目的 探究microRNA-223-3p(MiR-223-3p)对坏死性小肠结肠炎(NEC)新生大鼠肠组织的炎症反应和细胞损伤的影响及可能的机制。方法 构建新生大鼠NEC模型,随机分为NEC组、阴性对照组、MiR-223-3p过表达组,每组20只。另取相同数量正常大鼠作为正常组。苏木精-伊红(HE)染色、TUNEL染色检测肠组织病理学变化和细胞凋亡情况;ELISA法检测血清和肠组织中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)水平;SYBR Green I实时荧光定量PCR法检测肠组织中MiR-223-3p表达;Western Blot检测肠组织中NLRP3和caspase-1蛋白表达情况。双荧光素酶报告基因实验验证MiR-223-3p与NLRP3是否存在靶向关系。结果 与正常组比较,NEC组和阴性对照组大鼠血清及肠组织中IL-6、IL-1β、IL-18含量明显升高(F=215.525、276.499、354.826、204.410、261.474、280.667,P＜0.05),肠组织中NLRP3和caspase-1蛋白表达及细胞凋亡率均明显升高(F=181.745、137.553,P＜0.05),肠组织中MiR-223-3p表达明显降低(F=170.180,P＜0.05),且肠组织存在明显病理学损伤。与阴性对照组比较,MiR-223-3p过表达组能够上调肠组织中MiR-223-3p表达,降低大鼠血清及肠组织中IL-6、IL-1β、IL-18含量及细胞凋亡率,抑制NLRP3和caspase-1蛋白表达(P＜0.05),且肠组织病理学损伤明显改善。双荧光素酶报告基因实验分析结果显示,MiR-223-3p与NLRP3能够靶向结合。结论 MiR-223-3p通过靶向NLRP3减轻NEC新生大鼠肠组织的炎症反应和细胞损伤。
Abstract
Objective To explore the effects of microRNA-223-3p(MiR-223-3p) on the inflammatory response and cell damage in the intestinal tissue of neonatal rats with necrotizing enterocolitis(NEC) and its possible mechanism. Methods The NEC model of neonatal rats was constructed and randomly divided into a model group, a negative control group, and a MiR-223-3p overexpression group. The normal rats of same number were taken as the normal group. Hematoxylin-eosin(HE) staining and TUNEL staining were used to detect intestinal histopathological changes and cell apoptosis; ELISA method was used to detect the levels of interleukin-6(IL-6), interleukin-1β(IL-1β) and interleukin-18(IL-18) in serum and intestinal tissues; SYBR Green Ⅰ real-time fluorescent quantitative PCR method was used to detect the expression of MiR-223-3p in intestinal tissues; Western Blot was used to detect the expression of NLRP3 and caspase-1 proteins in intestinal tissue. Dual luciferase reporter gene experiment was used to verify the target relationship between MiR-223-3p and NLRP3. Results Compared with the normal group, the levels of IL-6, IL-1β, IL-18 in the serum and intestinal tissues of rats in the model group and the negative control group were all significantly increased(F=215.525, 276.499, 354.826, 204.410, 261.474, 280.667, P<0.05), the expression of NLRP3 and caspase-1 proteins in the intestinal tissues and the apoptosis rate were all significantly increased(F=181.745, 137.553, P<0.05), the expression of MiR-223-3p in the intestinal tissue was significantly reduced(F=170.180, P<0.05), and there was obvious pathological damage in the intestinal tissue. Compared with the negative control group, the MiR-223-3p overexpression group was able to up-regulate the expression of MiR-223-3p in intestinal tissues, reduce the levels of IL-6, IL-1β, IL-18 and cell apoptosis in rat serum and intestinal tissues, inhibit the expression of NLRP3 and caspase-1 proteins(P<0.05), and the pathological damage of intestinal tissue was significantly improved. The results of the dual luciferase reporter gene experimental analysis showed that MiR-223-3p and NLRP3 was able to targetingly be combined. Conclusion MiR-223-3p reduces inflammation and cell damage in the intestinal tissue of NEC neonatal rats by targeting NLRP3.
关键词
MiR-223-3p /
NLRP3 /
坏死性小肠结肠炎 /
新生大鼠 /
炎症反应 /
细胞凋亡
Key words
MiR-223-3p /
NLRP3 /
necrotizing enterocolitis /
neonatal rat /
inflammatory response /
apoptosis
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基金
2016年度海南省卫生计生行业科研项目(15A200076)
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