Ca2+在化脓隐秘杆菌溶血素致炎过程中的作用研究——聚焦calcineurin和calpain

Pyolysin (PLO) is one of the major factors that cause inflammation during the process of Trueperella pyogenes infection. However, most of the studies until now have investigated the virulent effect of PLO based on its hemolytic activity. While how PLO could cause inflammation had not been intensively studied. Taking the fact that the concentration of PLO might be sublytic to host cells at the early stage of Trueperella pyogenes infection into consideration, in current study, we will investigate the mechanism of PLO-induced inflammation based on a model established using mouse macrophages and sublytic dose PLO. Firstly, we will determine the way of interaction between macrophages and sublytic dose PLO through analyzing the changes of cytokine secreting levels and patterns in macrophages. And then, fluorescent probe or chemical colorimetry technologies will be used combining with different blockers of the Ca2+ channel to determine the changes of intercellular Ca2+ concentration induced by sublytic dose PLO and the mechanism. Finally, we will focus on two Ca2+ dependent enzymes, named calcineurin and calpain, and their downstream signal pathways which might relate to the sublytic dose PLO-induced proinflammatory cytokines secretion of macrophages. Western blot technology and different enzyme inhibitors will be used to investigate the influences of sublytic dose PLO on the activity of the two enzymes and the signal transduction through the following pathways. The results of our study will uncover partial of the mechanisms underlying the events of inducing proinflammatory cytokines secretion and inflammation by PLO.

化脓隐秘杆菌的溶血素蛋白（PLO）是导致其感染性炎症的重要原因。但已有研究多基于PLO的溶细胞功能探讨其对宿主的毒力效应，而对其致炎症效应及机制少见研究。考虑到自然感染早期PLO对宿主细胞更可能处于亚裂解剂量，本研究拟以亚裂解剂量PLO与巨噬细胞的相互作用为模型，探讨PLO的部分致炎机制。首先，我们将通过分析亚裂解剂量下PLO及其衍生物对炎症相关细胞因子表达水平及格局的影响，确定PLO与巨噬细胞相互作用的主要方式；随后，利用荧光探针或化学显色技术配合多种阻滞剂，探讨亚裂解剂量PLO对细胞内Ca2+浓度的影响及机制；最后，以Ca2+依赖酶类calcineurin和calpain为切入点，以western blot技术为主，配合多种酶抑制剂，研究亚裂解剂量PLO对两种酶及它们介导的下游信号通路的影响。本研究将确定上述事件与PLO导致的促炎性细胞因子分泌之间的关系，阐明PLO介导炎症的部分机制。

化脓隐秘杆菌是一种重要的动物条件性致病菌，主要引起动物发生化脓性炎症，其中肺炎是其导致的重要临床疾病之一，但该菌导致炎症性疾病的原因目前尚未充分阐明。溶血素（PLO）是化脓隐秘杆菌的主要毒力因子之一，缺失PLO编码基因，化脓隐秘杆菌致病性显著降低。溶血素是成孔毒素家族（PFT）成员之一，而表达PFT的细菌在造成肺感染的过程中可以藉由PFT在感染早期迅速杀伤肺泡巨噬细胞（Mφ）。但在PFT杀伤Mφ的同时，Mφ也会做出相应的反应。由于Mφ是介导炎症反应的重要免疫细胞之一，本研究拟以小鼠Mφ为模型，探讨PLO对细胞中促炎性细胞因子表达情况的影响，并解析过程中的部分分子机制。研究结果表明，亚裂解剂量PLO主要通过其成孔活性介导Mφ中促炎性细胞因子的转录，而PLO发挥病原相关分子模式（PAMP）样活性时主要诱导IL-10这种抑炎性细胞因子的表达；亚裂解剂量PLO使Mφ中游离Ca2+浓度下降，Mφ钙调神经磷酸酶活性无明显变化，钙蛋白酶活性受到显著抑制，转录因子NF-κb p65的磷酸化水平下降、NFATc1表达量降低；亚裂解剂量PLO可以使Mφ中NIK分子、磷酸化NF-κb p100、磷酸化IKK α/β的含量上升，同时，使细胞中NF-κb p52分子含量增加。上述结果表明，亚裂解剂量PLO作用的Mφ中，IL-1α和IL-1β基因的转录主要通过非经典NF-κb信号通路的活化而实现的，而不是通过PLO的PAMP样活性刺激Toll样受体，进而激活经典NF-κb信号通路的活化而实现的。本研究证实了PLO介导Mφ中促炎性细胞因子表达的部分分子机制。