目的 分析核苷酸结合寡聚化结构域样受体3(NLRP3)及相关炎性因子在新生大鼠坏死性小肠结肠炎(NEC)的表达水平,为临床上防治新生儿NEC提供科学依据。方法 选择3日龄新生SD大鼠24只,随机分为对照组(n=12)及实验组(n=12),采用配方奶人工喂养+缺氧+冷刺激的方法诱导NEC产生。在生后第7天断头处死大鼠,取回盲部肠组织进行相关指标检测。HE 染色观察回盲部肠组织病理学变化同时行双盲法进行病理学评分。TUNEL染色观察回盲部组织的细胞凋亡情况。电镜观察细胞内超微结构;ELISA 法检测 IL-18、IL-1β、IL-6 水平; Western-blot 检测 Caspase-1、Bcl-2、Bim 表达水平;RT-PCR 检测 NLRP3 mRNA 水平。结果 1)HE染色显示实验组大鼠肠壁绒毛损伤程度较重,大部分绒毛中部、根部断裂,部分隐窝无法识别、可见透壁坏死。对照组肠壁绒毛损伤情况较实验组轻。2)TUNEL染色显示实验组回盲部组织细胞凋亡数目增加,对照组回盲部组织细胞凋亡数目减少(t＝30.54,P＜0.05)。3)电镜下显示实验组线粒体肿胀,脊结构排列紊乱,并可出现部分脆性裂解等现象;对照组的线粒体损伤情况较实验组明显减轻。4)实验组大鼠回盲部肠组织中IL-18、IL-1β、IL-6水平较对照组显著升高(t=23.38、29.78、26.90,P＜0.05)。5)实验组Caspase-1、Bim 水平较对照组升高(t=15.12、5.82,P＜0.05),Bcl-2较对照组明显降低(t＝6.03,P＜0.05)。6)实验组的NLRP3 mRNA 表达水平明显高于对照组(t＝9.21,P＜0.05)。结论 NLRP3 在新生大鼠NEC的发病过程中发挥重要作用,阻断NLRP3的活化有望成为预防新生儿NEC的有效途径。
Abstract
Objective To investigate the expression level of nucleotide-binding oligomerization domain like receptor protein 3(NLRP3) and related inflammatory factors in newborn rats with necrotizing enterocolitis. Methods Twenty-four 3-day-old Sprague-Dawley (SD) rats were selected and randomly divided into control group (n=12) and experimental group (n=12). The rats were decapitated on the seventh day of life, their intestinal tissues of the caecal part were retrieved for related indexes. HE staining was used to observe the changes of ileocecal intestinal histopathology and double-blind method was used to score the pathology. Apoptosis in ileocecal tissue was observed by TUNEL staining. The ultrastructure of cells was observed by electron microscopy. IL-18, IL-1β and IL-6 levels were detected by ELISA. The expression levels of Caspase-1, Bcl-2 and Bim were detected by Western-blot, and NLRP3 mRNA level was detected by RT-PCR. Results 1) HE staining showed that the intestinal wall villi of rats in the experimental group were seriously damaged, most of the villi were broken in the middle and roots, and some of the crypts could not be recognized with visible wall necrosis. The intestinal wall villi damage in the control group was less than that in the experimental group. 2) TUNEL staining showed that the number of apoptosis increased in the ileocecal tissue of the experimental group and decreased in the control group (t＝30.54,P<0.05). 3) Under electron microscope, mitochondria of the experimental group were swollen, the ridge structure was disordered, and some brittle fractures were observed. Mitochondrial injury in control group was significantly reduced compared with that in experimental group. 4) The expression levels of IL-18, IL-1β and IL-6 in the ileocecal tissues of rats in the experimental group was significantly increased compared with those in the control group (t=23.38, 29.78, 26.90, P<0.05). 5) The expression levels of Caspase-1 and Bim in the experimental group were significantly higher than those in the control group (t=15.12, 5.82, P<0.05), while Bcl-2 expression was significantly lower in the experimental group(t＝6.03, P<0.05). 6) The mRNA expression level of NLRP3 in the experimental group was significantly higher than that in the control group (t＝9.21, P<0.05). Conclusions NLRP3 plays a crucial role in the development of NEC in newborn rats. Blocking the activation of NLRP3 is expected to be an effective way to prevent neonatal NEC.
关键词
核苷酸结合寡聚化结构域样受体3 /
炎性因子 /
新生大鼠 /
坏死性小肠结肠炎
Key words
nucleotide-binding oligomerization domain like receptor protein 3 /
inflammatory factor /
neonatal rats /
necrotizing enterocolitis
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参考文献
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基金
山东省医药卫生科技发展项目(2016WS0267)
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