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咖啡酸在重症急性胰腺炎小鼠模型中的作用及其机制
DOI: 10.12449/JCH250418
伦理学声明：本研究方案经由上海中医药大学附属龙华医院实验动物伦理委员会审批，批号：LHERAW-23045，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：续嗣钰、兰路路、薛怡宁负责实验部分及数据整理；柳涛、卫威、韩奕、穆素成参与收集数据，资料分析；续嗣钰负责撰写论文；杜施霖、宋海燕负责课题设计，指导撰写文章并最后定稿。
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摘要:
目的 研究咖啡酸（CA）对雨蛙素联合脂多糖诱导的重症急性胰腺炎的影响及潜在机制，以期为重症急性胰腺炎治疗提供潜在新型药物的研究基础。 方法 将6周龄C57BL/6J小鼠分为4组：对照组、模型组、CA组及醋酸奥曲肽（OA）组，每组6只。除对照组注射生理盐水外，其余各组通过腹腔注射雨蛙素联合脂多糖构建重症急性胰腺炎小鼠模型，在初次注射雨蛙素1 h后，CA组及OA组小鼠分别给予CA腹腔注射或OA皮下注射3次，每次间隔8 h。造模24 h后观察小鼠一般情况，收集血清和胰腺、肺、结肠组织。通过HE染色观察胰腺、肺组织病理变化。检测小鼠血清α-淀粉酶、脂肪酶、TNF-α、IL-6、ALT、AST和Cr水平。RT-PCR检测胰腺、肺组织促炎因子表达，通过髓过氧化物酶（MPO）免疫组化观察中性粒细胞浸润程度，Western Blot检测胰腺和肺组织核因子-κB（NF-κB）活化，中性粒细胞胞外陷阱（NET）形成标志物瓜氨酸化组蛋白H3（CitH3）水平，以及结肠组织ZO-1表达水平。计量资料多组间数据比较采用单因素方差分析，进一步两两比较采用Dunnett’s t检验。 结果 与对照组比较，模型组小鼠胰腺及肺组织损伤严重，血清α-淀粉酶、脂肪酶活性，以及血清、肺组织中促炎因子IL-6、IL-1β和TNF-α水平均显著升高（P值均<0.05），胰腺及肺组织中NF-κB活化、中性粒细胞浸润及NET形成均显著增加（P值均<0.05）。与模型组相比，CA组小鼠胰腺及肺组织病理损伤减轻，血清α-淀粉酶活性以及血清、肺组织中促炎因子IL-6、IL-1β和TNF-α水平均下调（P值均<0.05），胰腺及肺组织NF-κB活化、中性粒细胞浸润及NET形成均减少（P值均<0.05）。 结论 CA可减轻雨蛙素联合脂多糖诱导的SAP小鼠模型的症状，抑制中性粒细胞募集及NET形成可能是其部分作用机制。 -
关键词:
- 胰腺炎 /
- 小鼠， 近交C57BL /
- 咖啡酸 /
- 模型， 动物
Abstract:Objective To investigate the effect and potential mechanism of caffeic acid （CA） on severe acute pancreatitis （SAP） induced by caerulein combined with lipopolysaccharide （LPS）， and to provide a basis for the research on novel drugs for the treatment of SAP. Methods C57BL/6J mice， aged 6 weeks， were divided into control group， model group， CA group， and octreotide acetate （OA） group， with 6 mice in each group. The mice in the control group were given injection of normal saline， and those in the other groups were given intraperitoneal injection of caerulein combined with LPS to establish a mouse model of SAP. At 1 hour after the first injection of caerulein， the mice in the CA group and the OA group were given intraperitoneal injection of CA or subcutaneous injection of OA at an interval of 8 hours. The general status of the mice was observed after 24 hours of modeling， and serum， pancreas， lung， and colon samples were collected. HE staining was used to observe the histopathological changes of the pancreas and lungs， and the serum levels of α-amylase， lipase， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， alanine aminotransferase， aspartate aminotransferase， and creatinine were measured. RT-PCR was used to measure the expression of proinflammatory factors in the pancreas and lungs； myeloperoxidase （MPO） immunohistochemistry was used to observe the degree of neutrophil infiltration； Western blot was used to measure the activation of nuclear factor-kappa B （NF-κB） and the level of citrullinated histone H3 （CitH3）， a marker for the formation of neutrophil extracellular traps （NETs）， in the pancreas and lungs， as well as the expression level of ZO-1 in colon tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the Dunnett’s t-test was used for further comparison between two groups. Results Compared with the control group， the model group had severe injury in the pancreas and lungs and significant increases in the activity of serum α- amylase and lipase and the levels of the proinflammatory cytokines IL-6， interleukin-1β （IL-1β）， and TNF-α in serum and lung tissue （all P<0.05）， as well as significant increases in NF-κB activation， neutrophil infiltration， and the formation of NETs in the pancreas and lungs （all P<0.05）. Compared with the model group， the CA group had alleviated pathological injury of the pancreas and lungs and significant reductions in the activity of serum α-amylase and the levels of the proinflammatory cytokines IL-6， IL-1β， and TNF-α in serum and lung tissue （all P<0.05）， as well as significant reductions in NF-κB activation， neutrophil infiltration， and the formation of NETs in the pancreas and lungs （all P<0.05）. Conclusion CA can alleviate SAP induced by caerulein combined with LPS in mice， possibly by inhibiting neutrophil recruitment and the formation of NETs. -
Key words:
- Pancreatitis /
- Mice， Inbred C57BL /
- Caffeic Acid /
- Models， Animal
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注： a， 胰腺组织HE染色；b，胰腺组织病理评分。
图 1 胰腺组织HE染色及病理评分
Figure 1. HE staining and pathological scores of pancreatic tissues
注： a，小鼠肺组织HE染色；b，肺组织IL-6、IL-1β、TNF-α mRNA表达水平。
图 4 肺组织HE染色及炎症因子水平
Figure 4. HE staining and levels of inflammatory cytokines of lung tissues
注： a，胰腺；b， 肺。
图 5 胰腺及肺组织NF-κB蛋白表达和活化水平
Figure 5. Protein expression and activation levels of NF-κB in pancreatic and lung tissues
图 6 胰腺和肺组织MPO免疫组化染色
Figure 6. MPO immunohistochemical staining of pancreatic and lung tissues
注： a，胰腺；b，肺。
图 8 胰腺及肺组织CitH3的蛋白表达水平
Figure 8. Protein expression levels of CitH3 in pancreatic and lung tissues
图 9 结肠组织炎症因子和ZO-1表达水平
Figure 9. Expression levels of inflammatory cytokines and ZO-1 in colon tissues
表 1 胰腺炎组织病理评分表
Table 1. Histopathological scale of pancreatitis
评分 水肿 坏死 炎性细胞浸润 0 无水肿 无坏死 炎性细胞数0～1个 1 局限性小叶间水肿 坏死细胞1～4个 炎性细胞数2～10个 2 弥漫性小叶间水肿 坏死细胞5～10个 炎性细胞数11～20个 3 腺泡增大，小叶间隔增宽 坏死细胞11～16个 炎性细胞数21～30个 4 明显小叶分隔 坏死细胞>16个 炎性细胞数>30个 
下载: 导出CSV
表 2 PCR引物序列
Table 2. PCR primer sequence
基因 种属 序列 IL-6 小鼠 上游：5´-GGGACTGATGCTGGTGACAAC-3´ 下游：5´-CAACTCTTTTCTCATTTCCACGA-3´ IL-1β 小鼠 上游：5´-GCTTCAGGCAGGCAGTATCA-3´ 下游：5´-TGCAGTTGTCTAATGGGAACG-3´ TNF-α 小鼠 上游：5´-CCCTCCAGAAAAGACACCATG-3´ 下游：5´-CACCCCGAAGTTCAGTAGACAG-3´ β-actin 小鼠 上游：5´-GAGACCTTCAACACCCCAGC-3´ 下游：5´-ATGTCACGCACGATTTCCC-3´
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