副猪嗜血杆菌损伤猪浆膜间皮细胞紧密连接的分子机制研究及靶标药物筛选

Polyserositis caused by pathogenic Haemophilus parasuis (HPS) can lead to the death of piglets, resulting in great economical loss. Structural and functional alterations of tight junctions and tight junction associated proteins in mesothelial cells have been observed in HPS-caused polyserositis. We have previously shown that HPS induces apoptosis, reactive oxygen species (ROS) over-production, and inflammatory cytokines generation in pig peritoneal mesothelial cells. We hypothesize that HPS infection activates the TLRs/PKC/MAPK and NLRP3/HMGB1/RAGE signaling pathways leading to polyserositis by damaging the tight junctions of mesothelial cells. We further hypothesize that anti-inflammatory drugs, such as baicalin and geniposide, can inhibit the activation of the signaling pathways induced by HPS in mesothelial cells and subsequently reduce the damage in polyserositis by inhibiting the activation of NF-κB and the generation of ROS. In this project, we will use both in vitro and in vivo piglet models to test our hypotheses on (1) the influence of HPS on TLRs/PKC/MAPK, MAPK/MLCK/MLC, and NLRP3/HMGB1/RAGE signaling pathways in pig serosal mesothelial cells; (2) the effect of baicalin, geniposide, and other drugs on the secretion of inflammatory cytokines and mediators induced by HPS; (3) the structural and functional changes of tight junctions in serosal mesothelial cells and the animal histopathology; and (4) the key molecules of the related signaling pathways. This study sets not only to figure out the molecular mechanisms of tight junction damage induced by HPS in porcine serosal mesothelial cell, but also to screen effective target drugs for the control of serositis in HPS disease.

副猪嗜血杆菌所引起多发性浆膜炎，造成猪只的死亡，带给养猪业严重的经济损失。多发性浆膜炎是副猪嗜血杆菌病的特征病变与间皮细胞紧密连接的结构和功能变化有关。我们前期观察到副猪嗜血杆菌能诱导猪浆膜间皮细胞的活性氧生成、炎性因子表达和细胞凋亡，推测副猪嗜血杆菌可能激活间皮细胞TLRs/PKC和NLRP3/HMGB1等信号通路，通过损伤间皮细胞的紧密连接引起浆膜炎；黄芩苷和栀子苷等抗炎药物可通过抑制NF-κB和减少活性氧，抑制副猪嗜血杆菌激活的间皮细胞损伤的信号通路，减轻浆膜炎症。本项目采用细胞培养和动物攻毒模型，研究副猪嗜血杆菌对猪浆膜间皮细胞TLRs/PKC/MAPK、MAPK/MLCK/MLC和NLRP3/HMGB1/RAGE信号通路的影响；黄芩苷和栀子苷等药物对副猪嗜血杆菌诱导后炎性因子释放；间皮细胞紧密连接的结构和功能变化、动物体征与病理组织学；信号通路关键分子等的影响，藉此阐明副猪嗜血杆菌损伤猪浆膜间皮细胞紧密连接的分子机制，进而筛选控制副猪嗜血杆菌病浆膜炎的靶标药物。

副猪嗜血杆菌病也称格拉泽氏病，是严重危害养猪业的主要细菌性疾病之一，以纤维素性多发性浆膜炎和败血症为主要病理特征。多发性浆膜炎和败血症的发生可能与浆膜间皮细胞和血管内皮细胞紧密连接的结构和功能变化有关。本研究建立了副猪嗜血杆菌感染猪腹膜间皮细胞的细胞感染模型，研究了副猪嗜血杆菌感染猪腹膜间皮细胞后对细胞凋亡的影响，细胞紧密连接occludin、ZO-1、claudin-1和JAM-1的mRNA表达，紧密连接蛋白结构和分布的影响，PKC-MAPK和MLCK-MLC信号通路的影响，并且筛选了对其紧密连接有保护作用的药物。结果表明副猪嗜血杆菌感染猪腹膜间皮细胞后，上调凋亡基因和核转录因子的mRNA表达导致细胞凋亡，通过激活PKC-MAPK和MLCK-MLC信号通路下调细胞紧密连接的mRNA表达，破坏紧密连接蛋白结构和分布。筛选出的药物黄芩苷能够通过上调凋亡基因和核转录因子AP-1的mRNA表达发挥抗凋亡作用，通过抑制PKC-MAPK和MLCK-MLC信号通路的激活，上调细胞紧密连接的mRNA表达，保护细胞紧密连接蛋白结构和分布。本研究还建立了副猪嗜血杆菌感染仔猪模型，研究了副猪嗜血杆菌感染仔猪后对仔猪腹膜和血管紧密连接mRNA表达，蛋白结构和分布的影响，PKC和MLCK-MLC信号通路的影响，以及黄芩苷对腹膜和血管紧密连接的保护作用。结果表明仔猪感染副猪嗜血杆菌后，激活PKC和MLCK-MLC信号通路，下调腹膜和血管紧密连接mRNA表达，破坏紧密连接蛋白结构和分布。黄芩苷处理组仔猪腹膜和血管紧密连接mRNA表达显著上调至正常水平，紧密连接蛋白结构和分布正常。综上所述，副猪嗜血杆菌通过激活紧密连接相关的信号通路导致紧密连接受损，黄芩苷在体内和体外对副猪嗜血杆菌诱导的PKC-MAPK和MLCK-MLC信号通路激活有显著的抑制作用，保护紧密连接的正常形态和功能。