南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (3): 376-383.doi: 10.12122/j.issn.1673-4254.2021.03.09

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鸟苷酸环化酶C在重症急性胰腺炎大鼠相关性肠损伤中的变化

王 冰，黄启林，李 帅，吴 俊，原小惠，孙红玉，汤礼军   

  • 出版日期:2021-03-20 发布日期:2021-04-06

Changes of guanylate cyclase C in colon tissues of rats with intestinal injury associated with severe acute pancreatitis

  • Online:2021-03-20 Published:2021-04-06

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摘要： 目的 探讨鸟苷酸环化酶C（GC-C）在重症急性胰腺炎（SAP）相关性肠损伤中的变化规律及作用。方法（1）36只成年雄性SD大鼠用随机数字表法分为假手术组（n=18，SO组，开腹后仅翻动胰腺数次后关腹）和重症急性胰腺炎组[n=18，SAP组，开腹后经胰胆管逆行微量泵入5%牛磺胆酸钠（0.1 mL/100 g）制备SAP模型]。各组根据用药时间再次随机分为12 h组，24 h组和48 h 组（n=6）。于建模后12、24、48 h分批处死大鼠收集结肠组织进行免疫蛋白印迹、免疫组织化学和RT-PCR实验明确GC-C在SAP相关性肠损伤中的变化规律，选取GC-C表达最低的时间点作为肠损伤最严重的大概时间点，即作为干预时间点进行后续实验；（2）18只成年雄性SD大鼠用随机数字表法分为SO组（n=6，处理方法同前）、SAP组（n=6，处理方法同前）和Linaclotide组 [n=6，诱导SAP后立即用GC-C激动剂利那洛肽（Linaclotide）水溶液（10 μg/kg/d）灌胃1次]。根据第一部分实验结果，选取12h节点进行检测。以HE染色评估胰腺与结肠病理改变；ELISA法测定血清中淀粉酶（AMY）、二胺氧化酶（DAO）、D-乳酸（DLac）和TNF-α浓度；通过RT-PCR、免疫蛋白印迹法、免疫组织化学染色法和透射电镜检测结肠组织中GC-C和紧密连接蛋白Claudin-1的表达。结果 GC-C在SAP组大鼠结肠中表达显著降低，于SAP建模后12 h表达最低（P<0.05），但随着时间推移GC-C表达逐渐升高。而结肠中Claudin-1表现出相似的趋势。与SO组相比，SAP组和Linaclotide组结肠表现为不同程度的粘膜不连续、间质水肿及炎性细胞浸润，病理学评分均升高（P<0.05）；血清淀粉酶、二胺氧化酶、D-乳酸和TNF-α均显著升高（P< 0.05）；结肠中GC-C和Claudin-1表达下降。与SAP组相比，Linaclotide组结肠组织病理评分降低，血清中上述各因子水平降低，而结肠组织中GC-C和Claudin-1表达水平升高。结论 GC-C在SAP相关性肠损伤中呈现出先降低后升高的表达趋势，于诱导SAP后12 h表达最低；通过激活GC-C可以提高GC-C和Claudin-1的表达水平并缓解肠道损伤，表明GC-C或许通过调节紧密
连接蛋白的表达来维持肠屏障功能的完整性。

关键词: 鸟苷酸环化酶C；重症急性胰腺炎；肠屏障

Abstract: Objective To explore the dynamic changes of guanylate cyclase C (GC-C) in the colon tissues of rats with intestinal injury associated with severe acute pancreatitis (SAP). Methods Thirty-six SD rats were randomized equally into two groups to receive either sham operation or retrograde pumping of 5% sodium taurocholate (0.1 mL/100 g) into the pancreaticobiliary duct following laparotomy to induce SAP. At 12, 24, and 48 h after modeling, 6 rats from each group were euthanized and the colon tissues were collected for Western blotting, immunohistochemistry and RT-PCR to determine the changes in GC-C expression, and the lowest GC-C expression was deemed to indicate the most serious intestinal injury and the time window for intervention. Another 18 SD rats were randomized into 3 groups for sham operation, SAP modeling or intragastric administration of linaclotide (a GC-C agonist) solution once daily at the dose of 10 μg/kg. At 12 h after modeling, the pathological changes in the pancreas and colon were observed with HE staining; the serum level of AMY, DAO, D-Lac and TNF-α were measured with ELISA, and the expressions of GC-C and claudin-1 were detected using Western blotting, immunohistochemical and transmission electron microscopy. Results The expression of GC-C was significantly reduced in the colon of rats in SAP group, and its lowest expression occurred at 12 h after modeling (P<0.05) followed by gradual increase over time. Claudin-1 showed a similar trend in the colon. Compared with the sham-operated rats, the rats in SAP and Linaclotide groups showed significantly increased pathological scores of the colon tissues (P<0.05) and serum levels of AMY, DAO, D-Lac and TNF-α and decreased expressions of GC-C and claudin-1 in the colon (P<0.05). Compared with those in SAP group, the rats in linaclotide group had significantly lower colonic histopathological scores, lower serum levels of AMY, DAO, D-Lac and TNF-α, and higher expression levels of GC-C and claudin-1 in the colon tissue. Conclusion In rats with SAP-related intestinal injury, the expression of GC-C in the colon tissue decreases to the lowest level at 12 h after SAP onset followed by gradual increase. activating GC-C can increase the expression levels of GC-C and claudin-1 and alleviate intestinal injury, suggesting the role of GC-C in maintaining intestinal barrier integrity by regulating the expression of tight junction proteins.

Key words: guanylate cyclase C; severe acute pancreatitis; intestinal barrier