摘要： 目的·探究沉默信息调节因子1（silent information regulator transcript 1，SIRT1）在坏死性小肠结肠炎（necrotizing enterocolitis，NEC）肠管组织中的表达特点，初步探讨SIRT1对NEC的影响及可能机制。方法·纳入2018年6月—2020年10月经浙江大学医学院附属儿童医院新生儿外科手术治疗的NEC患儿80例，分为观察组和对照组。观察组组织样本为炎症坏死肠管；对照组为经保守治疗后发生肠狭窄，再次选择外科手术治疗的NEC患儿，组织样本为切缘肠管。收集2组患儿术前24 h内的血清中降钙素原（procalcitonin，PCT）、超敏C反应蛋白（hypersensitive C reactive protein，hs-CRP）、细胞因子［白细胞介素-2（interleukin-2，IL-2）、IL-4、IL-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、γ干扰素（interferon-γ，IFN-γ）］以及性别、孕周等临床资料。采用免疫组织化学法检测2组患儿组织样本中的SIRT1、核因子κB（nuclear factor-κB，NF-κB）、转化生长因子β1（transforming growth factor β1，TGF-β1）和Smad3蛋白的表达水平。体外培养SD雌性大鼠小肠上皮IEC-6细胞，采用干扰小RNA（small interfering RNA，siRNA）技术抑制IEC-6细胞中的SIRT1表达后，使用蛋白质印迹检查NF-κB表达变化，通过Cell Counting Kit-8（CCK8）和Transwell迁移实验检测SIRT1对IEC-6增殖和迁移能力的影响。结果·2组患儿的性别、孕周、出生体质量和分娩方式等一般资料差异无统计学意义。与对照组相比，观察组术前24 h内血清hs-CRP、PCT、IL-6、IL-10升高（均
P=0.000）。与对照组NEC肠狭窄切缘肠管组织相比，观察组NEC坏死肠管组织中的SIRT1呈低表达，NF-κB呈过表达（均
P=0.000）。Smad3蛋白和TGF-β1蛋白表达2组差异无统计学意义。NEC坏死肠管组织中SIRT1与NF-κB表达呈负相关（
r=-0.592，
P=0.000）。抑制SIRT1的mRNA和蛋白表达后，IEC-6细胞中的NF-κB相对表达量升高，细胞的增殖能力和迁移能力明显降低。结论·SIRT1可能通过抑制NF-κB的表达来降低NEC中炎症因子的表达，从而减轻NEC的进展。另一个可能的机制是SIRT1通过保护肠上皮细胞，减少其凋亡和迁移，从而减轻NEC的进展。
关键词: 沉默信息调节因子1, 核因子κB, 坏死性小肠结肠炎, 炎症细胞因子, 新生儿 Abstract: Objective·To explore the expression characteristics of silent information regulator transcript 1 (SIRT1) in intestinal tissues of necrotizing enterocolitis (NEC), and preliminarily discuss the effect and possible mechanisms of SIRT1 in NEC.
Methods·From June 2018 to October 2020, 80 children with NEC who were treated by neonatal surgery in the Children's Hospital, Zhejiang University School of Medicine were divided into observation group and control group. The tissue samples of the observation group were inflammatory necrotic intestinal tubes, while those of the control group were incised intestinal tubes. The NEC children in the control group were with intestinal strictures after conservative treatment, and then were treated by surgery again. The clinical data of the two groups were collected 24 h before surgery, including procalcitonin (PCT), hypersensitive C reactive protein (hs-CRP), cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)] in the serum, gender, gestational age and so on. The expression characteristics of SIRT1, nuclear factor-κB (NF-κB), transforming growth factor β1 (TGF-β1) and Smad3 proteins of the tissues in the two groups were detected by immunohistochemistry. IEC-6 cells of small intestinal epithelium of SD female rats were cultured in vitro. After SIRT1 expression in IEC-6 cells was inhibited by small interfering RNA (siRNA) technology, the protein expression of NF-κB was detected by Western blotting and the effects of SIRT1 on IEC-6 cells were detected by Cell Counting Kit-8 (CCK8) and Transwell migration assay.
Results·There was no significant difference in gender, gestational age, birth weight and delivery mode between the two groups. Compared with the control group, the levels of hs-CRP, PCT, IL-6 and IL-10 in the serum in the observation group increased within 24 h before surgery (all P=0.000).Compared with the margin tissues of narrow intestines in the control group, the positive expression of SIRT1 in NEC necrotic intestinal tissues in the observation group showed low expression, and the positive expression of NF-κB was overexpressed (both P=0.000). There was no significant difference in the expression of Smad3 and TGF-β1 between the two groups. The expression of SIRT1 in NEC necrotic intestinal tissue was negatively correlated with the expression of NF-κB (r=-0.592, P=0.000). After inhibiting the mRNA and protein expression of SIRT1, the relative expression of NF-κB in IEC-6 cells was increased, and the proliferation and migration ability of the cells was significantly decreased.
Conclusion·The mechanism for the SIRT1 reducing progression of NEC may be that SIRT1 can inhibit the expression of NF-κB to reduce the expression of inflammatory factors in NEC, and another possible mechanism may be that SIRT1 can protect intestinal epithelial cells by promoting cell proliferation and migration.
Key words: silencing information regulator factor 1 (SIRT1), nuclear factor-κB (NF-κB), necrotizing enterocolitis (NEC), inflammatory cytokine, neonate PDF全文下载地址:
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