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Sonic Hedgehog信号通路在重症急性胰腺炎大鼠模型肠黏膜屏障损伤中的作用探讨
DOI: 10.3969/j.issn.1001-5256.2023.05.020
伦理学声明：本研究方案于2022年3月9日经由西南医科大学动物保护与福利委员会批准，批号：swmu20220019，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：文琳负责课题设计，资料分析，撰写论文；曾家月、马风雨参与收集数据，修改论文；陈霞负责拟定写作思路，指导撰写文章并最后定稿。
Role of the Sonic Hedgehog signaling pathway in intestinal mucosal barrier injury in rats with severe acute pancreatitis
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摘要:
目的 探讨Sonic Hedgehog(Shh)信号通路在重症急性胰腺炎(SAP)大鼠肠道黏膜屏障损伤中的表达及作用。 方法 48只SD大鼠按随机数字表法分为：假手术组(Sham组)、SAP模型组(SAP组)、SAP模型组+Shh信号通路特异性激动剂嘌呤胺组(PUR+SAP组)、SAP模型组+Shh信号通路特异性抑制剂环巴胺组(CYC+SAP组)，各组又分为12 h、24 h两个亚组，每个亚组6只大鼠。SAP造模采用5%牛磺胆酸钠胰胆管逆行注射，干预组分别在造模前腹腔注射0.69 mg/kg嘌呤胺及0.69 mg/kg环巴胺。在造模后12 h和24 h取材。HE染色观察大鼠胰腺及回肠病理学变化；ELISA法检测大鼠血清淀粉酶、脂肪酶、DAO和EndoCAb的表达水平；TUNEL法检测肠上皮细胞凋亡情况；Western Blot检测回肠组织Shh、Ptch1和Gli1的表达。计量资料符合正态分布时多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验法；计量资料不符合正态分布时多组间比较及进一步两两比较均采用Kruskal-Wallis H检验法。 结果 与Sham组相比，SAP组胰腺和回肠组织病理学评分均明显增加，血清脂肪酶、淀粉酶、DAO和EndoCAb水平明显升高，肠上皮细胞凋亡增加，回肠组织Shh、Ptch1和Gli1蛋白表达升高(P值均＜0.05)。与SAP组对比，PUR+SAP组胰腺和肠道病理损伤及功能障碍减轻，肠道上皮细胞凋亡显著减少，回肠组织中的Shh、Ptch1和Gli1蛋白表达明显升高(P值均＜0.05)。与SAP组对比，CYC+SAP组胰腺和肠道病理损伤及功能障碍加重，肠道上皮细胞凋亡显著增加，回肠组织中的Shh、Ptch1和Gli1蛋白表达明显降低(P值均＜0.05)。 结论 Shh信号通路可能参与了SAP肠黏膜屏障损伤并且发挥保护作用。 Abstract:Objective To investigate the expression and role of the Sonic Hedgehog (Shh) signaling pathway in intestinal mucosal barrier injury in rats with severe acute pancreatitis (SAP). Methods A total of 48 Sprague-Dawley rats were divided into sham-operation group (Sham group), SAP model group (SAP group), SAP+Shh signaling pathway-specific agonist purmorphamine group (PUR+SAP group), and SAP+Shh signaling pathway-specific antagonist cyclopamine group (CYC+SAP group) using a random number table, with 12 rats in each group, and each group was further divided into 12-hour and 24-hour subgroups, with 6 rats in each subgroup. Rats were given retrograde injection of 5% sodium taurocholate into the pancreatic and bile ducts to establish a model of SAP, and rats in the intervention groups were given intraperitoneal injection of 0.69 mg/kg purmorphamine and 0.69 mg/kg cyclopamine before modeling. Related samples were collected at 12 and 24 hours after modeling. HE staining was used to observe the pathological changes of the pancreas and the ileum; ELISA was used to measure the serum levels of amylase, lipase, diamine oxidase (DAO), and endotoxin-core antibody (EndoCAb); the TUNEL method was used to observe the apoptosis of intestinal epithelial cells; Western blot was used to measure the expression levels of Shh, Ptch1, and Gli1 in ileal tissue. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and further comparison between two groups. Results Compared with the Sham group, the SAP group had significant increases in the pathological scores of pancreatic and ileum tissue, the serum levels of lipase, amylase, DAO, and EndoCAb, the apoptosis of intestinal epithelial cells, and the protein expression levels of Shh, Ptch1, and Gli1 in ileal tissue (all P < 0.05). Compared with the SAP group, the PUR+SAP group had significantly alleviated pathological injury and dysfunction of the pancreas and intestine, a significant reduction in the apoptosis of intestinal epithelial cells, and significant increases in the protein expression levels of Shh, Ptch1, and Gli1 in ileal tissue (all P < 0.05). Compared with the SAP group, the CYC+SAP group had significant aggravation of the pathological injury and dysfunction of the pancreas and intestine, a significant increase in the apoptosis of intestinal epithelial cells, and significant reductions in the protein expression levels of Shh, Ptch1, and Gli1 in ileal tissue (all P < 0.05). Conclusion The Shh signaling pathway may be involved in intestinal mucosal barrier injury in SAP and exerts a protective effect. -
Key words:
- Pancreatitis /
- Intestinal Mucosal Barrier /
- Signal Transduction /
- Apoptosis
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图 1 胰腺组织病理学改变(HE染色，×200)
Figure 1. Histological analyses of pancreatic tissues(HE, ×200)
图 3 血清中淀粉酶、脂肪酶、DAO和EndoCAb水平
Figure 3. Serum levels of amylase, lipase, DAO and EndoCAb
注：光镜观察，原始放大倍数×200；凋亡细胞的特征是细胞核呈绿色。
图 4 TUNEL法检测回肠黏膜细胞凋亡水平
Figure 4. Apoptosis of ileal mucosal cells detected by TdT-mediated dUTP nick end labeling assay
注：a，Western Blot检测Shh、Ptch1、Gli1通路相关蛋白表达，GAPDH作为对照; b、c，Shh、Ptch1和Gli1蛋白相对表达量。
图 6 大鼠回肠组织中Shh、Ptch1和Gli1的蛋白表达水平
Figure 6. The protein expression levels of Shh、Ptch1and Gli1in ileum
表 1 胰腺和回肠组织的病理学评分
Table 1. The pancreas and ileum tissue pathology score
组别 动物数(只) 胰腺组织12 h 胰腺组织24 h 回肠组织12 h 回肠组织24 h Sham组 6 0.50(0.50~0.63) 0.50(0.50~0.63) 0.50(0.38~0.50) 0.75(0.50~1.00) SAP组 6 7.76(7.36~8.50)1) 8.75(8.38~9.50)1) 3.62(3.53~3.73)1) 4.25(4.07~4.73)1) PUR+SAP组 6 2.25(1.88~2.50)1)2) 2.75(2.38~3.13)1)2) 2.16(2.08~2.29)1)2) 1.20(1.08~1.60)1)2) CYC+SAP组 6 13.25(12.38~13.63)1)2) 15.50(15.00~15.63)1)2) 4.75(4.50~5.00)1)2) 5.00(5.00~5.00)1)2) H值 21.84 21.88 21.78 21.73 P值 ＜0.05 ＜0.05 ＜0.05 ＜0.05 注: 与Sham组比较，1)P＜0.05；与SAP组比较，2)P＜0.05。 
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