HIF-1α调控miR-105加重急性胰腺炎病情的机制研究

The increased expression of hypoxia inducible factor-1α(HIF-1α) is a hallmark of the transition from acute pancreatitis(AP) to severe acute pancreatitis(SAP). However, the mechanism of this phenomenon has not been fully elucidated. Our preliminary study found that patients who had history of alcohol abuse tended to have SAP, thus, we fed rats with ethanol to simulate the condition before we established the AP model. We found that the expression of HIF-1α, the autophagy activity and the pathological score increased comparing with the control rats. Moreover, we found that simply stimulating or inhibiting autophagy activity can not change the severity of AP. Then we found out miR-105 was both abnormally expressed under hypoxia and acted ULK1 as its target by the miRNA chip and Targetscan database analysis. Based on the above, our project aims to discover the effects of autophagy and miR-105 on the severity of AP. Meanwhile, we detect the exact relationship among HIF-1α, miR-105 and ULK1. Finally, we want to find out the important cellular factor that play indispensable role in the metabolism and death pathway of acinar cells, which exerts its effects with ULK1. In conclusion, we desire to find out the molecular mechanism based on “HIF-1α-miR-105-ULK1” modulating axis in order to provide a warning molecular and potential therapeutic targets of the transition from AP to SAP.

缺氧诱导因子（HIF）-1α表达升高是急性胰腺炎（AP）病情进展为重症急性胰腺炎（SAP）的重要标志，但其具体调控机制尚不明确。我们研究发现长期饮酒患者易发生SAP。利用大鼠模拟饮酒后造模发现，与对照组大鼠相比，饮酒大鼠胰腺组织HIF-1α表达升高，自噬活性增强，病理学评分升高；并观察到单独刺激或抑制自噬活性并不能改变AP病情。利用miRNA芯片与Targetscan数据库分析后，我们找到既在低氧状态表达异常，又以ULK1为靶基因的miR-105。在此基础上，本课题拟研究自噬及miR-105对AP病情的影响，同时检测HIF-1α，miR-105与ULK1三者之间的关系，并寻找与ULK1相结合并影响腺泡细胞代谢及死亡方式的重要细胞因子。明确AP基于HIF-1α-miR-105-ULK1调控轴的分子机制，为AP病理生理过程的转化预警提供标志物和干预治疗靶点。

目的：探讨急性胰腺炎发病早期缺氧状态下miRNA-195通过对LAMP2的调节加重缺陷型自噬进而加重疾病病情的机制。.方法：Wistar大鼠分为四组：对照组，低氧组，胰腺炎组，低氧联合胰腺炎组，造模6h后取材进行病理分析，电镜检测，Western blot和Realtime PCR检测，分析HIF-1α，miRNA-195，LAMP2的表达相关性，及其相关性与病理分析及电镜下缺陷型自噬之间的关系，确定急性胰腺炎发病早期低氧调节缺陷型自噬的大体机制。利用AR42J细胞系进行体外实验，观察缺氧状态和雨蛙素刺激状态下HIF-1α和miRNA-195的表达。通过对miRNA-195的过表达和沉默调控观察下游LAMP2的表达情况，利用荧光素酶报告基因系统研究miRNA-195与LAMP2的直接作用关系。同时进行调控轴的回复实验。.结果：①体内实验发现除对照组外其余三组HIF-1α均表达升高，但组间存在差异，miRNA-195与HIF-1α表达成正相关，LAMP2表达与HIF-1α，miRNA-195表达负相关；胰腺的病理评分与炎症因子水平与HIF-1α表达成正相关；②体外实验发现缺氧及雨蛙素刺激并不能影响细胞活性，但细胞内缺陷性自噬蛋白表达存在差异。缺氧状态下AR42J细胞HIF-1α，miRNA-195，LAMP2表达与体内实验相同；雨蛙素刺激情况下细胞HIF-1α，miRNA-195，LAMP2表达并没有显著差异；③ 过表达或抑制miRNA-195后并没有影响细胞活性，但是影响了LAMP2及其他缺陷性自噬相关蛋白的表达。进一步通过荧光素酶报告基因系统发现miRNA-195直接调控LAMP2表达；④回复实验发现，转染miRNA-195 inhibitor细胞系在缺氧状态下培养，虽然HIF-1α表达升高但LAMP2表达未见降低。此外，对转染了miRNA-195 mimics的细胞系进行LAMP2的过表达操作后，发现LAMP2表达升高，缺陷性自噬缓解。.结论：急性胰腺炎发病初期存在局部缺氧状态，HIF-1α调控下游miRNA-195表达，且miRNA-195直接调控下游分子LAMP2表达，加重缺陷性自噬的发生，加重急性胰腺炎病情。对此调控轴的研究为早期干预急性胰腺炎，控制病情向重症急性胰腺炎的转化提供了新的治疗靶点与思路。