肠道碱性磷酸酶对肠道通透性的作用及其机制研究

Intestinal alkaline phosphatase (IAP) can detoxify lipopolysaccharide (LPS) and maintain gut barrier function. In our previous study, IAP was found to be able to attenuate LPS-induced permeability increasing of intestinal epithelial cells by regulating the expression and function of tight junction protein (TJP). We also found that IAP overexpressing and knockout lead to upregulation and downregulation of TJP expression respectively, indicating that IAP regulates TJP through mechanism independent of detoxifying LPS，which need to be elucidated. As an important pathway of regulating TJP, MLC phosphorylation caused by NFκB-enhanced MLCK activity is the key step for LPS or TNF-α induced intestinal hyper-permeability. Based on the knowledge that IAP can inhibit the activity of NFκB, we hypothesize that IAP regulate TJP and intestinal permeability through inhibiting NFκB and MLCK pathway. In this study, we will first prove that there is alternative mechanism other than detoxifying LPS involved in regulating intestinal permeability by IAP, then investigate the role of NFκB and MLCK pathway in the mechanism of IAP regulating intestinal permeability in physiological condition and LPS stressing, followed by evaluating the effect of IAP on intestinal hyper-permeability induced by TNF-α to further confirm that IAP works through NFκB and MLCK pathway. This study will provide valuable information for further investigating intestinal permeability related disease by identifying the effect and mechanism of IAP on intestinal permeability.

肠粘膜屏障受损与多种疾病相关，肠道碱性磷酸酶（IAP）具有降低内毒素（LPS）毒性和维护肠粘膜屏障的作用。我们前期研究发现IAP能抑制LPS导致的肠上皮细胞通透性增加和TNF-α的产生并上调紧密连接蛋白（TJP），还发现高表达和敲除IAP都能够影响TJP的表达，提示IAP调节TJP不依赖于其对LPS的直接作用，但机制有待探讨。NFκB增强肌球蛋白轻链激酶（MLCK）表达使MLC磷酸化是LPS和TNF-α导致肠道通透性增加的重要环节。IAP能抑制NFκB活性，我们推测IAP可能通过抑制NFκB和MLCK影响肠道通透性。本研究包括：明确IAP调节肠道通透性不依赖于对LPS的直接作用；探讨NFκB和MLCK通路在IAP影响肠道通透性机制中的作用；观察IAP对TNF-α所致肠道通透性增加的影响，验证其作用机制。本研究将初步明确IAP对肠道通透性的作用和机制，为相关疾病的研究提供重要参考。

背景:已有报道称肥胖患者存在肠道通透性受损。Roux-en-Y胃分流术(RYGB)已被证明是治疗严重肥胖最有效的方法，但其分子作用机制尚不完全清楚。RYGB手术将消化道重新排列，从根本上改变了食物消化吸收的过程。然而，RYGB对肠道通透性的影响尚未清楚。.方法:8周龄的普通C57BL/6J雄鼠高脂饮食12周，然后进行RYGB手术和假手术。术后8周检测血清脂多糖(LPS)和炎症因子。采用Ussing-chamber检测肠道通透性。qPCR检测toll样受体(TLR)、肠道炎症因子、紧密连接蛋白(TJP)、抗菌肽、肠道碱性磷酸酶(IAP)和胰高血糖素原的表达情况。.结果:与假手术相比，RYGB显著减轻了体重，改善了糖耐量和胰岛素抵抗，而且RYGB手术降低了血清中脂多糖、白细胞介素6和TNFα的水平。RYGB组小鼠肠道的共同肢和结肠支的通透性较假手术组明显改善。与假手术组相比，RYGB组小鼠小肠中白细胞介素1β、白细胞介素 6和Toll样受体4的信使RNA表达水平明显降低。RYGB组小鼠的抗菌肽（REG3β和REG3γ）和肠道碱性磷酸酶的表达均高于假手术组。.结论:在饮食诱导的肥胖小鼠模型中，RYGB通过增加肠道碱性磷酸酶和抗菌肽，抑制肠道先天性免疫和降低炎症反应，从而改善肠道通透性导致减轻全身炎症，最终达到改善机体肥胖和代谢的目的。