具有任意伸展结构两栖类皮肤抗菌肽chensinin-1b抗LPS引起的感染性休克与免疫调节的机制研究

Antimicrobial peptides can bind to lipopolysaccharide (LPS) and thereby reduces LPS-induced pro-inflammatory responses by decreasing NF-kB activation in vitro. Our previous studies have revealed that chensinin-1b with random coil structure, an antimicrobial peptide from amphibian skin, uptake into Macrophages and monocytes mediated by atypical endocytic processes and modulate the inflammatory response by the interaction with the intracellular target. But the molecular level mechanism of action remains unknown. In this present study, the intracellular molecular target will be identified by using a combined stable isotope labeling by amino acids in cell culture-advanced proteomics methodology, and the interaction of chensinin-1b with the direct binding partner will be determined by the several biochemical and biophysical binding experiments. Here, we set out to investigate the effects of chensinin-1b on the expression of signal molecules and pro-inflammatory factors by using microarray and using gene silencing of target molecule with small interfering RNA (siRNA) in vitro. Our present study aims at revealing the selective immune modulation of chensinin-1b on pro- and anti-inflammatory cytokines and chemokines in sepsis and septic shock. These investigations may contribute to the development of amphibian skin antimicrobial peptides as new therapeutic modalities using in protecting from LPS-mediated clinical infectious and autoimmune disease.

抗菌肽可以在细胞外与脂多糖（LPS）竞争性结合，阻断LPS诱导的炎症信号传导通路，减轻过度炎症反应给机体带来的损伤。但我们的前期研究表明具有任意伸展结构抗菌肽chensinin-1b是在细胞内，通过与细胞内靶点分子相互作用，降低促炎因子的表达，抑制感染性休克的发生。这提示chensinin-1b通过细胞内分子介导了新的抗炎反应信号通路。本研究采用基于质谱的细胞体内稳定同位素标记定量蛋白质组学技术，及表面等离子共振和荧光能量共振转移等现代生物物理技术，研究chensinin-1b在免疫细胞内的作用靶点及与靶点分子相互作用动力学；探讨肽与靶点的相互作用对NF-κB信号通路中信号分子和细胞因子表达的影响，提出一种新的通过细胞内靶点介导的炎症反应信号传导通路，揭示具有任意伸展结构的两栖类抗菌肽预防或阻断炎症反应过程的新途径，以及选择性免疫调节功能的分子机制，为从新的视点揭示炎症发生的机制奠定基础。

本研究探讨了具有任意伸展结构的两栖类抗菌肽chensinini-1b在预防及阻断LPS诱导的炎症反应的信号通路以及可能的细胞内作用靶点，揭示其选择性免疫调节功能的分子机制，从新的视点为揭示炎症的发生机制奠定基础。基于质谱的细胞体内稳定同位素标记定量蛋白质组学技术(AACT/SILAC)和生物信息学分析是筛选相互作用蛋白的新技术，本研究利用该技术筛选chensinin-1b在免疫细胞内的作用靶点；重点研究chensinin-1b与靶点的相互作用对MAPKs、I-κB和NOD信号通路中信号分子和细胞因子表达的影响，探讨chensinin-1b在抗感染性休克与免疫调节和保护之间的关系。我们进一步地建立了小鼠败血症模型，通过对促炎症因子（如TNF-α, IL-1β等）表达和释放的研究，了解chensinin-1b在体内选择性地调节炎症抑制细胞因子以及免疫细胞趋化因子的表达和释放，对于全身性和致死性败血症的预防和保护作用，为两栖类皮肤抗菌肽用于临床感染性和免疫性疾病治疗药物的研发提供新的思路和科学依据。

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