硌利普兰对脓毒性休克NF-kappaB信号通路负调节机制的研究

Sepsis is a systemic inflammatory response syndrome caused by bacterial infections,when further developing it can lead to septic shock, multiple organ failure(MOF),which are the most important cause of death of the clinical critically ill patients. Althrough the antibiotics have been widely used in the clinical,but with the emergence of antibiotic-resistant bacterial strains, its clinical effect for sepsis has not been significantly improved. Consepuently,development of novel therapeutic strategies is urgently needed for the treatment of septic shock and multiple organ failure. Our previous studies found that the mitogen-activated protein kinase phosphatase-1 (MKP-1) can regulate the production of inflammatory mediators and reduce the mortality of septic shock animals through the negative regulator of NF-kappaB activation. Our results also indicated that Rolipram, which has already been used for chronic obstructive pulmonary disease (COPD), inhibits the activation of NF-kappaB, reduces inflammation response and lung tissue damage by enhancing the expression of MKP-1. Therefore, Rolipram may act as an anti-sepsis agent for septic shock. Based on these interesting and important findings, the primary objectives of this study are targeted toward completing the preclinical development of Rolipram as a novel therapeutic agent in reducing mortality of septic shock and multiple organ failure, as well as determining the mechanism responsible for its beneficial effect both in vitro and in vivo by using gene knockout, RNA interference, gene transfection, liquid chips and other technology. These proposed studies should provide useful mechanisms and preclinical information that will allow us to initiate clinical studies for Rolipram in victims with septic shock and multiple organ failure.

脓毒症指由感染引起的全身炎症反应综合征，进一步发展可导致脓毒性休克、多器官功能衰竭，是临床危重病患者最主要的死亡原因。尽管抗菌素在临床上广泛使用，但随着细菌抗药性的产生，临床治疗脓毒症的效果至今未见明显改善，迫切需要发展新的治疗脓毒症的思维和方法。我们前期的研究发现丝裂原活化蛋白激酶磷酸酶 1（MKP-1）可以通过负性调节NF-kappaB的活化，控制炎症介质的产生，降低脓毒性休克动物的死亡率；抗炎药物硌利普兰（Rolipram）可通过增强MKP-1的表达抑制NF-kappaB的活化，减轻炎症反应和肺组织损伤。本项目拟在上述发现的基础上，分别从分子、细胞和整体水平，利用基因敲除、RNA干扰、基因转染、液相芯片等技术，进一步探讨MKP-1在脓毒症中的作用机理，深入研究新型抗炎药物硌利普兰治疗脓毒症的效果及其机理，为脓毒症和脓毒休克的临床治疗寻求新的治疗药物和策略。

脓毒症（Sepsis）是由感染引起的全身炎症反应综合征，可导致多器官功能障碍甚至死亡。脓毒症是当前重症监护病房（ICU）面临的棘手难题，一方面因为脓毒症由不同的微生物引起，导致相应的疫苗难以开发；另一方面，由于抗生素的滥用，耐药菌株不断产生，应用抗生素控制感染越发困难。因此，尽管对于脓毒症的发病机制的研究取得了很大的进展，目前对于脓毒症的治疗，仍然没有特效的药物。.本研究构建了一个含MKP-1的启动子驱动的荧光素酶报告基因的RAW264.7细胞稳定表达细胞系，发现Rolipram协同LPS增强MKP-1启动子活性，同时能抑制LPS引起的RAW264.7细胞释放炎症细胞因子。在动物水平，不管是预防性给药还是治疗性给药，Rolipram都能使LPS引起的脓毒症小鼠的存活率从36.4%提高到100%。HE染色显示Rolipram能减轻小鼠肺组织的损伤。肺组织Western-blot结果显示Rolipram能抑制LPS引起的NF-κB p65磷酸化以及 ERK, JNK和p38 MAPK的磷酸化，肺组织免疫荧光结果显示Rolipram 能抑制NF-κB p65入核。.本项目研究结果提示，上调MKP-1可能成为炎症性疾病如脓毒症和脓毒性休克的治疗策略。Rolipram已被美国FDA批准为治疗COPD的临床药物，我们发现其对脓毒症和脓毒症休克小鼠具有保护作用，这对于拓展Rolipram的临床新用途具有重要的实践意义。