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松果菊苷对雨蛙素诱导的急性胰腺炎大鼠模型胰腺及肝损伤的影响与机制
DOI: 10.12449/JCH240422
伦理学声明：本研究方案经由青海大学附属医院伦理委员会审批，批准号：P-SL-2022-018，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：刘川川、樊海宁负责课题设计；马明和、达哇卓玛、刘川川参与收集数据，资料分析，撰写论文，修改论文；樊海宁负责拟定写作思路，指导撰写文章并最后定稿。
Effect of echinacoside intervention on liver and pancreas injury in rats with acute pancreatitis and its mechanism
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摘要:
目的 通过建立急性胰腺炎（AP）大鼠模型，研究松果菊苷（ECH）对雨蛙素诱导的AP大鼠模型胰腺及肝损伤的改善作用及机制。 方法 24只SD大鼠随机分为空白组（Con组）、对照组（Con+ECH组）、AP组和AP+ECH组4组，每组各6只。AP模型构建前7 d给予10 mg/kg ECH腹腔注射，雨蛙素末次给药24 h后，腹主动脉取血，分离血清行ALT、AST、乳酸脱氢酶（LDH）、GGT、ALP、Alb、TBil、胆碱酯酶（ChE）、血淀粉酶（Amy）、脂肪酶（LPS）等生化检测；HE染色检测胰腺和肝组织病理学改变；透射电镜观察胰腺和肝组织微观结构改变；ELISA检测肝组织匀浆中IL-1β、IL-6、TNF-α、IL-10水平；免疫组化分析胰腺和肝组织中TNF-α和p-p65 NF-κB水平；Western Blot检测肝组织中NF-κB通路蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用SNK-q或Dunnett’s T3检验。 结果 与Con组相比，AP组大鼠ALT、AST、GGT、LDH、ALP、TBil、AMY、LPS指标均明显增高（P值均<0.01）；肝组织匀浆液IL-1β、IL-6、IL-10、TNF-α水平均增加（P值均<0.01）。ECH干预降低AP大鼠ALT、AST、GGT、LDH、ALP、TBil、AMY、LPS水平，并能抑制IL-1β、IL-6、TNF-α的分泌。HE染色观察可见ECH干预后胰腺组织中腺泡细胞空泡变性和炎性细胞浸润较AP组减轻，肝细胞坏死较AP组减轻。透射电镜检查镜下见ECH干预后肝和胰腺细胞内线粒体肿胀程度较AP组减轻。ECH干预后能部分逆转AP大鼠胰腺和肝组织中p-p65 NF-κB、TNF-α的表达增加。此外，AP大鼠肝组织中MyD88、p-IκBα、p-IKKα、p-p65表达上调，而ECH干预后能部分逆转。 结论 松果菊苷可通过抑制TLR4/MyD88/NF-κB途径来改善AP诱发的胰腺和肝损伤。 -
关键词:
- 松果菊苷 /
- 胰腺炎 /
- 大鼠， Sprague-Dawley /
- NF-κB
Abstract:Objective To investigate the effect and mechanism of echinacoside （ECH） in improving liver injury in rats with acute pancreatitis by establishing a rat model of acute pancreatitis and liver injury. Methods A total of 24 Sprague-Dawley rats were randomly divided into blank group （Con group）， control group （Con+ECH group）， acute pancreatitis group （AP group）， and acute pancreatitis+ECH intervention （AP+ECH group）. The rats were given intraperitoneal injection of 10 mg/kg ECH on day 7 before the establishment of the model of acute pancreatitis； at 24 hours after the last administration of cerulein， blood samples were collected via the abdominal aorta， and serum was separated for biochemical analysis including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， lactate dehydrogenase （LDH）， gamma-glutamyl transpeptidase （GGT）， alkaline phosphatase （ALP）， albumin （Alb）， total bilirubin （TBil）， cholinesterase， blood amylase （Amy）， and lipase （LPS）. HE staining was used to observe the histopathological changes of the pancreas and the liver； transmission electron microscopy （TEM） was used to observe the microstructural changes of pancreas and liver tissue； ELISA was used to measure the levels of interleukin-1β （IL-1β）， interleukin-16 （IL-6）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） in liver tissue homogenate； immunohistochemistry was used to measure the levels of TNF-α and p-p65 NF-κB in pancreas and liver tissue； Western blot was used to measure the expression levels of NF-κB pathway proteins in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the SNK test or the Dunnett’s T3 method was used for further comparison between two groups. Results Compared with the Con group， the AP group had significant increases in ALT， AST， GGT， LDH， ALP， TBil， Amy， and LPS （all P<0.01）， as well as significant increases in the levels of IL-1β， IL-6， IL-10， and TNF-α in liver tissue homogenate （all P<0.01）. ECH intervention reduced the levels of ALT， AST， GGT， LDH， ALP， TBil， AMY， and LPS and inhibited the secretion of IL-1β， IL-6， and TNF-α in rats with acute pancreatitis. HE staining showed that ECH intervention alleviated the vacuolar degeneration of acinar cells， inflammatory cell infiltration in pancreatic tissue， and the necrosis of hepatocytes compared with the AP group. TEM showed that compared with the AP group， there was a reduction in the degree of mitochondrial swelling in liver and pancreatic cells after ECH intervention. ECH intervention partially reversed the elevated expression levels of p-p65 NF-κB and TNF-α in liver and pancreatic tissue. In addition， the expression levels of MyD88， p-IκBα， p-IKKα， and p-p65 were upregulated in liver tissue of rats with acute pancreatitis， which could be partially reversed after ECH intervention. Conclusion Echinacoside can alleviate liver and pancreatic injury induced by acute pancreatitis by inhibiting the TLR4/MyD88/NF-κB pathway. -
Key words:
- Echinacoside /
- Pancreatitis /
- Rats， Sprague-Dawley /
- NF-kappa B
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图 1 ECH对AP大鼠模型血清AMY和LPS活性的影响
Figure 1. The effects of Echinacoside on serum amylase and lipase activity in rats with Cerulein-induced acute pancreatitis
图 3 ECH对AP大鼠胰腺组织病理学改变的影响（HE染色，×400）
注： a，Con组；b，AP组；c，ECH组；d，AP+ECH组
Figure 3. The effect of Echinacoside on the pancreatic morphology of rats with AP（HE staining，×400）
图 4 ECH对AP大鼠肝脏组织病理学改变的影响（HE染色，×400）
注： a，Con组；b，AP组；c，ECH组；d，AP+ECH组
Figure 4. The effect of Echinacoside on the hepatic morphology of rats with AP （HE staining，×400）
图 5 TEM观察肝组织微观结构变化
注： a，Con组（×25 000）；b，AP组（×8 000）；c，ECH组（×25 000）；d，AP+ECH组（×8 000）。N：细胞核；Mi：线粒体；APG：自噬体。
Figure 5. Changes of hepatic microscopic structure under TEM
图 6 TEM观察胰腺组织微观结构变化
注： a，Con组（×25 000）；b，AP组（×8 000）；c，ECH组（×25 000）；d，AP+ECH组（×8 000）。ZG：酶原颗粒；ER：内质网。
Figure 6. Changes of pancreatic microscopic structure under TEM
图 7 大鼠IL1β、IL-6、TNF-α、IL-10炎症因子变化水平
Figure 7. Change levels of inflammatory factors of IL-1β， IL-6， TNF-α and IL-10 in rats
图 8 免疫组化分析胰腺和肝组织中NF-κB p65、TNF-α表达水平
Figure 8. The expression levels of NF-κB p65 and TNF-α in pancreatic and hepatic tissues with Immunohistochemical analysis
图 9 Western Blot分析肝组织中NF-κB信号通路蛋白表达水平
Figure 9. The expression levels of NF-κB p65 signaling pathway protein in hepatic tissues with Western blot
表 1 ECH对雨蛙素诱导AP大鼠肝酶水平的影响
Table 1. The effects of Echinacoside on hepatic enzyme in rats with Cerulein-induced acute pancreatitis
项目 Con组（n=6） Con+ECH组（n=6） AP组（n=6） AP+ECH组（n=6） F值 P值 ALT（U/L） 37.35±11.04 32.95±9.90 185.07±33.581） 101.05±21.883） 66.53 <0.01 AST（U/L） 138.67±45.21 155.72±52.61 588.58±91.761） 329.28±85.663） 50.93 <0.01 GGT（U/L） 27.13±5.98 27.92±5.35 74.78±22.351） 43.80±11.653） 17.03 <0.01 Alb（U/L） 38.05±2.66 38.72±1.80 15.40±3.821） 27.82±3.343） 79.53 <0.01 ChE（U/L） 114.60±19.36 117.87±21.63 58.35±12.251） 93.95±8.193） 16.96 <0.01 LDH（U/L） 389.72±125.35 381.48±120.51 642.20±155.95 440.87±120.55 5.17 <0.01 ALP（U/L） 26.43±4.56 26.70±5.22 55.88±14.371） 35.77±7.333） 14.88 <0.01 TBil（μmol/L） 5.02±1.00 5.46±1.17 11.56±2.731） 6.25±1.723） 17.34 <0.01 注：与Con组比较，1）P<0.01，2）P<0.05；与AP组比较，3）P<0.01。 
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