腺苷抑制GSK-3β活性对大鼠急危重症胰腺炎肠屏障功能障碍的影响

李旻昊; 袁梅; 夏加增; 巢琳

doi:10.13898/j.cnki.issn.1000-2200.2022.10.005

腺苷抑制GSK-3β活性对大鼠急危重症胰腺炎肠屏障功能障碍的影响

Effect of adenosine inhibition of GSK-3β activity on intestinal barrier dysfunction in rats with severe acute pancreatitis

摘要

摘要:
目的研究腺苷对大鼠急危重症胰腺炎(SAP)肠屏障功能障碍(IBD)的保护途径。
方法将64只健康雄性SD大鼠随机分为假手术组8只、SAP组40只(分为0、3、6、12、24 h不同时间点处理组，每组8只)、腺苷处理组(Ade-Ⅳ组)8只、线粒体膜通透性转移孔(mPTP)抑制剂环孢素A(CsA)组8只。大鼠SAP造模完成后，血清胰淀粉酶升高及镜下胰腺组织病理学改变提示造模成功，HE染色观察大鼠小肠组织损伤情况，Western blotting法检测大鼠小肠组织P-GSK-3β表达水平，应用电镜观察小肠线粒体超微结构的变化，采用差速离心法分离线粒体，并在Ca²⁺诱导下完成线粒体肿胀实验。
结果大鼠SAP造模成功后，SAP组40只大鼠血清胰腺淀粉酶水平随着0~12 h时间点的延长逐渐升高，且12 h达到高峰，24 h下降至最低点，差异均有统计学意义(P < 0.01)；切片染色观察，SAP组小肠黏膜充血、水肿，并伴有柱状上皮细胞坏死，甚至出现部分绒毛的断裂、缺损，而Ade-Ⅳ组肠黏膜上皮及绒毛形态基本完整，绒毛排列基本整齐；P-GSK-3β的表达，Ade-Ⅳ组较SAP组显著增加(P < 0.01)；小肠线粒体超微结构在SAP组肿胀明显，还出现了空泡变性，而Ade-Ⅳ组肿胀程度减轻，空泡变性减少；线粒体肿胀实验发现520 nm处线粒体吸光度值CsA组 < Ade-Ⅳ组 < SAP组，差异有统计学意义(P < 0.01)。
结论腺苷对SAP并发的IBD有保护作用，其机制可能与抑制GSK-3β活性，进而阻止mPTP的开放有关。

Abstract:
ObjectiveTo explore the protection pathway of adenosine against intestinal barrier dysfunction(IBD) in rats with severe acute pancreatitis(SAP).
MethodsA total of 64 male SD rats were randomly divided into 4 groups: sham group, adenosine treatment group(adenosine), SAP group and mPTP inhibitor cyclosporin A group.Serum amylase level and pancreatic pathology were tested to confirm the success of SAP model; the damage of small intestine tissue was observed by HE staining; the expression of P-GSK-3β in the small intestine of rats was detected by Western blotting; electronmicroscope was used to observe the changes of mitochondrial ultrastructure; differential centrifugation method was used to separate mitochondria, then, mitochondrial swelling experiments were conducted under Ca²⁺ induction.
ResultsWith the success of SAP model, the serum pancreatic amylase level was gradually increased with the extension of the time point from 0 to 12 h, which reached the peak at 12 h and decreased to the lowest at 24 h.The difference was statistically significant(P < 0.01).Staining of sections showed that SAP group had hyperemia and edema of intestinal mucosa, accompanied by necrosis of columnar epithelial cells, and even fracture and defect of some villi, while the intestinal mucosal epithelium and fluffy form of the Ade-Ⅳ group were basically complete, and the velvet arrangement was basically neat.Compared to the SAP group, the P-GSK-3β expression of the Ade-Ⅳ group increased significantly(P < 0.01).In SAP group, intestinal mitochondrial ultra-micro structure was significantly swollen, even empty bubble degeneration appeared, while in Ade-Ⅳgroup, this significantly reduced.Mitochondrial swelling experiment showed that the mitochondrial absorbance value at 520 nm was the highest in SAP group, secondary was Ade-Ⅳ group and CSA group was the lowest, and the differences were statistically significant(P < 0.01).
ConclusionsAdenosine prevents SAP intestinal barrier injury presumably by targeting the mPTP through inactivation of GSK-3β.

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