AngⅡ/AT1R-RIPK1/3-MLKL通路调控肠上皮细胞程序性坏死参与NEC发生的机制研究

Necrotizing enterocolitis (NEC) refers to a spectrum of acute necrotic inflammatory disease with unclear mechanisms, which could be induced by multiple perinatal factors. Our preliminary research found that the expression of AT1R, a component of the renin-angiotensin system (RAS) and RIPK1, a necroptotic protein, elevated in NEC patients and murine models. Activation of RAS would aggravate bowel inflammation, possibly through another form of intestinal epithelial cell death-necroptosis. Based on the above, we hypothesized that the multiple factors that induced NEC (prematurity, mesenteric vascular obstruction, inflammation, hypoxia, ischemia, etc.) could activate RAS and subsequently stimulate intestinal necroptosis, contributing to the cascade of intestinal damage in NEC. RAS blockers and necroptosis inhibitors will be given to neonatal NEC rats to demonstrate the relationship between Ang Ⅱ/AT1R and RIPK1/3-MLKL pathway. In vitro studies using AT1R and RIPK1 up- and down-regulated intestinal cells will be performed to confirm the findings. Once confirmed, our conclusions would add new knowledge to the mechanism of NEC and facilitate early detection and timely treatment.

新生儿坏死性小肠结肠炎（NEC）是由围产期多种致病因素导致的急性坏死性肠道疾病，确切发病机制目前尚不清楚。申请者通过前期研究发现：①NEC患儿及新生鼠肠粘膜RAS相关蛋白AT1R和程序性坏死蛋白RIPK1表达增加；②RAS活化导致NEC小鼠肠炎损伤加重与其诱导肠上皮细胞死亡的另一模式——“程序性坏死”有关。因此，申请者提出：NEC多重因素（早产、肠系膜血流障碍、感染、缺氧等）引起RAS活化，诱导肠上皮细胞程序性坏死可能参与NEC的发病机制。本课题拟通过临床NEC患儿肠道组织标本、建立NEC新生鼠模型、靶向抑制RAS及程序性坏死通路、构建AT1R和RIPK1表达上调及下调的肠上皮细胞系，从人体组织、动物模型、分子及细胞水平共同阐明AngⅡ/AT1R通过调控RIPK1/3-MLKL通路导致肠上皮细胞程序性坏死可能是NEC的重要发病机制，为临床早期预测及治疗NEC提供新思路。

新生儿坏死性小肠结肠炎（NEC）是由围产期多重致病因素综合所致的急性严重肠道坏死性疾病。我们既往研究发现：临床上NEC相关的多重致病因素（早产、肠系膜血流障碍、感染、缺氧等）均可引起RAS活化，据此，我们发现NEC患儿及新生鼠肠粘膜RAS相关蛋白和程序性坏死蛋白表达增加；RAS活化可导致NEC新生鼠肠炎加重与其诱导肠上皮细胞程序性坏死有关。在此基础上，我们进一步深入本课题研究，发现：①NEC患儿及新生鼠肠上皮细胞程序性坏死显著增加，给予程序性坏死小分子抑制剂Nec-1s干预能够抑制肠上皮细胞程序性坏死、减轻NEC肠炎；②NEC患儿及新生鼠RAS活化及TLR4表达显著增加，血管紧张素受体AT1R抑制剂Losartan能够抑制TLR4表达，减轻肠上皮细胞程序性坏死，改善NEC肠炎；③我们进一步构建TLR4敲除的NEC新生鼠模型，发现TLR4敲除抑制肠上皮细胞程序性坏死，NEC肠炎显著减轻；④通过构建siRNA TRIF和siRNA TNFR1的HCT-116细胞系，证实TLR4通过TRIF和TNFR1通路调控程序性坏死途径。综上，我们得出：NEC多重致病因素相互促进，共同激活RAS通路，继而引起TLR4活化，促进肠上皮细胞程序性坏死，最终导致NEC的发生。为今后临床上NEC 的早期诊断及治疗提供新的靶点。