双功能融合蛋白CRISPLD2-anti-HMGB1治疗脓毒症的效果和机制研究

The nature of sepsis is excessive inflammation caused by infection. LPS and HMGB1 are two critical pro-inflammatory factors which emerge in the early and late stages of sepsis respectively, as well as lead to lethality. Furthermore, LPS and HMGB1 will form LPS-HMGB1 complex which has stronger pro-inflammatory properties. In addition, LPS-HMGB1 complex can mediate a special programmed cell death (pyroptosis) restricted to monocytes and macrophages. We designed and synthesized a two functional fusion protein named “CRISPLD2-anti-HMGB1” which can simultaneously antagonize the inflammation triggered by LPS, HMGB1 and LPS-HMGB1 complex, as well as may reverse the process of pyroptosis induced by LPS-HMGB1 complex. We will further explore the effect and the underlying mechanism of this fusion protein against inflammation and its impact on downstream inflammatory signaling pathways, including MAPK pathway and NF-kB pathway. We will also investigate whether this fusion protein can reverse the process of pyroptosis induced by LPS-HMGB1 complex, and its impact on activation and expression of a series of molecules which were involved in the pathway of pyroptosis, including Cathepsin B, Caspase1. In animal experiment, we will determine the pharmacokinetics and safety of this fusion protein in vivo. Next, we will test the effects of this fusion protein on serum inflammation levels, sepsis induced organ disfunction and survival rates. To determine whether this fusion protein can be applied in clinical patients with sepsis, we will test its performance in human serum specimen. In all, this new artificial fusion protein is a promising method for the treatment of sepsis. Further, the exploration of its effects on inflammation and the process of pyroptosis will provide insight into the mechanism and the pathophysiologic process of sepsis.

脓毒症的本质是感染引起的过度炎症反应。LPS和HMGB1是脓毒症早期和晚期最重要的促炎和致死性因子，二者还可形成LPS-HMGB1复合物，促进炎症反应扩大化并介导单核巨噬细胞的程序性死亡（Pyroptosis）。我们国内外首先设计并合成了可同时拮抗LPS、HMGB1及LPS-HMGB1复合物的双功能融合蛋白CRISPLD2-anti-HMGB1。我们将探索该融合蛋白的抗炎效应，具体机制，对炎症信号通路（MAPK、NF-kB）的影响；探索其能否逆转LPS-HMGB1复合物介导的Pyroptosis，及对该通路中Cathepsin B、Caspase1等分子表达、激活的影响。评价该融合蛋白在小鼠体内代谢动力学和安全性，对脓毒症小鼠血清炎症水平，器官功能及生存率的影响。验证该融合蛋白在人类血清中的工作性能。为脓毒症治疗提供新的方法及机制。

脓毒症的本质是感染引起的过度炎症反应。LPS和HMGB1是脓毒症早期和晚期最重要的促炎和致死性因子，二者还可形成LPS-HMGB1复合物，促进炎症反应扩大化。我们国内外首先设计并合成了可同时拮抗LPS、HMGB1及LPS-HMGB1复合物的双功能融合蛋白CRISPLD2-anti-HMGB1，该蛋白大小约 250kD，其与 LPS 和HMGB1结合的亲和力与天然状态下CRISPLD2 与 anti-HMGB1与LPS和HMGB1的亲和力类似。本课题证实了CRISPLD2-anti-HMGB1能够有效的在体内和体外拮抗LPS及HMGB1导致炎症反应，抑制对炎症信号通路（MAPK、NF-kB）的激活，从而保护脓毒症小鼠器官功能及生存率，为脓毒症治疗提供新的方法及靶点。