment of testicular swelling. However, bioactive investigations on AO of this plant have not been reported. Thus, bioassays on experimental autoimmune orchitis (EAO) in mice were performed to evaluate the effect of this medicine, and it was demonstrated that the EtOAc and H2O parts from the ethanol extracts of Semen citri reticulatae decreased obviously on levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Moreover, the testicular histology was observed by haematoxylin-eosin (HE) staining, pathological changes had a weak tendency as treated by Semen citri reticulatae. However, the effective substances and the mechanism remain unclear. Inspired by the anti-inflammatory effects of the active fractions, we carried out a bioassay-guided isolation and characterization of the chemical constituents of this medicine. Thirteen compounds, including limonoids, flavonoids and sesquiterpene, were isolated, and compound 1 was a novel sesquiterpene glucoside. The purpose of the work was to evaluate the effect of Semen citri reticulatae on EAO. Based on the fact, this project, guided by active tracking, intends to clarify the effective substances responsible for the antagonism on EAO by methods of various modern purification and structure-identificated techniques. Furthermore, to seek for effective and antagonistic lead compounds on AO, we will study the possible mechanisms of those active compounds through toll-like receptors and tyrosine kinase receptors (TAM) pathways. Those works may provide material basis for the research and development of anti-AO drugs.

自身免疫性睾丸炎(AO)是导致男性不育的重要原因之一，目前尚无理想的治疗药物。中药橘核长期用于睾丸肿痛的治疗，但缺乏相关药理学研究的报道。我们前期研究发现，橘核乙酸乙酯和水萃取部位能明显降低实验性自身免疫性睾丸炎(EAO)小鼠血清中白细胞介素-1β及肿瘤坏死因子-α的水平。苏木素-伊红(HE)染色观察睾丸组织结构变化发现，橘核能明显减轻小鼠睾丸组织病变，然而其药效物质基础及作用机制尚不明确。现已从橘核两个活性部位得到13个化合物，类型涉及柠檬苦素类、黄酮类和倍半萜类，其中化合物1为结构新颖的倍半萜苷类。本项目旨在评价橘核抗EAO作用的基础上，以活性追踪为指导，运用多种现代分离方法及波谱分析技术研究其化学成分，明确其药效物质基础。同时从Toll样受体(TLRs)及酪氨酸激酶受体(TAM)信号通路探索其可能的作用机制，以期从橘核中发现高效的抗AO活性先导化合物，可为抗AO药物研发提供物质基础。

自身免疫性睾丸炎(AO)是导致男性不育的重要原因之一，目前尚无理想的治疗药物。中药橘核长期用于睾丸肿痛的治疗，但缺乏相关药理学研究的报道。因此，本项目在评价了橘核抗氧化及抗炎镇痛作用，验证其乙醇提取物有活性，明确其乙酸乙酯部位和水部位有明显抑制活性基础上。利用液相色谱-串联质谱技术（LC-MS/MS）对橘核主要成分及其类型进行分析，初步判断主要为柠檬苦素类及黄酮类化合物。采用多种色谱技术对橘核的化学成分进行分离纯化，根据化合物的理化性质及波谱数据鉴定其结构。从橘核中分离鉴定化合物57个，其中5个为新化合物（包括1个结构新颖的倍半萜苷类化合物），13个为首次从柑橘属中分离，29个为首次从橘核中分离得到，通过电子圆二色谱技术（ECD）确定了新化合物的绝对构型。此外，通过评价橘核对小鼠睾丸支持细胞Sertoli细胞（SC）和睾丸间质细胞Leydig细胞（LC）炎症活性，及对小鼠实验性自身免疫性睾丸炎（EAO）的作用，检测与自身免疫性睾丸炎密切相关的炎症因子、抗精子抗体及雄激素等指标，验证了橘核有抗EAO的作用，明确了乙酸乙酯及水部位为其活性部位，筛选出有明显抗小鼠 EAO 作用的化合物柠檬苦素等成分，初步明确柠檬苦素四降三萜类及黄酮类成分可能是其主要药效物质。在此基础上，通过转录组高通量测序、实时荧光定量PCR和蛋白质免疫印迹等技术，明确橘核可能通过调控小鼠睾丸Toll样-核因子受体κB（TLR-NF-κB）受体信号通路发挥抗EAO的作用。. 本项目系统揭示了橘核的物质基础，从现代药理学角度验证了橘核具有抗EAO的作用，得到了活性明显成分，初步明确了其可能的作用机制。为橘核的临床应用提供科学依据，为AO天然药物开发利用提供了物质基础。