瘦素在肥胖合并重症急性胰腺炎肠粘膜屏障中的作用机制及途径研究

As the initial change and critical process in the pathogenesis of severe acute pancreatitis (SAP), the increased permeability of intestinal mucosa characterized by reduced proliferation and increased apoptosis of intestinal epithelial cell (IEC), as well as the aberrant expression and distribution of tight junction proteins including occludin, claudin, ZO-1 have been observed. In our former study, we found that adipose tissue could further exacerbate the permeability of intestinal mucosa. Leptin as the brain-gut peptide was reported to play a key role in ameliorating pancreatic injury by down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), inhibiting apoptosis of IEC, promoting proliferation and regulating tight junction. MEK1-ERK1/2/ MAPK has been regarded as the essential pathway of leptin. However, the mechanism of leptin in regulation of intestinal mucosa permeability under the condition of obesity combined with SAP and whether the MEK1-ERK1/2/ MAPK signal path is the corresponding pathway are still uncertain. Our research intends to explore this mechanism and pathway through construction of the SAP models by using high-fat diet induced fat rats and leptin-deficient C57BL/6Job/ob mice, assessment of pathological injury in intestinal mucosa, D - lactic acid and endotoxin levels in portal vein, and application of Ussing chambers connected to a voltage-clamp apparatus, immunofluorescence, laser co-focus microtechnique, Western-blot and real-time fluorescent quantitative PCR et al to further explore the mechanism of leptin in intestinal mucosal barrier.

重症急性胰腺炎（SAP）进展中肠粘膜通透性（IP）增高存在肠上皮细胞（IEC）增殖减少、凋亡增加、细胞间紧密连接蛋白表达和分布异常等重要表现。我们前期发现：肥胖者SAP状态下IP进一步增高。已知脑肠肽瘦素通过下调促炎介质改善胰腺损伤；并抑制IEC凋亡、促进增殖、调节紧密连接蛋白表达分布，对肠粘膜屏障起关键保护作用。MEK1-ERK1/2/ MAPK途径是瘦素发挥作用的重要信号通路。然而在肥胖合并SAP状态下瘦素是否通过MEK1-ERK1/2/ MAPK途径改善IP的作用还亟待阐明。本课题拟利用高脂饮食致肥胖鼠和C57BL/6J瘦素基因缺陷的ob/ob种系先天性肥胖小鼠构建SAP模型结合细胞实验，采用测门静脉D-乳酸、内毒素水平、尤斯室灌流系统、免疫荧光、激光共聚焦显微技术、Western-blot、荧光定量PCR等技术，探讨瘦素在肥胖合并SAP肠粘膜屏障中的作用机制及相关途径。

摘要：.背景：. 重症急性胰腺炎（SAP）进程中出现肠粘膜屏障损伤，而肥胖可加重SAP程度，肥胖合并SAP肠粘膜屏障的变化尚待研究，瘦素可能参与其中作用。.方法：. 雄性SD大鼠予普通或高脂饲料喂养。采用胆胰管逆行注射5%牛磺胆酸钠建立急性胰腺炎模型。28只大鼠随机分配于普通饲料+假手术组（N），普通饲料+SAP组（NA），高脂饲料+假手术组（O）及高脂饲料+SAP组（OA）。检测动物血脂、血淀粉酶、肠道异硫氰酸荧光素-葡聚糖（FD4）吸收、门静脉内毒素水平，观察胰腺、回肠、结肠病理改变，检测血清、回肠、结肠TNF-α、IL-1β水平，观察回肠、结肠超微结构，采用免疫印迹及实时定量PCR检测肠道紧密连接蛋白occludin、claudin-1、瘦素及其受体的表达水平，变性梯度胶电泳（DGGE）基因测序分析肠道菌群改变。.结果：. 肥胖及SAP模型建立成功。SAP模型组血清FD4浓度及门静脉内毒素水平高于假手术组。SAP组结肠病理评分高于假手术组（P<0.05），回肠评分OA组高于O组（P<0.05）。各组间肠道超微结构观察上皮紧密连接无明显差异，同时occludin、claudin-1在SAP造模后无明显改变（P>0.05）。SAP建立后血清促炎因子（TNF-α、IL-1β）上升。在NA组肠道促炎因子水平较N组更高，然而O组与OA组肠道促炎因子水平无明显改变（除外回肠IL-1β，O>OA，P<0.05）。NA组回肠瘦素及其受体水平均N组升高，而OA组较O组下降。OA组结肠瘦素水平低于O组，但所有大鼠SAP造模后结肠瘦素受体无明显变化。肠道菌群分析提示NA组回肠细菌丰度高于N组，而OA组低于O组。.结论：. 肥胖可加重SAP进展及其引起的肠道通透性的增加。SAP可导致肠道局部促炎因子、回肠瘦素与其受体水平升高以及回肠菌群丰度的升高，然而肥胖可影响甚至逆转该变化，这可能与肥胖状态下回肠局部瘦素及其受体水平降低有关。