【作者】 李彬；

【导师】 胡国勇；

【作者基本信息】 上海交通大学 ， 消化内科， 2018， 硕士

【摘要】 背景与目的:白三烯B4（leukotriene B4,LTB4）是一种强效的趋化因子,可以招募多种免疫细胞包括中性粒细胞和巨噬细胞等,在多种炎症性疾病中发挥着重要作用。P物质（substance P,SP）作为一种神经递质主要调控伤害性感受和神经性炎症,多种研究表明SP促进了急性胰腺炎（acute pancreatitis,AP）时胰腺炎症和肺损伤的发生,但其具体机制尚不明确。体内外研究均发现SP可促进LTB4的合成。本课题旨在研究LTB4在急性胰腺炎及相关肺损伤中的作用以及LTB4合成的分子机制。方法:体内建立雨蛙肽加LPS或左旋精氨酸诱导的小鼠急性胰腺炎模型。LTB4特异性受体（BLT1）拮抗剂LY293111或SP特异性受体（NK1R）拮抗剂CP96345均在造模前0.5小时腹腔注射。分析LTB4/BLT1系统在AP中的变化。通过检测血清脂肪酶、淀粉酶和胰腺组织病理学评分评估AP严重程度。检测胰腺组织Ly6G⁺中性粒细胞和F4/80⁺巨噬细胞浸润以及炎症因子（IL-6和IL-1β）mRNA水平评估AP局部炎症反应。通过组织病理学评分、中性粒细胞浸润和炎症因子（IL-6、IL-1β和TNF-α）mRNA水平评估急性肺损伤。流式细胞学检测反向迁移中性粒细胞(ICAM-1^highCXCR1^low)比例。体外以SP刺激胰腺腺泡细胞（pancreatic acinar cells,PACs）,检测上清中LTB4含量及PKCα/MAPK信号通路改变。结果:在两种AP模型中,LTB4及其特异性受体BLT1水平均显著增高。BLT1特异性受体拮抗剂LY293111显著减轻了AP严重程度,减少了反向迁移至外周血的中性粒细胞比例,减轻了急性胰腺炎相关肺损伤。以SP刺激PACs后,其合成LTB4的能力显著增强,PKCα和MAPK（ERK1/2、P38和JNK）的磷酸化水平依次增高。此外,体内应用CP96345阻断NK1R,可减轻AP的严重程度并抑制LTB4合成。结论:SP通过PKCα/MAPK信号通路调控了急性胰腺炎中LTB4合成;LTB4通过招募中性粒细胞和巨噬细胞促进急性胰腺炎局部炎症反应,通过调控中性粒细胞反向迁移促进急性胰腺炎相关肺损伤。更多还原

【Abstract】 Background and PurposeLeukotriene B4（LTB4）is a potent chemoattractant for immune cells,including neutrophils and macrophages,which play a major role in inflammatory responses.Substance P（SP）is a neuropeptide mainly involved in nociception and neurogenic inflammation.Research increasingly suggests that SP contributes to inflammation in acute pancreatitis（AP）and associated acute lung injury（ALI）,but the molecular mechanism remains unclear.Substance P（SP）has been reported to promote the production of LTB4 in vivo and in vitro.Here,we investigated the role of LTB4 in AP,AP-associated ALI and the related mechanisms of LTB4 production in AP.MethodIn vivo,murine AP model was induced by caerulein and lipopolysaccharide（LPS）or L-arginine（L-Arg）.The specific BLT1 antagonist LY293111 or the selective NK1R antagonist CP96345 was injected intraperitoneally 0.5 h before the first injection of caerulein or L-Arg.Change of LTB4/BLT1 system was analysed.The severity of AP was assessed by amylase and lipase levels and histopathological scoring.The local inflammation of AP was assessed by the infiltration of Ly6G⁺neutrophils and F4/80⁺macrophages and the mRNA levels of inflammatory cytokines（IL-6 and IL-1β）in the pancreatic tissue.ALI was assessed by histopathological scoring,Ly6G⁺neutrophils infiltration and the mRNA levels of inflammatory cytokines（IL-6,IL-1βand TNF-α）in lung.The proportion of reverse migrated neutrophils,which have a distinctive ICAM-1^highCXCR1^low phenotype,was detected by flow cytometry.In vitro,pancreatic acinar cells（PACs）were stimulated with SP and the supernatants were collected for LTB4 measurement.The PKCα/MAPK signaling pathway was evaluated.ResultsThe levels of LTB4 and its specific receptor BLT1 were markedly upregulated in both AP models.Blockade of BLT1 by LY293111 attenuated the severity of AP,decreased neutrophil reverse transendothelial cell migration（rTEM）into the circulation and alleviated the severity of ALI.Treatment of PACs with SP increased LTB4production.Furthermore,SP treatment increased phosphorylation of protein kinase C（PKC）αand mitogen activated protein kinases（MAPKs）,including extracellular signal-regulated kinase（ERK）,p-38 MAPK and c-Jun NH2-terminal kinase（JNK）.Finally,blockade of NK1R by CP96345 significantly attenuated the severity of AP and decreased LTB4 production.ConclusionSubstance P regulates LTB4 production in AP via PKCα/MAPK pathway;LTB4increases local inflammation of AP by attracting neutrophils and macrophages and promotes AP-associated lung injury through neutrophil reverse migration.更多还原