猪肝羧酸酯酶调控机体炎症机制研究

Pig liver carboxylesterases play an important role in pharmacology and most probably also put a powerful finger in the regulation of inflammation, but the underlying mechanism is waiting for illumination. Thus, this project plan to study on the correlation mechanism of PLE among PLE, cannabinoids and inflammation mediators in vivo and in vitro model. LC-MS/MS will be used to analyze the enzymatic dynamics of each major PLE isoenzyme in cannabinoids hydolysis. The cell model will be used to study PLE effects of hydrolyzed cannabinoids on inflammation with the detection of inflammation mediators by protein array and hydrolytic products by LC-MS/MS. BNPP which is the PLE specific inhibitor will be used to treat cell model and study the correlation among PLE activities, cannabinoids and inflammation mediators. The PLE over expression, PLE gene silence or gene knock out will be done in Hepli cell line, and the cell line will be used to test the contribution of PLE for the regulation of inflammation and the major PLE isoenzymes will be identified. In vivo study will be adopted to demonstrate the correlation of PLE level, cannabinoids level and inflammation level. BNPP will be use to inhibit PLE in LPS induced Large white swine inflammation model, the PLE level and activities, cannabinoids level, activation and translocation of nucleus of NF-κB and STAT1、infiltration of inflammatory cells,inflammatory mediators will all be detected. Finally, inflammation control mechanism of PLE will be uncovered and this will act as the theory basis for the control of pig inflammatory diseases and the research results will provide targets for the development of new anti-inflammatory drugs.

猪肝羧酸酯酶（PLE）除具有药理学作用外，很有可能参与机体炎症调控，但机制不清。本项目拟在体外和体内模型上解析PLE/炎症介质/大麻素关联性及机制。以LC-MS/MS解析PLE主要亚型水解大麻素的酶促动力学；在细胞模型上研究PLE水解后的大麻素抑炎活性，以蛋白质芯片等检测炎症介质水平、LC-MS/MS检测水解产物；用PLE抑制剂处理细胞模型，研究PLE酶活力、大麻素、炎症介质相关性；在猪肝细胞系上实现PLE过表达、沉黙、基因敲除，试验PLE各亚型水解大麻素、调控炎症的贡献、鉴定调控炎症介质的关键PLE亚型；最后在活体上研究PLE酶活力、大麻素、炎症介质相关性，用PLE特异抑制剂处理LPS诱导大白猪炎症模型，检测PLE水平和活力、大麻素水平、NF-κB和STAT1激活入核、炎症细胞浸润、炎症介质。最终阐明PLE调控机体炎症机制，为猪炎症性疾病的防控提供理论依据，为抗炎新药开发提供靶标。

申请人按计划高质量完成了项目设定的研究任务和研究目标。建立了检测大麻素2-AG、AEA及其水解产物的LC-MS/MS方法，检测了PLE水解大麻素的活力，发现PLE可水解2-AG和AEA生成促炎介质AA，PLE对2-AG水解活力高，且对2-AG水解活力远高于AEA；已知机体内2-AG含量远大于AEA，说明PLE主要通过水解2-AG而发挥促炎作用。建立了原代PAM和原代猪肝细胞的共培养炎症模型，研究了PLE/促炎因子/2-AG关联性，发现2-AG能抑制促炎因子TNF-α/IL-1β/IL-6表达，而PLE可水解2-AG大幅增加TNF-α/IL-1β/IL-6表达，且具有剂量依赖性，而PLE特异性抑制剂（BNPP）、在PAM细胞上进行的PLE干扰均可显著下调TNF-α/IL-1β/IL-6水平，证明了PLE可通过水解 抑炎介质2-AG发挥促炎作用。通过BNPP和经典大麻素降解酶抑制剂JZL184，抑制原代猪肝细胞裂解液等样品水解活性的比较研究，发现了PLE水解2-AG及其氧化产物PGF2α-G的贡献比经典大麻素降解酶的还要高、PLE是大麻素代谢的限速酶。LPS诱导的仔猪体内炎症模型上的研究结果表明：PLE特异性抑制剂BNPP能使机体组织中的AA和PGs水平明显降低，能明显减轻在体炎症反应和组织损伤，说明PLE是体内AA和PGs的主要贡献者，在体内炎症反应中发挥关键作用。另外，还通过PLE原核表达、过表达以及SiRNA干扰试验，鉴定出了猪体内降解大麻素2-AG、调控炎症反应的关键PLE亚型是PLE-B6和PLE-A1。本项目阐明了PLE调控机体炎症的机制，为猪炎症性疾病的防控提供了理论依据，为抗炎新药开发提供了新的靶标。. 在完成本项目预设的研究任务和目标后，申请人还进行了延伸研究，发现LPS、TNF-α/IL-1β等促炎因子，又可反过来抑制PLE的表达。进一步说明了PLE既是炎症的调控者又是被调控者，在炎症反应中发挥着不可忽视的作用。