miR-301a对小鼠坏死性小肠结肠炎的影响及作用机制

doi:10.11958/20220254

摘要/Abstract

摘要：

目的观察miR-301a对小鼠坏死性小肠结肠炎（NEC）的影响及作用机制。方法将60只新生BALB/c小鼠采用随机数字表法分为对照组、NEC组、miR-301a拮抗剂组,每组20只。NEC组、miR-301a拮抗剂组通过人工喂养、缺氧和冷刺激5 d建立NEC模型,miR-301a拮抗剂组给予miR-301a拮抗剂。实验期间记录小鼠体质量,HE染色观察各组肠道组织病理改变,并对肠组织进行损伤评分;采用酶联免疫吸附试验（ELISA）检测各组小鼠血清肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-8的含量。采用TUNEL法测定不同组小鼠肠组织的细胞凋亡情况,qPCR检测miR-301a和胱天蛋白酶（Caspase）-1基因表达水平,Western blot检测Caspase-1蛋白表达水平。结果与对照组相比,NEC组小鼠体质量减轻,肠组织出现明显的炎症损伤,损伤评分升高;血清中TNF-α、IL-6和IL-8的含量升高,肠组织细胞凋亡指数增加,miR-301a和Caspase-1的表达水平升高（均P<0.05）。与NEC组相比,miR-301a拮抗剂组小鼠NEC症状明显减轻,损伤评分及血清TNF-α、IL-6和IL-8含量明显下降,肠组织细胞凋亡指数下调,miR-301a和Caspase-1的表达水平降低（均P<0.05）。结论 miR-301a在小鼠NEC肠组织中表达增加,下调miR-301a可以在一定程度上抑制小鼠的NEC进展。

关键词: 小肠结肠炎,坏死性, 细胞凋亡, 炎症, 小鼠,近交BALB C, miR-301a

Abstract:

Objective To investigate the effect and mechanism of miR-301a on necrotizing enterocolitis (NEC) in mice. Methods Sixty newborn BALB/c mice were randomly divided into the control group, the NEC group and the antagonist group (miR-301a antagonist group), with 20 mice in each group. NEC model was established in the NEC group and the miR-301a antagonist group by artificial feeding, hypoxia and cold stimulation for 5 days. The miR-301a antagonist group was given miR-301a antagonist on the basis of the NEC group. During the experiment, the body mass of mice was recorded, the histopathological changes of intestinal tissues were observed by HE staining in each group, and the inflammatory damage in intestinal tissues was scored. Serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) in each group. Apoptosis was measured by TUNEL method, and the protein expression levels of miR-301a and Caspase-1 were measured by qPCR and Western blot assay. Results Compared with the control group, mice in the NEC group showed reduced body mass, obvious inflammatory injury in intestinal tissue and increased intestinal tissue injury score. Serum levels of TNF-α, IL-6 and IL-8 were increased, apoptosis index of intestinal tissues were increased and mRNA and protein expression levels of miR-301a and Caspase-1 were also increased (all P < 0.05). Compared with the NEC group, mice in the miR-301a antagonistic group showed significantly reduced NEC symptoms, significantly decreased inflammatory injury score and serum levels of TNF-α, IL-6 and IL-8 in intestinal tissue, down-regulated apoptotic index of intestinal tissue and decreased expression levels of miR-301a and Caspase-1 (all P < 0.05). Conclusion miR-301a expression is increased in mouse NEC intestinal tissue, and downregulation of miR-301a could inhibit NEC progression in mice to a some extent.

Key words: enterocolitis,necrotizing, apoptosis, inflammation, mice,inbred BALB C, miR-301a

中图分类号:

R363.1

图/表 4

图1 3组小鼠体质量和病理评分变化 A：各组小鼠在第1~5天的体质量变化;F（1 d）=2.213,P=0.119;F（2 d）=9.544,F（3 d）=6.618,F（4 d）=3.411,F（5 d）=36.284,均P<0.05;B：肠组织HE染色后病理损伤评分;a与对照组比较,b与NEC组比较,P<0.05。

Fig.1 Variations of body weight and pathological score indexes in three groups of mice

图2 3组小鼠肠组织病理改变（HE染色,×200）

Fig.2 Variations of HE staining in 3 groups of mice (HE staining, ×200)

图3 3组小鼠肠组织炎性因子表达及细胞凋亡水平比较 A~C：ELISA法检测各组小鼠血清中TNF-α、IL-6和IL-8的表达水平;D：TUNEL法测定各组小鼠肠组织的细胞凋亡情况;a与对照组比较, b与NEC组比较,P<0.05。

Fig.3 Comparison of inflammatory factor expression and apoptosis level in intestinal tissue between three groups of mice

图4 3组小鼠肠组织miR-301a和Caspase-1 mRNA及Caspase-1蛋白表达水平比较 A、B：qPCR检测各组小鼠肠道组织中miR-301a和Caspase-1的mRNA表达水平;a与对照组比较,b与NEC组比较,P<0.05。C：Western blot检测各组小鼠肠道组织中Caspase-1的蛋白表达水平。

Fig.4 Comparison of miR-301a and Caspase-1 protein expression levels in intestinal tissue between the three groups of mice

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