Data_Sheet_2_Expression and Possible Role of Silent Mating Type Information Regulation 2 Homolog 1 in Post-necrotizing Enterocolitis Stricture in vivo and in vitro.ZIP

PurposeTo investigate the expression and possible role of Sirtuin1 or Silent mating–type information regulation 2 homolog-1 (SIRT1) in post-necrotizing enterocolitis stricture.
Materials and MethodsThe expression characteristics of SIRT1 and TGF-β1 in post-necrotizing enterocolitis stricture were detected by immunohistochemistry. The siRNA-SIRT1 was used to inhibit the expression of SIRT1 in intestinal epithelial cells-6 (IEC-6), and qRT-PCR, WB, and ELISA were utilized to detect the changes of Transforming growth factor-β1 (TGF-β1), nuclear factor (NF)-κB, tumor necrosis factor-α (TNF-α), tight junction protein-1 (ZO-1), and vascular endothelial growth factor (VEGF) expressions. The IEC-6 cell proliferation and migration ability were tested via CCK8 kit and Transwell test. The expression of E-cadherin and Vimentin in cells was detected by immunofluorescence.
ResultsThe CRP, IL-6, IL-10, and IFN-γ in the serum of Necrotizing enterocolitis (NEC) intestinal stenosis patients were significantly higher than the reference values. The SIRT1 protein was under-expressed and the TGF-β1 protein was overexpressed in NEC intestinal stenosis tissue. And the expression of SIRT1 was negatively correlated with TGF-β1. At the time of diagnosis of NEC, the expression of SIRT1 decreased in children with respiratory distress syndrome and CRP level increased. After inhibiting the expression of SIRT1 in IEC6 cells, the expression levels of TGF-β1, Smad3, and NF-κB were decreased, and the expression of ZO-1 was also decreased. The proliferation and migration ability of IEC6 cells was decreased significantly, and the expression of E-cadherin and Vimentin proteins in IEC6 cells did not change significantly.
ConclusionPromotion of intestinal fibrosis by inflammation may be the mechanism of post-necrotizing enterocolitis stricture. SIRT1 may be a protective protein of NEC. The probable mechanism is that SIRT1 can regulate intestinal fibrosis and can protect the intestinal mucosal barrier function to participate in the process of post-necrotizing enterocolitis stricture.

**研究目的**：探究沉默信息调节因子2同源物1（Silent mating–type information regulation 2 homolog-1，SIRT1，又名Sirtuin1）在坏死性小肠结肠炎后狭窄中的表达情况及其潜在作用。
**材料与方法**：采用免疫组织化学法（immunohistochemistry）检测坏死性小肠结肠炎后狭窄组织中SIRT1与转化生长因子-β1（TGF-β1）的表达特征。通过靶向SIRT1的小干扰RNA（siRNA-SIRT1）抑制肠上皮细胞-6（IEC-6）中SIRT1的表达，利用实时荧光定量聚合酶链反应（qRT-PCR）、蛋白质免疫印迹（WB）及酶联免疫吸附测定（ELISA）检测转化生长因子-β1（TGF-β1）、核因子κB（NF-κB）、肿瘤坏死因子-α（TNF-α）、紧密连接蛋白-1（ZO-1）及血管内皮生长因子（VEGF）的表达变化。采用CCK8试剂盒与Transwell小室实验分别检测IEC-6细胞的增殖与迁移能力。采用免疫荧光法（immunofluorescence）检测细胞中E-钙粘蛋白（E-cadherin）与波形蛋白（Vimentin）的表达水平。
**研究结果**：坏死性小肠结肠炎（NEC）肠狭窄患者血清中的C反应蛋白（CRP）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）及干扰素-γ（IFN-γ）水平均显著高于参考值。NEC肠狭窄组织中SIRT1蛋白表达下调，而转化生长因子-β1（TGF-β1）蛋白表达上调，且SIRT1的表达与TGF-β1的表达呈负相关。在确诊NEC时，合并呼吸窘迫综合征的患儿SIRT1表达水平降低，且CRP水平升高。抑制IEC-6细胞中SIRT1的表达后，TGF-β1、Smad3及NF-κB的表达水平均下调，ZO-1的表达水平亦出现下降。IEC-6细胞的增殖与迁移能力显著降低，而细胞中E-钙粘蛋白与波形蛋白的表达水平无明显变化。
**研究结论**：炎症介导的肠道纤维化进程可能是坏死性小肠结肠炎后狭窄的发生机制。SIRT1或可作为NEC的保护性蛋白，其潜在作用机制为通过调控肠道纤维化进程、保护肠黏膜屏障功能，参与坏死性小肠结肠炎后狭窄的发生发展过程。