异类叶升麻苷通过阻断TLR4二聚体化抑制MyD88/TRIF信号通路减轻重症急性胰腺炎急性肺损伤的作用机制研究

It is the key of the treatment of severe acute pancreatitis (SAP)-associated acute lung injury (ALI)by controlling early excessive inflammatory response in SAP-associated ALI. The inflammatory responses can be blocked by inhibition of inflammation-related signaling pathways,and the mechanism is still unclear.It was found in our study that TLR4-related signaling pathway plays a key role in inflammatory response of SAP-associated ALI. TLR4 dimerization is a key target of activation of inflammation-associated signaling pathways, and there is no study about the role of it in SAP-associated ALI.It was found that Isoacteoside (ISO) which is anti-inflammatory traditional Chinese medicine,can block the formation of TLR4 dimer completely. Moreover,we found that ISO can alleviate the inflammatory reaction and lung injury in rats of SAP-associated ALI. We speculate that ISO can inhibit releasing of inflammatory factors and reducing lung injury of SAP-associated ALI by inhibiting the MyD88/TRIF signaling pathway by blocking the formation of TLR4 dimer in lung tissue. In our study,we intend to establish model of SAP-associated ALI of rats and cells, with different doses of ISO intervention, and through the promotion and inhibition of formation of TLR4 dimer,to clarify the mechanism of ISO in treatment of SAP-associated ALI, and new targets and drugs may be found in the treatment of SAP-associated ALI.

控制早期过度炎症反应是重症急性胰腺炎（SAP）并发急性肺损伤（ALI）的治疗关键，抑制相关信号通路可阻断炎症反应，相关机制仍未明确。我们前期研究发现：TLR4相关信号通路在SAP并发ALI炎症反应中起到关键作用。TLR4二聚体形成是激活炎症相关信号通路的关键靶点，尚未有TLR4二聚体化在SAP并发ALI中作用的相关研究。抗炎中药单体-异类叶升麻苷(ISO)可完全阻断TLR4二聚体形成，我们研究发现ISO可减轻SAP并发ALI大鼠炎症反应和肺损伤。我们推测：ISO能够通过阻断SAP并发ALI肺组织中TLR4二聚体化，抑制其介导的MyD88/TRIF信号通路，抑制炎症因子的释放，减轻肺损伤。本研究拟建立大鼠和细胞SAP并发ALI模型，用不同剂量ISO干预，通过促进和阻断TLR4二聚体化进行对照验证，阐明ISO在SAP并发ALI中的具体作用机制，可为SAP并发ALI治疗发掘新的靶点和药物。

背景：重症急性胰腺炎 (Severe acute pancreatitis，SAP）表现为胰腺坏死,并伴有多器官功能障碍综合征（multiple organ disfunction syndrome, MODS）。急性肺损伤(acute lung injury, ALI) 和急性肾损伤（acute kidney injury，AKI) 的发病率占SAP合并MODS的前两位。控制早期过度炎症反应, 阻断 (systemic inflammatory reaction syndrome，SIRS) 发生是治疗SAP并发ALI和AKI的关键，抑制相关信号通路可阻断炎症反应，但相关机制仍未明确，也尚未有特异性治疗药物。Toll样受体4(Toll-like receptors 4, TLR4) 在SAP并发ALI的小鼠模型中起到重要的促炎作用，异类叶升麻苷（Isoacteoside，ISO) 是一种从鹿茸草中分离出的苯乙醇苷，研究表明ISO有确切的抗炎、抗氧化作用，但其在SAP并发ALI和AKI中的作用未见有文献报道。.研究内容：本研究通过建立SAP 合并ALI和AKI大鼠模型及ALI体外模型，先用不同剂量ISO干预体内模型组，通过其对血清淀粉酶（AMY）和肝功能检测确定最佳作用剂量，随后用最佳剂量ISO干预SAP并发ALI和AKI大鼠和细胞模型，检测炎症因子水平及胰腺、肺和肾损伤程度，并检测肺组织中髓过氧化物酶（MPO）和肾组织中一氧化氮（NO）的表达水平。检测肺和肾组织中Toll样受体4(Toll-like receptors 4, TLR4) 和磷酸化核因-kB(nuclear factor kappa-Bp65，NF-kB p65)的表达水平。在体外用TLR4特异性抑制剂和ISO干预ALI模型细胞，检测ALI模型细胞中炎症因子的表达。.结果：ISO可显著减少炎症因子的释放，并减轻胰腺、肺和肾组织损伤，降低肺和肾组织中TLR4和NF-kB p65的表达。用TLR4特异性抑制剂和ISO干预联合干预ALI模型细胞较TLR4特异性抑制剂单独干预ALI模型细胞，TNF-α表达减少。.结论：ISO可通过抑制TLR4/NF-kbp65信号通路减轻SAP并发的ALI和AKI,可能还通过其他信号通路起到抑制炎症反应的作用，ISO可能成为治疗SAP诱发的ALI和AKI的潜在治疗药物。