Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.
寒凝血瘀证是妇科临床常见证候。然而目前关于妇科寒凝血瘀证的分子机制研究较少。本研究旨在探讨妇科寒凝血瘀证的发生和调控机制，为妇科寒凝血瘀证的治疗提供理论依据。运用Label-free定量蛋白质组学筛选原发性痛经寒凝血瘀证患者、子宫内膜异位症寒凝血瘀证患者和健康女性的血浆蛋白。基于“异病同证”理论，将各组获得的差异表达蛋白进行交集映射以获取共有的差异表达蛋白。将交集部分的妇科寒凝血瘀证差异表达蛋白与课题组前期依据“同病异证”理论所获取的妇科寒凝血瘀证差异表达蛋白再次进行交集映射以获取两部分所共有的妇科寒凝血瘀证差异表达蛋白。将共有的差异表达蛋白进行后续生物信息学分析，并用酶联免疫吸附测定(ELISA)法对所挑选的目标差异表达蛋白进行验证。共筛选出67个共有的妇科寒凝血瘀证差异表达蛋白，包括33个上调差异表达蛋白和34个下调差异表达蛋白。对差异表达蛋白进行功能分类统计主要涉及代谢过程、能量的产生与转化、免疫过程、抗氧化活性、对刺激的反应、生物黏附等。亚细胞定位主要在细胞质、细胞核、细胞外等。GO富集分析表明，上调的差异表达蛋白主要与脂质转运、细胞迁移和炎症反应有关，下调的差异表达蛋白主要与细胞连接、代谢和能量反应有关。蛋白结构域富集分析和聚类分析显示，差异表达蛋白的功能主要涉及细胞增殖和分化、细胞形态、代谢、免疫等。KEGG通路富集分析和聚类分析表明，上调差异表达蛋白主要富集在炎症、氧化损伤等，下调差异表达蛋白主要富集在炎症、细胞黏附、细胞凋亡与代谢等。ELISA结果显示，与健康女性相比，妇科寒凝血瘀证患者差异表达蛋白中黑色素瘤细胞黏附分子(MCAM)、载脂蛋白C1(APOC1)水平显著升高；血管舒张剂刺激磷蛋白(VASP)、脂肪酸结合蛋白5(FABP5)、纽蛋白(VCL)水平显著降低。本研究为妇科寒凝血瘀证的物质基础提供了依据。寒冷可能通过影响免疫过程、炎症反应、代谢过程、能量产生与转化、氧化损伤、内皮细胞功能障碍等差异蛋白的表达，从而导致妇科寒凝血瘀证的发生。.
Keywords: cold coagulation and blood stasis syndrome; endometriosis; primary dysmenorrhea; proteomics; traditional Chinese medicine; 中医; 原发性痛经; 子宫内膜异位症; 寒凝血瘀证; 蛋白质组学.
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