西咪替丁对坏死性小肠结肠炎新生大鼠 CaMKIV/ CREM 通路及肠上皮完整性的影响
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河北北方学院附属第二医院儿科,河北 张家口 075100

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R-33


The effects of cimetidine on the calmodulin-dependent protein kinase IV / cyclic AMP response element modulator pathway and intestinal epithelial integrity in necrotizing enterocolitis in neonatal rats
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Department of Paediatrics, the Second Affiliated Hospital of Hebei North University, Zhangjiakou 075100, China

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    摘要:

    目的 探讨西咪替丁对坏死性小肠结肠炎(NEC)肠上皮完整性及钙调蛋白依赖性蛋白激酶 IV (CaMKIV) / 环磷酸腺苷反应元件调节蛋白(CREM)通路的影响。 方法 实验分为对照组、模型组、西咪替丁组和 CaMKIV 抑制剂组。 除对照组外,其余各组出生 1 d 缺氧+复氧+冷刺激建立 NEC 新生大鼠模型,连续 3 d。 每天刺激后西咪替丁组静脉注射 30. 5 mg / kg 西咪替丁,CaMKIV 抑制剂组插管灌胃 0. 72 mg / kg CaMKIV 抑制剂 KN62,对照组和模型组静脉注射和灌胃生理盐水处理相同天数。 观察大鼠临床情况;异硫氰酸荧光素-葡聚糖( FITC- Dextran)示踪法检测肠道通透性;苏木精-伊红(HE)染色观察肠道组织病理学变化;酶联免疫吸附(ELISA)检测大鼠肠组织中血小板活化因子(PAF)、肿瘤坏死因子 α(TNF-α) 水平;蛋白免疫印迹检测大鼠肠组织中 CaMKIV、 CREM 蛋白水平。 结果 模型组出现活动减少、反应迟钝现象,喂养时出现不耐受现象,大便形状改变;肠壁充气扩张、弹性差,部分出现穿孔现象,显微镜下观察到黏膜下层或固有层分离严重,绒毛脱落、腺体紊乱。 西咪替丁组和 CaMKIV 抑制剂组较模型组活动量有所增加,喂养状态缓和;肠壁充气扩张,显微镜下观察黏膜下层或固有层部分分离,出现部分绒毛脱落现象。与对照组相比,模型组肠道 FITC-Dextran 水平,组织学评分,肠组织中 PAF、 TNF-α水平,CaMKIV、CREM 蛋白水平升高(P<0. 05);与模型组相比,西咪替丁组、CaMKIV 抑制剂组肠道 FITC- Dextran 水平,组织学评分,肠组织中 PAF、TNF-α 水平,CaMKIV、CREM 蛋白水平降低(P<0. 05)。 结论 西咪替丁可能通过抑制 CaMKIV/ CREM 通路缓解 NEC 中炎症进而改善肠上皮,从而实现对 NEC 新生大鼠的保护。

    Abstract:

    Objective To investigate the effect of cimetidine on intestinal epithelial integrity and the calmodulin- dependent protein kinase IV ( CaMKIV) / cyclic AMP ( cAMP ) response element modulator ( CREM ) pathway in necrotizing enterocolitis (NEC). Methods Rats were divided into a control group, model group, cimetidine group, and CaMKIV inhibitor group. Except for the control group, the NEC newborn rat model was established by hypoxia + reoxygenation + cold stimulation on the first day of birth in each group over 3 consecutive days. After daily cold stimulation, 30. 5 mg / kg cimetidine was injected intravenously in the cimetidine group. In the CaMKIV inhibitor group, 0. 72 mg / kg of the CaMKIV inhibitor, kn62, was administered by intubation, and the control group and model group were treated with normal saline for the same number of days. The rats were then observed clinically. Intestinal permeability was measured by the fluorescein isothiocyanate-dextran ( FITC-dextran) tracing method ; hematoxylin eosin ( HE) staining was used to observe the histopathological changes of the intestinal tract; the concentrations of platelet activating factor ( PAF) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA); and the protein levels of CaMKIV and CREM were detected by Western blot. Results In the model group, we observed decreased activity and slow responses, low appetite, and change of stool shape. The intestinal wall was thickened and edematous, with poor elasticity and some perforation, microscopically; the submucosa or lamina propria were markedly separated; villi were missing; and glands were disordered. Compared with the activity in the model group, activity in the cimetidine group and CaMKIV inhibitor group increased, and appetite was moderate; the intestinal wall was thickened and edematous, microscopically; and the submucosa or lamina propria were partially separated, with some partially denuded villi. Compared with the control group, the FITC-dextran levels, histological scores, and the levels of PAF, TNF-α, CaMKIV, and CREM proteins in the intestinal tissue of the model group mice were higher (P< 0. 05). Compared with the model group, the levels of FITC- dextran, histological scores, and the concentrations of PAF, TNF-α, and CaMKIV and CREM proteins in the intestinal tissue in the cimetidine group and CaMKIV inhibitor group were lower ( P< 0. 05). Conclusions Cimetidine may alleviate inflammation in NEC and improve the intestinal epithelium by inhibiting the CaMKIV/ CREM pathway, which may protect NEC newborn rats.

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孙子梅,林晓燕,苏 琴,毕胜利.西咪替丁对坏死性小肠结肠炎新生大鼠 CaMKIV/ CREM 通路及肠上皮完整性的影响[J].中国比较医学杂志,2021,31(3):49~54.

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  • 收稿日期:2020-06-04
  • 在线发布日期: 2021-04-30
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