炎症及炎症耐受模型筛选广谱趋化因子抑制肽(英文)

Screening for a Peptide That Inhibits Expression of a Broad-spectrum of Chemokines Using Models of Endotoxin Tolerance and LPS-induced Pro-inflammation

通过减少炎性组织或细胞趋化因子及炎性因子的表达量能将炎症性病理过程抑制在起始阶段.我们通过体外构建人外周血单个核细胞LPS激活的急性炎症模型及内毒素耐受模型,进行噬菌体肽库亲和筛选,ELISA检测与炎性PBMC的结合能力,分泌抑制实验筛选抑制性噬菌体克隆,经趋化抑制、竞争结合及生成抑制实验检测体外活性,大鼠足肿胀及关节炎模型检验多肽体内作用,SqRT-PCR检测趋化因子及TTP的mRNA水平,探讨其作用机制.筛选到的目标多肽CI-S5趋化抑制率达到48.72%,明显抑制噬菌体阳性克隆P15与LPS激活PBMC的结合,动物试验能明显降低大鼠足肿胀及关节炎症.机制研究显示,CI-S5多肽能降低3种趋化因子的表达量,并调节TTP使其表达增加,提示CI-S5能够靶向炎症前期PBMC,为炎症治疗提供了针对早期急性炎症反应的广谱小分子抑制肽.

The goal of this study was to screen bioactive peptides to identify an efficient antagonist of multiple pro-inflammatory chemokines that inhibits the pathological process of inflammatory diseases. A phage display library was sequentially screened by binding phages. The binding properties of individual phage clones to LPS-activated PBMCs were determined using cell-based ELISAs. The positive clones were selected and determined by chemotaxis assays. A high-activity peptide was determined to inhibit carrageenan-induced paw oedema and formaldehyde-induced arthritis in Wistar rats in vivo. A possible mechanism of inflammation inhibition involving chemokine mRNA by the peptide was determined by analyzing mRNA expression levels of chemokines and tristetraprolin （TTP） by SqRT-PCR.Nineteen phage clones were selected after four rounds ofbiopanning with a cut-off of 3-fold higher binding to LPS-activated PBMCs than to normal PBMCs. Nine of the phage clones inhibited IL-8, MCP-1, and MIP-1β production in vitro. Five clones displayed the same peptide（CI-S5）most robustly inhibited the chemotactic activity in vitro and reduced paw oedema and arthritis in Wistar rats in v ivo. SqRT-PCR results indicated that mRNA expression of IL-8, MCP-1, and MIP-1β were reduced and TTP mRNA expression was increased in the CI-S5 treatment group. Our data demonstrate that CI-S5 is a broad-spectrum antagonist of pro-inflammatory chemokines as it enhances the expression of TTP to reduce chemokine mRNA expression. This study provides a basis for the development of new peptide-based therapies for the treatment of inflammatory diseases.