靶向抑制法尼基转移酶通过PD-L1/PD-1信号通路调控脓毒症小鼠机体免疫功能的机制研究

Severe sepsis and septic shock are major causes of morbidity and mortality. Our preliminary data demonstrated that statin significantly reduced LPS-induced mortality and increased survival rate in mouse xenograft. Our group first reported that the induction of sepsis by cecal ligation and puncture (CLP) resulted in increases in farnesyltransferase activity, farnesylated proteins in the spleen, bacteria and macrophages in peripheral blood, concentration of Tregs relative to sham operation. Nevertheless, inhibition of farnesyltransferase activity increases overall survival rate in patients with sepsis via specific signaling pathway was not investigated. In this study, by cecal ligation and puncture (CLP) and after treatment of FTI, we examined changes of pathophysiological characteristics in CLP-induced sepsis mouse model. The immune suppression of septic mice will be analyzed by Immunohistochemistry, flow cytometry and ELISA with spleen、thymus、blood and peritoneal lavage. The expression levels of PD-L1/PD-1 signaling pathway and apoptosis related genes were investigated to identify that inhibition of farnesyltransferase activity lead to impaired cellular immune response and regulation due to deactivation of PD-L1/PD-1 signaling pathway. We expected that targeting farnesyltransferase would serve as a valuable addition and important tool for the treatment of patients with sepsis.

脓毒症是由感染引发的全身性炎症反应，由于缺乏有效治疗方法，是危重病人死亡的重要原因之一。本课题组前期研究发现他汀类药物能提高动物感染模型的存活率，且首次发现脓毒症小鼠脾脏内的法尼基化蛋白总量及法尼基转移酶活性、外周血及腹腔灌洗液中细菌含量和巨噬细胞数量、调节T细胞的含量都显著升高，接受法尼基转移酶抑制剂治疗的脓毒症小鼠，其生存率也明显升高。FTI抑制法尼基转移酶活性是否能通过影响特定信号通路提高脓毒症患者生存时间，至今尚无研究报道。本研究拟通过结肠结扎-穿孔手术构建脓毒症小鼠模型，使用FTI治疗脓毒症小鼠，检验脓毒症小鼠病理生理特性较治疗前的系列变化，检测FTI治疗后PD-L1/PD-1信号通路相关基因表达水平，明确FTI是否通过引起PD-L1/PD-1信号通路抑制，提高脓毒症小鼠生存率，从而深入探讨FTI与脓毒症引发的免疫调节的关系。为临床治疗脓毒症、改善脓毒症的预后提供理论和实验基础。

法尼基转移酶抑制剂已经在临床试验中用于治疗肿瘤。在败血症中，PD-1与PD-L1的结合促进淋巴细胞凋亡并降低细胞因子表达，从而影响存活率。 PD-1 / PD-L1途径在慢性病毒感染，细菌感染和败血症中起重要作用,然而，我们对这个途径的确切的免疫抑制和抗炎功能仍然知之甚少。在我们以前的研究中，通过盲肠结扎和穿刺（CLP）来诱导败血症模型。其相对于假处理的方式，法尼基转移酶活性和法尼基蛋白水平增加了。然而，抑制法尼基转移酶活性对脓毒症患者总体生存率的影响以及所涉及的特定信号通路仍有待研究。在此，用法尼基转移酶抑制剂（FTI-277）处理患有CLP诱导的败血症的小鼠，并检查脓毒性脾淋巴细胞上的PD-L1表达。流式细胞术分析显示PD-L1在淋巴细胞上显著性地表达，并且在CLP之后PD-L1蛋白表达强烈地上调。 FTI-277是以剂量依赖方式下调脓毒症脾淋巴细胞PD-L1 mRNA和蛋白的表达，并且这种效应与NF-κB密切相关。此外，FTI-277对PD-L1信号的显着阻止作用促进小鼠中抗CD3 + CD28 +抗体的IFN-γ分泌，白细胞介素-2产生和脾细胞增殖。此外，FTI-277降低了脓毒症小鼠的脾淋巴细胞凋亡。因此，FTI-277通过PD-L1信号通路调节脾淋巴细胞活性，具有显著的抗NF-κB通路抑制作用。法尼基转移酶将会作为一种有效地脓毒血症的治疗方式。