以B型利钠肽代谢干预和基因调控为基础的心衰创新治疗靶点研究

In patients with heart failure (HF),plasma B-type natriuretic peptide (BNP) levels are increased, but their anti-HF effects are weakened. Being called as BNP paradox internationally, this phenomenon has perplexed people for many years due to its unclear mechanisms. The possible explanation is that with the full-length form of BNP decreasing, its truncated forms are increased in patients with HF, which weakens the activation of BNP functional receptor (NPR-A). Meanwhile, it is possible that there is down-regulated NPR-A in patients with HF. We found：①lacking C-terminal amino acid of BNP weakened the binding of BNP to NPR-A and its biological functions, suggesting that insulin-degrading enzyme (IDE) is a key enzyme of BNP metabolism; ②the mutation of BNP precursor(NPPB) gene single nucleotide polymorphism (SNP) site rs198389 significantly inhibited the transcription of NPPB gene,suggesting that it induced the deterioration of cardiac function by reducing the synthesis of BNP；③there was NPPB gene site with different degree of methylation in patients with HF, suggesting that methylation was related to the development of HF. This project will first enhance the functions of BNP from the perspective of metabolic intervention through decreasing the C-terminal truncated from of BNP with the inhibitors of IDE (ML-345 and 6bk), and through up-regulating NPR-A. Moreover, in the aspect of gene regulation, gene mutation (SNP) and epigenetics (DNA methylation and miRNA/proteins in exosomes) will be used to explore molecular mechanisms and promote gene transcription. These will help us clarify the mechanisms of BNP paradox, understand the process of HF, and explore innovative therapeutic targets.

心衰（HF）患者B型利钠肽（BNP）增高，抗HF作用却减弱。这困扰人们多年，国际上称BNP悖论，机制不明。可能因全长BNP减少，截短形式增多，后者对BNP功能受体（NPR-A）作用减弱，或NPR-A下调。申请人前期发现：①BNP缺失C端抑制BNP结合NPR-A和发挥功能，即胰岛素降解酶（IDE）是BNP代谢关键酶；②B型利钠肽前体（NPPB）基因SNP位点rs198389突变抑制NPPB基因转录，即其通过减少BNP合成致心功能恶化；③HF患者NPPB基因有甲基化差异位点，即其甲基化与HF发病相关。本项目从代谢干预角度增强BNP功能，一是通过IDE抑制剂ML-345和6bk减少BNP截短C端，二是转基因上调NPR-A。从基因调控方面，通过基因突变（SNP）和表观遗传学(DNA甲基化和外泌体miRNA/蛋白)探索分子机制和促进基因转录。这些有助于阐明BNP悖论机制，解释HF过程和探索治疗靶点。

心衰患者B型利钠肽（BNP）增高，抗心衰作用却减弱。这困扰人们多年，国际上称BNP悖论，机制不明。可能因全长BNP减少，截短形式增多，后者对BNP功能受体（利钠肽A型受体，NPR-A）作用减弱，或NPR-A下调。本研究分析BNP全长肽和截短肽与过表达NPR-A细胞模型的相互作用和下游活性，发现BNP的C端氨基酸缺失抑制BNP受体结合、下游活性和生物功能，缓解大鼠左心室收缩压的下降，改善了心、脑和胰腺组织的炎症状态，减轻了组织坏死，明确了BNP的C端氨基酸对BNP生物作用的重要意义，说明胰岛素降解酶（IDE）可能是影响BNP活性的关键酶；通过生物大分子模拟和转录组学测序在分子结构和通路靶点上阐明了BNP1-32 的C端氨基酸影响BNP相互作用和生物功能的具体机制；应用IDE抑制剂ML-345和6bk减少了IDE对BNP的C端清除而增加了BNP的下游活性，而NPR-A转基因过表达增强了BNP的生物功能，包括增加了细胞活力、促进了细胞增殖和抑制了细胞凋亡，为NPR-A过表达治疗心衰提供了理论基础和应用潜力，而且有助于阐明BNP悖论机制、解释心衰发展过程和探索心衰治疗靶点。