Card9对重症急性胰腺炎NF-κB、p38MAPK信号通路的作用及机制

Macrophage activation and infiltration are initiation steps in the development of severe acute pancreatitis (SAP), although the mechanism of macrophage activation remains vague. Card9 is an upstream regulatory signaling molecule of NF-κB and p38MAPK activation in macrophages. In the present study, we found that Card9 mRNA expression was upregulated in the pancreatic tissues of a rat SAP model the NF-κB and p38MAPK pathways were subsequently activated, and thereby increased the expression of proinflammatory cytokines. Thus, upregulated Card9 expression in macrophages is suspected of NF-κB and p38MAPK pathway activation, and the subsequent induction of inflammatory responses in SAP. Herein, we established a Card 9 shRNA and overexpression macrophage model and a siRNA-Card9 SAP rat model to elucidate the effect of Card9 on SAP using signal transduction chip technology, reverse blocking, and co-immunoprecipitation, etc. and uncovered novel ITAM-Card9-NF-κB and TLRs-Card9-MAPK pathways in SAP. The role of Card9 in treatment of SAP was investigated to discover key factors in the control of inflammatory responses to block SAP progression. Hence, our findings should advance the development of a novel Card9-targeted SAP treatment strategy.

巨噬细胞激活是重症急性胰腺炎（SAP）发展早期的启动因子，但SAP的巨噬细胞激活机制尚不清楚。Card9是炎症相关因子，是巨噬细胞NF-κB和MAPK激活的上游信号分子。本课题组首次发现：SAP大鼠胰腺组织Card9 mRNA表达升高，NF-κB、p38MAPK活化，促炎细胞因子表达增加。推测，SAP中巨噬细胞Card9上调，激活NF-κB、p38MAPK，激活SAP炎症反应。本项目拟建立shRNA-Card9和过表达巨噬细胞模型、siRNA-Card9 SAP大鼠模型、应用信号转导芯片、反向阻断及免疫共沉淀等技术，阐明Card9对SAP的作用、揭示在SAP中存在ITAM-Card9-NF-κB和TLRs-Card9-MAPK信号通路、探讨Card9对SAP的治疗作用。有望发现控制SAP炎症反应的关键点，阻断SAP进程，为Card9成为SAP新的治疗靶点提供理论依据。

巨噬细胞激活是重症急性胰腺炎（SAP）发展早期的启动因子，是SAP从胰腺局部病变迅速发展为全身炎症反应综合征和多器官功能障碍综合征的关键所在。本课题收集SAP患者外周血单核细胞，发现单核细胞Card9表达水平与SAP疾病严重程度密切相关，ROC曲线分析Card9敏感性高达87.5%，可作为评估SAP 严重程度的一个新的、早期、有效的临床监测指标。其次构建siRNA干扰沉默Card9基因的SAP大鼠，显著减轻SAP大鼠胰腺组织炎症反应，提高SAP大鼠生存率，证实Card9为靶点治疗SAP的可行性。分离siRNA干扰的SAP大鼠腹腔巨噬细胞，发现腹腔巨噬细胞NF-κB和p38MAPK活化被抑制， TNF-α、IL-1β等炎症因子也明显下降，揭示巨噬细胞内存在Card9/NF-κB和Card9/p38MAPK炎症信号通路参与了SAP疾病的发生发展。SAP大鼠腹腔巨噬细胞体外siRNA干扰，构建Card9-/-的腹腔巨噬细胞，TLR4受体（脂多糖）刺激的Card9-/-腹腔巨噬细胞，NF-κB和p38MAPK显著下调， TNF-α、IL-1β等炎症因子释放降低，明确SAP巨噬细胞活化存在TLR4-Card9-NF-κB和TLR4-Card9-MAPK的分子机制。最后探讨靶向Card9的药物治疗SAP，发现吡格列酮可通过抑制SAP大鼠的Card9表达，继而抑制NF-κB、p38MAPK活化、促进胰腺腺泡细胞凋亡减轻SAP炎症反应，提高SAP大鼠生存率，可能为SAP提供有效的靶向药物。以上研究成果发表于SCI收录杂志Cell Death Dis（IF 5.965）、J Cell Mol Med（IF：4.499）以及中华消化杂志等多篇论文。