基于白三烯C4代谢途径研究肝脏IR损伤致LTC4异常增加及IP调控的分子机制

Hepatic ischemia-reperfusion (IR) injury is an importantclinical issue and its mechanisms remain to be further clarified. Increasing literatures indicate that hepatic IR injury is related to leukotriene (LT). That the LTC4 synthase (LTC4S) catalyzes LTA4 and GSH to generate LTC4 is the first committed step in the synthesis of cysteinyl LTs, LTC4, LTD4 and LTE4. Our previous findings suggest that abnormal LTC4 accumulation during hepatic IR injury can be partially caused by LTC4S over-expressions and its activities augment. Ischemic preconditioning(IP), which defined as a brief period of liver ischemia followed by reperfusion,has been demonstrated to protect against a following prolonged IR injury. However,some contradictory reports also presented and the mechanisms underlie IP protecting against hepatic IR not fully clear. Our recent document demonstrated that the decline in LTC4 production following hepatic IR could be partially resulted from IP induced suppression of the expressions and activities of LTC4S. It is well known that endogenous NO acted as an important role in the mechanisms of IP protecting against IR injury. But we don’t know whether IP can regulate LTC4 products via an endogeneous NO activation pathway. LTC4 are arachidonic acid-derived preinflammatory factors which are mainly metabolized in liver. LTC4 is predominantly influenced by its synthesis, uptake and excretion in the liver.However, there is no report involved the relationships among the abnormal accumulated LTC4 and its uptake and excretion, as well as IP action on them in IR rat liver. Here using the models of hepatic IR injury in vivo and liver slice hypoxia-reoxygenation(HR) in vitro , we will further study the following items: 1.How about the relationships between the abnormal accumulated LTC4 and the changes in the expressions,distributions and activite of LTC4S in the late phase of hepatic IR injury in rats? What’s the effects of IP on them？2. How about the relationships among LTC4 accumulation and the alterations in the expressions,distributions and activites of organic anion transporting polypeptides(Oatp)1,Oatp2 and Oatp4 that responsible for hepatic LTC4 uptake? What’s the effects of IP on them？3. How about the relationships among the abnormal increased LTC4 and the changes in the expressions, distributions and activites of multidrug resistance protein(Mrp)2、Mrp3 and Mrp4 which responsible for hepatic LTC4 excretion? What’s the effects of IP on them? All these topics if completed will further elucidate the mechanisms underlying the relations between LTC4 accumulation, IP reducing LTC4 products and the balance of hepatic LTC4 synthesis, uptake and excretion during hepatic IR injury in rats. We also explore whether IP can regulate LTC4 products via an endogeneous NO activation pathway using NO antagonist.These findings will provide a new experimental and theoretic evidences for the treatment of liver transplantation and liver resectional surgery.

白三烯C4（LTC4）是主要经肝脏代谢的前炎症因子。肝脏LTC4含量受其合成、摄取和分泌排泄的代谢平衡调节。我们前期报道肝缺血再灌注损伤(IR)早期LTC4堆积与LTC4合酶（LTC4S）表达上调和酶活性增强致LTC4合成增加有关；最近我们发现缺血预处理（IP）通过降低LTC4S表达和酶活性抑制LTC4生成，减轻大鼠肝IR损伤。本课题拟用肝IR损伤和肝切片缺氧/复氧模型，研究LTC4堆积及其IP调控作用1.与LTC4S表达、分布定位和酶活性改变的相关性；2.与肝有机阴离子转运肽Oatp1、Oatp2和Oatp4摄取LTC4功能改变的相关性；3.与肝多药耐药相关蛋白Mrp2、Mrp3和Mrp4分泌排泄LTC4功能变化相关性。从在体和离体水平围绕LTC4合成、摄取和分泌排泄的代谢平衡调节，阐释肝IR损伤早、晚期LTC4异常增加及其IP调控机制。为临床治疗肝移植和肝肿瘤切除等相关疾病提供新依据。

肝脏缺血再灌注（IR）损伤是一个重大临床问题，在创伤、内毒素休克、重建血管外科、肝移植和肝肿瘤切除外科手术中均存在IR损伤，是导致肝叶切除、肝移植术后器官功能障碍和衰竭的重要因素。白三烯C4（LTC4）是主要经肝脏代谢的前炎症因子。我们前期报道IR早期LTC4堆积与LTC4合酶（LTC4S）表达上调和酶活性增强致LTC4合成增加有关。有文献报道认为肝脏中主要以LTC4为底物的有机阴离子转运肽（Oatp）1、Oatp2和Oatp4以及多药耐药相关蛋白（Mrp）2、Mrp3和Mrp4分别摄取和排泄LTC4。缺血预处理（IP）是指肝脏短暂IR能对随后较长时间IR损伤产生保护作用并提高其再生能力。IP被证明在肝切除术中通过改变一氧化氮（NO）的生成保护肝实质IR损伤，但IP是否经刺激内源性NO调控LTC4失衡尚不清楚。本项目采用在体70%肝脏IR损伤模型，应用NOS抑制剂，进一步探索肝脏IR损伤期间LTC4合成、摄取和分泌排泄的平衡调节改变导致LTC4异常增加以及IP调控LTC4代谢失衡的机制。结果发现：①IP在肝脏IR早期可能通过NO激活途径下调LTC4S基因和蛋白表达，并抑制LTC4S活性,减少LTC4异常堆积，进而保护肝脏免于IR损伤。②肝脏I/R期间LTC4含量异常增加可能与肝Mrp2,Mrp3和Mrp4表达下调导致其排泄障碍有关。③IP减少LTC4储留可能涉及其上调Mrp2和Mrp4表达促进LTC4排泄。L-NAME仅逆转了Mrp4的作用，对Mrp3和Mrp2无明显影响，表明IP可能经NO信号通路调节Mrp4的作用。④肝脏I/R期间LTC4含量异常增加可能与肝Oatp1,Oatp2和Oatp4表达显著上调增加LTC4摄取有关。⑤IP减少LTC4储留可能主要涉及其下调肝Oatp1和Oatp4表达减少LTC4摄取。L-NAME可明显逆转Oatp1和Oatp4的作用，对Oatp2无明显影响，提示IP经NO信号通路的调控Oatp1和Oatp4摄取LTC4。⑥IP抑制LTC4产物增加可能也与其改善肝功能损伤、膜脂质过氧化、氧化应激和组织结构损伤从而增加LTC4代谢产物排泄有关，该作用可能受IP对NO信号通路的调控。这些研究成果进一步阐释了肝IR期间LTC4异常增加及IP调控的分子机制，为临床治疗肝移植和肝肿瘤切除等重大疾病提供了新的实验理论依据，以及药物干预新策略和手段。