运用差异蛋白组学方法研究虫草益肾方多靶点调控肾间质纤维化关键信号转导通路防治慢性肾衰竭

Renal Interstitial Fibrosis(RIF) is a process and pathological presentation shared by many types of chronic kidney disease(CKD),which contributes to the development of the terminal phase of Chronic Renal Failure (CRF).Using drugs to slow down the process of RIF is crucial for protection against CRF. Currently, in order to interfere RIF, anti-RAS system drugs, antioxidants, immunosuppressive agents and statin drugs have been adopted as main methods in treatments, but the results are not satisfactory. On the base of summarizing the previous research of the theory of Qi Transformation, the applicant points out that Chongcaoyishen Prescription can effectively slow down or even reverse the process of RIF and downregulate the TGF-β and NF-κB signaling pathway in the transcriptional level. However, the identification of drug targets and the complex regulation mechanism between these drug targets remain unclear. This study employes unilateral ureteral obstruction to establish rat RIF model and interfere it with Chongcaoyishen Prescription and fosinopril. In order to identify target proteins related to RIF and the drug targets of Chongcaoyishen Prescription, we use proteomics mass spectrometry technology to measure the expression of proteins related to these key signaling pathways. The applicant try to validate the advantage of Chongcaoyishen Prescription in preventing and treating RIF by systematically analyzing the way Chongcaoyishen Prescription regulates proteins related to RIF and the differences between drug targets of Chongccaoyishen Prescription and fosinopril. The completion of this research will widen the theory basis of treating RIF with Chinese medicine and provide new insight into the treating CRF with Chinese medicine.

肾间质纤维化（RIF）是各种慢性肾脏病进展至慢性肾衰竭（CRF）终末阶段的共同病理表现和途径，药物延缓RIF是防治CRF的关键。目前多以抗RAS系统药物、抗氧化剂、免疫抑制剂、他汀类药等干预，但效果不甚理想。申请人基于中医气化理论的研究表明，虫草益肾方可有效延缓或逆转RIF，转录水平研究证实其对TGF-β、NF-κB信号通路有下调作用，但其确切的作用靶点及调控机制不清。本项目采用单侧输尿管结扎法制备大鼠RIF模型，以虫草益肾方和抗RAS系统药物福辛普利干预，采用蛋白组学质谱技术检测肾脏组织中上述关键信号通路及其上下游蛋白的表达，鉴定RIF关联蛋白及药物靶蛋白。系统分析虫草益肾方调控RIF关联蛋白的作用，及与福辛普利作用靶点的差异，探究其与药效的关系。从而阐明虫草益肾方多途径多靶点防治RIF的优势。本项目的完成将拓宽中医药防治RIF的理论基础，开辟临床中药防治CRF的新途径。

据国际肾脏病学会和肾脏基金联合会在2015年肾脏病日公布的统计数字显示，目前全球有超过5亿的肾脏病患者，并且这一数字正随着糖尿病、高血压发病率的提高而不断上升。如何延缓肾衰竭的进展，已经成为肾病医师和患者十分关注的问题。肾间质纤维化是慢性肾病患者肾脏功能逐渐减退的主要原因，与预后密切相关。虫草益肾方在前期的临床应用中取得了较好的疗效，可改善肾功能，延缓肾衰竭的进展；实验研究亦显示虫草益肾方可通过抑制TGF-β、NF-κB等达到延缓肾病进展的目的，但其作用机制尚不完全清楚。本研究通过建立单侧输尿管梗阻（UUO）大鼠模型，研究虫草益肾方对肾间质纤维化大鼠肾功能和组织形态学的影响，利用iTRAQ联合质谱分析的蛋白质组学方法探讨肾间质纤维化机制以及虫草益肾方抗肾间质纤维化的作用机制，并通过体内、体外实验验证部分筛选出的差异蛋白及信号通路。参照中药新药标准，对虫草益肾方的颗粒制剂进行了新药研究。实验结果显示虫草益肾方可改善大鼠一般状态，增加UUO大鼠体重、尿量；虫草益肾方可减少UUO大鼠尿蛋白，降低血清肌酐、尿素氮水平，改善肾脏功能；虫草益肾方可减轻UUO大鼠肾脏病理改变；UUO大鼠肾间质纤维化的机制与多个细胞因子、多条信号通路、多个生物过程有关，而虫草益肾方延缓肾间质纤维化的作用除调节TGF-β、NF-κB信号通路外，还可能是通过抑制Wnt/β-catenin、megsin、p38MAPK等信号通路实现的。本研究运用差异蛋白组学研究肾间质纤维化的机制，从整体角度研究病理状态下机体内蛋白质的变化，了解蛋白质之间的相互作用和联系，从而揭示肾间质纤维化的作用机制和潜在药物作用点，进一步阐明虫草益肾方防治肾间质纤维化的机制，为中医药学防治肾间质纤维化提供理论依据，对揭示中药复方复杂药效的作用机制提供了新思路，为抗肾脏纤维化的治疗提供新的可能途径及干预靶点，具有广阔的应用潜力和市场前景。