异常黏液质型阳痿病证及其方药干预与体内蛋白表达调控的关系研究

Impotence is a predominant disease in Uygur Medicine, and its main type is abnormal phlegmatic syndrome. In our previous study, we investigated the serum proteomics of abnormal phlegmatic syndrome of impotence disease with and without drug intervention with Yimusake and found 23 protein as common candidate biomarkers. In this study, we will quantify the expression of these proteins in serum of impotence patients and animals to identify common index shared by clinical patients and animal models. Using the above animal models with and without drug intervention, we will collect potential target organs of abnormal phlegmatic syndrome, such as hypothalamus, pituitary, testis, penis. According to the candidate protein expression level and protein phosphorylation level in these organs, we will define the relationship between the changes of serum protein in the whole body and gene expression and regulation in the target organs. Furthermore, we will treat corpora cavernosa endothelial cells with the serum of animals with abnormal phlegmatic syndrome of impotence disease with and without drug intervention. By analyzing the candidate protein levels and endothelial functions, we are able to explore the effect of syndrome and drug intervention on target cell function of impotence syndrome. In the present study, we are going to establish protein biomarkers of impotence disease syndrome, to find the potential target for drug treatment, and to explore the relationship between the occurrence of impotence disease and the abnormal changes in functions of multiple systems and organs as a whole body. The present study will provide the scientific basis for the Uygur Medicine theory and clinical practice about "combination of disease and syndrome "and " prescriptions corresponding to syndromes ".

阳痿是维医优势病种，其主要证型是异常黏液质。前期工作中，我们通过异常黏液质型阳痿病证及其方药伊木萨克片干预的血清蛋白质组学研究，发现23种共性候选蛋白质标志物。本研究拟以上述蛋白质为指标，对阳痿患者及动物模型血清进行定量分析，明确临床和动物模型的共同指标；利用上述动物模型及其方药干预，收集下丘脑、垂体、睾丸、阴茎等阳痿病证的潜在靶器官组织，通过候选蛋白质表达和蛋白质磷酸化分析，明确血清水平的整体变化与特定组织内基因表达和调控的关系；收集阳痿病证动物模型及其方药干预后血清，对阴茎海绵体内皮细胞进行干预，通过候选蛋白质水平及内皮功能指标分析，探讨阳痿病证及方药干预对内皮细胞功能的影响。通过本研究，建立阳痿病证的蛋白质标志物，方药作用的潜在靶点，从整体观探讨阳痿病证发生与多系统和组织功能异常变化的关系及方药的多靶点作用机制，为诠释“病证结合”、“方证相应”等维医理论和临床实践的科学性提供依据。

本研究通过对ED病证大鼠模型与对应方药伊木萨克片相关23种特异性蛋白质开展临床、动物模型与细胞层面的研究，首先明确了ED患者不仅表现为ED，同时可伴发性欲减退，ED的发生发展中性腺轴出现以血清睾酮降低雌二醇升高为特征内分泌紊乱，其不仅可能是ED发生重要机制，同时和伴发性欲减退有着密切关系，而肾上腺轴出现COR显著升高，并且肝功能改变或损伤可能和ED存在着密切关系。经对23种特异性蛋白质基于MRM技术验证，初步确定TF、APCS、APOH、CRP、A1M和PF4共6种蛋白质为临床ED患者和ED动物模型共同的血清特异性蛋白质指标，经进一步与动物模型血清中验证结果进行综合分析，进一步确认APOH、CRP和A1M 3个指标作为临床ED患者和ED动物模型共同的蛋白质指标，TF、APCS、PF4需通过别的技术方法在ED模型中进一步验证，而ORM1、HPX、RBP4、TTR、ANTPT1 5个指标可作为该病证ED大鼠模型候选血清蛋白标志物。随后通过在病证动物模型阴茎、肝脏、下丘脑等靶器官、组织等层面上开展的mRNA和蛋白质表达水平改变的研究后得出，CRP、APOH、A1M、APCS、RBP4、ORM1、TTR、TF、PF4、HPX和ANGPT1等指标在上述潜在靶器组织中均有不同程度的表达，相关指标在血清中含量改变和上述阴茎组织、肝脏、垂体等不同器官组织中表达改变有关，而伊木萨克片的治疗作用亦和反向调控RBP4、CRP、ANGPT1等指标在阴茎、肝脏等相关组织中表达水平有关。随后在对大鼠阴茎海绵体内皮细胞（CCECs）分离、培养并鉴定的基础上，通过细胞活性蛋白干预研究，发现RBP4、CRP和APOH/ANGPT1、TF 5个特异性蛋白对细胞增值的抑制/促进作用，明确对细胞周期影响特征。最后对RBP4和CRP对CCECs影响机制研究表明，其可通过增加CCECs中ET-1表达，下调eNOS水平并降低NO含量来改变内皮细胞功能，并降低CCECs血管形成能力；而伊木萨克片及其提取物均可阻断RBP4和CRP对CCECs的影响，逆转RBP4和CRP干预CCECs后发生的抑制细胞增殖现象，同时显著促进RBP4、CRP干预后的CCECs的增殖，并提高细胞活力；其机制和降低ET-1的表达，提升eNOS表达水平和NO含量，并显著改善CCECs的血管形成能力有关。