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

文琳; 曾家月; 马凤雨; 陈霞

doi:10.3969/j.issn.1001-5256.2023.05.020

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

文琳¹,
曾家月¹,
马凤雨¹,
陈霞^{1, 2, , ,}

1.
西南医科大学附属医院消化内科，四川泸州 646000
2.
成都医学院第一附属医院消化内科，成都 610000

基金项目:

国家自然科学青年基金 (81600420);

泸州市人民政府-西南医科大学科技战略合作项目 (2019LZXNYDJ48)

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。

详细信息

通信作者:
陈霞，970217858@qq.com (ORCID: 0000-0003-3675-5336)

计量
- 文章访问数: 582
- HTML全文浏览量: 184
- PDF下载量: 49
- 被引次数: 0
出版历程
- 收稿日期: 2022-10-27
- 录用日期: 2022-12-17
- 出版日期: 2023-05-20

Role of the Sonic Hedgehog signaling pathway in intestinal mucosal barrier injury in rats with severe acute pancreatitis

Lin WEN¹,
Jiayue ZENG¹,
Fengyu MA¹,
Xia CHEN^{1, 2
, ,
,}

1.
Department of Gastroenterology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China
2.
Department of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu 610000, China

Research funding:

National Natural Science Foundation of China (81600420);

Luzhou Municipal People's Government-Southwest Medical University Science and Technology Strategic Cooperation Project (2019LZXNYDJ48)

More Information

Corresponding author: CHEN Xia, 970217858@qq.com (ORCID: 0000-0003-3675-5336)

摘要

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。
- 胰腺炎 /
- 肠黏膜屏障 /
- 信号传导 /
- 细胞凋亡
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.
- Pancreatitis /
- Intestinal Mucosal Barrier /
- Signal Transduction /
- Apoptosis

HTML全文

图 1 胰腺组织病理学改变(HE染色，×200)

Figure 1. Histological analyses of pancreatic tissues(HE, ×200)

下载: 全尺寸图片幻灯片

图 2 回肠组织病理学改变(HE染色，×200)

Figure 2. Histological analyses of ileum tissues(HE, ×200)

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 4 TUNEL法检测回肠黏膜细胞凋亡水平

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。

Figure 4. Apoptosis of ileal mucosal cells detected by TdT-mediated dUTP nick end labeling assay

下载: 全尺寸图片幻灯片

图 5 细胞凋亡的积分光密度值

Figure 5. Integral optical density values of apoptosis

下载: 全尺寸图片幻灯片

图 6 大鼠回肠组织中Shh、Ptch1和Gli1的蛋白表达水平

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。

Figure 6. The protein expression levels of Shh、Ptch1and Gli1in ileum

下载: 全尺寸图片幻灯片

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。

下载: 导出CSV

参考文献(29)

[1]	Chinese Society for Emergency Medicine; Beijing-Tianjin-Hebei Alliance of Emergency Treatment and First Aid; Emergency Medicine Branch, Beijing Medical Association, et al. Expert consensus on emergency diagnosis and treatment of acute pancreatitis[J]. J Clin Hepatol, 2021, 37(5): 1034-1041. DOI: 10.3969/j.issn.1001-5256.2021.05.012. 中华医学会急诊分会, 京津冀急诊急救联盟, 北京医学会急诊分会, 等. 急性胰腺炎急诊诊断及治疗专家共识[J]. 临床肝胆病杂志, 2021, 37(5): 1034-1041. DOI: 10.3969/j.issn.1001-5256.2021.05.012.
[2]	TERAO K, WAKE H, ADACHI N, et al. Histidine-rich glycoprotein suppresses hyperinflammatory responses of lung in a severe acute pancreatitis mouse model[J]. Pancreas, 2018, 47(9): 1156-1164. DOI: 10.1097/MPA.0000000000001153.
[3]	CHEN X, ZHAO HX, FU XS, et al. Glucagonlike peptide 2 protects intestinal barrier in severe acute pancreatitis through regulating intestinal epithelial cell proliferation and apoptosis[J]. Pancreas, 2012, 41(7): 1080-1085. DOI: 10.1097/MPA.0b013e31824966b0.
[4]	ZHAO HX, FU XS, ZHOU XY, et al. Endoplasmic reticulum stress may not be involved in intestinal epithelial cell apoptosis in experimental acute pancreatitis[J]. Dig Dis Sci, 2015, 60(6): 1690-1698. DOI: 10.1007/s10620-015-3542-y.
[5]	INGHAM PW, MCMAHON AP. Hedgehog signaling in animal development: paradigms and principles[J]. Genes Dev, 2001, 15(23): 3059-3087. DOI: 10.1101/gad.938601.
[6]	PERIDES G, van ACKER GJ, LAUKKARINEN JM, et al. Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct[J]. Nat Protoc, 2010, 5(2): 335-341. DOI: 10.1038/nprot.2009.243.
[7]	KUGLER MC, JOYNER AL, LOOMIS CA, et al. Sonic hedgehog signaling in the lung. From development to disease[J]. Am J Respir Cell Mol Biol, 2015, 52(1): 1-13. DOI: 10.1165/rcmb.2014-0132TR.
[8]	ZHOU X, LIU Z, JANG F, et al. Autocrine Sonic hedgehog attenuates inflammation in cerulein-induced acute pancreatitis in mice via upregulation of IL-10[J]. PLoS One, 2012, 7(8): e44121. DOI: 10.1371/journal.pone.0044121.
[9]	HUANG J, ZHENG YQ, ZHOU XY. The role of Hedgehog signal in mice with acute pancreatitis[J]. J Luzhou Med Coll, 2013, 36(2): 121-123. https://www.cnki.com.cn/Article/CJFDTOTAL-LXYB201302010.htm 黄娟, 郑英强, 周翔宇. 小鼠急性胰腺炎时Hedgehog信号通路mRNA的表达及意义[J]. 泸州医学院学报, 2013, 36(2): 121-123. https://www.cnki.com.cn/Article/CJFDTOTAL-LXYB201302010.htm
[10]	LAI K, LUO L, WANG F, et al. Expression of Shh in different tissues in acute pancreatitis[J]. J Southwest Med Univ, 2017, 40(1): 26-30. DOI: 10.3969/j.issn.1000-2669.2017.01.008. 赖坤, 罗澜, 王芳, 等. 小鼠急性胰腺炎中Shh在胰腺局部和远隔器官的表达变化[J]. 西南医科大学学报, 2017, 40(1): 26-30. DOI: 10.3969/j.issn.1000-2669.2017.01.008.
[11]	SINHA S, CHEN JK. Purmorphamine activates the Hedgehog pathway by targeting Smoothened[J]. Nat Chem Biol, 2006, 2(1): 29-30. DOI: 10.1038/nchembio753.
[12]	van den HEUVEL M, INGHAM PW. smoothened encodes a receptor-like serpentine protein required for hedgehog signalling[J]. Nature, 1996, 382(6591): 547-551. DOI: 10.1038/382547a0.
[13]	LIAO X, SIU MK, AU CW, et al. Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation[J]. Carcinogenesis, 2009, 30(1): 131-140. DOI: 10.1093/carcin/bgn230.
[14]	MAHESHWARI R, SUBRAMANIAN RM. Severe acute pancreatitis and necrotizing pancreatitis[J]. Crit Care Clin, 2016, 32(2): 279-290. DOI: 10.1016/j.ccc.2015.12.006.
[15]	DENG W, ABLIZ A, XU S, et al. Severity of pancreatitis-associated intestinal mucosal barrier injury is reduced following treatment with the NADPH oxidase inhibitor apocynin[J]. Mol Med Rep, 2016, 14(4): 3525-3534. DOI: 10.3892/mmr.2016.5678.
[16]	TIAN R, TAN JT, WANG RL, et al. The role of intestinal mucosa oxidative stress in gut barrier dysfunction of severe acute pancreatitis[J]. Eur Rev Med Pharmacol Sci, 2013, 17(3): 349-355.
[17]	CAPURSO G, ZERBONI G, SIGNORETTI M, et al. Role of the gut barrier in acute pancreatitis[J]. J Clin Gastroenterol, 2012, 46 (Suppl): S46-S51. DOI: 10.1097/MCG.0b013e3182652096.
[18]	Pancreas Study Group, Chinese Society of Gastroenterology, Chinese Medical Association, Editorial Board of Chinese Journal of Pancreatology, Editorial Board of Chinese Journal of Digestion. Chinese guidelines for the management of acute pancreatitis (Shanghai, 2013)[J]. J Clin Hepatol, 2013, 29(9): 656-660. DOI: 10.3969/j.issn.1001-5256.2013.09.006. 中华医学会消化病学分会胰腺疾病学组, 《中华胰腺病杂志》编辑委员会, 《中华消化杂志》编辑委员会. 中国急性胰腺炎诊治指南(2013年, 上海)[J]. 临床肝胆病杂志, 2013, 29(9): 656-660. DOI: 10.3969/j.issn.1001-5256.2013.09.006.
[19]	WISCHMEYER PE. Nutrition therapy in sepsis[J]. Crit Care Clin, 2018, 34(1): 107-125. DOI: 10.1016/j.ccc.2017.08.008.
[20]	LUAN ZG, ZHANG H, MA XC, et al. Role of high-mobility group box 1 protein in the pathogenesis of intestinal barrier injury in rats with severe acute pancreatitis[J]. Pancreas, 2010, 39(2): 216-223. DOI: 10.1097/MPA.0b013e3181bab5c5.
[21]	PAN LY, CHEN YF, LI HC, et al. Dachengqi Decoction attenuates intestinal vascular endothelial injury in severe acute pancreatitis in vitro and in vivo[J]. Cell Physiol Biochem, 2017, 44(6): 2395-2406. DOI: 10.1159/000486155.
[22]	LEE JH, CHUNG YC, BOK E, et al. Injury-stimulated Sonic hedgehog expression in microglia contributes to neuroinflammatory response in the MPTP model of Parkinson's disease[J]. Biochem Biophys Res Commun, 2017, 482(4): 980-986. DOI: 10.1016/j.bbrc.2016.11.144.
[23]	XIA YP, HE QW, LI YN, et al. Recombinant human sonic hedgehog protein regulates the expression of ZO-1 and occludin by activating angiopoietin-1 in stroke damage[J]. PLoS One, 2013, 8(7): e68891. DOI: 10.1371/journal.pone.0068891.
[24]	LV T, SHEN L, YANG L, et al. Polydatin ameliorates dextran sulfate sodium-induced colitis by decreasing oxidative stress and apoptosis partially via Sonic hedgehog signaling pathway[J]. Int Immunopharmacol, 2018, 64: 256-263. DOI: 10.1016/j.intimp.2018.09.009.
[25]	IKEDA H, SUZUKI Y, SUZUKI M, et al. Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium[J]. Gut, 1998, 42(4): 530-537. DOI: 10.1136/gut.42.4.530.
[26]	YAMAMOTO S, TANABE M, WAKABAYASHI G, et al. The role of tumor necrosis factor-alpha and interleukin-1beta in ischemia-reperfusion injury of the rat small intestine[J]. J Surg Res, 2001, 99(1): 134-141. DOI: 10.1006/jsre.2001.6106.
[27]	MENG QH, LIU HB, WANG JB. Polydatin ameliorates renal ischemia/reperfusion injury by decreasing apoptosis and oxidative stress through activating sonic hedgehog signaling pathway[J]. Food Chem Toxicol, 2016, 96: 215-225. DOI: 10.1016/j.fct.2016.07.032.
[28]	LIANG R, MORRIS P, CHO SS, et al. Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair[J]. J Pediatr Surg, 2012, 47(12): 2251-2263. DOI: 10.1016/j.jpedsurg.2012.09.016.
[29]	TIAN A, SHI Q, JIANG A, et al. Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells[J]. J Cell Biol, 2015, 208(6): 807-819. DOI: 10.1083/jcb.201409025.