PARP 在重症急性胰腺炎肾上腺损伤中对PI3K/Akt信号通路调控的作用及机制研究

Severe acute pancreatitis (SAP) complicated with adrenal insufficiency often cause rapid deterioration and bad prognosis, however, its pathogenic mechanism has not been exactly elucidated until now. Our preliminary work has demonstrated that Poly (ADP-ribose) polymerase-Nuclear factor-κB (PARP-NF-κB) inflammatory pathway may play an important role in the pathological process of adrenal injury associated with SAP. PARP blockade down-regulated NF-κB activity and its downstream inflammatory gene expression, attenuated adrenal pathological changes, and relieved the SAP condition. It was recently found that PARP is an important negative regulator of PI3K/Akt pathway. Our study showed that over-activation of PARP and reduced activation of Akt in adrenal injury after SAP model. The role of PI3K/Akt in PARP-mediated suicidal pathway is unclear. Based on previous work, the present project intends to establish SAP associated adrenal injury model, research on rat adrenocortical cells in vivo and in vitro, try to use PARP inhibitor, PI3K inhibitor, PI3K activator, and gene transfection technology to explore the regulation and mechanism of PARP towards PI3K/Akt, in order to explain that promoting the PI3K/Akt activation can activate its downstream signaling pathways, then regulate a large number of vital pathways (cellular inflammatory responses, apoptosis, energy metabolism), which can protect the structure and function of adrenocortical cells. The project aims to provide molecular mechanisms and potential strategies for prevention and treatment of SAP and associated adrenal injury.

重症急性胰腺炎(SAP)并发肾上腺功能不全导致患者病情恶化，预后凶险，具体机制尚不明确。本组前期研究证实PARP-NF-κB炎症通路参与相关肾上腺损伤病理过程，抑制PARP下调NF-κB活性及下游炎症基因表达，减轻肾上腺损伤，缓解SAP病情。新近发现PARP是PI3K/Akt信号通路的重要负向调控分子。我们研究显示SAP时肾上腺PARP过激活，Akt活化减少，但PI3K/Akt通路在肾上腺损伤时PARP引起细胞死亡中的作用尚不清楚。本项目拟在前期工作基础上，建立SAP肾上腺损伤模型，以体内及离体培养的肾上腺皮质细胞为研究对象，利用PARP抑制剂、PI3K抑制剂和激活剂、基因转染技术研究PARP对PI3K/Akt的调控作用及机制，阐释促进PI3K/Akt活化可激活下游信号通路，作用于细胞炎症反应、凋亡和能量代谢，保护肾上腺结构和功能，为临床防治SAP合并肾上腺损伤提供分子机制和潜在治疗方案。

重症急性胰腺炎（SAP）病情凶险，常发生多器官功能障碍。研究显示SAP病情恶化与肾上腺损伤相关，其具体机制尚未明确。聚腺苷二磷酸核糖聚合酶（PARP）与脓毒症、胰腺炎等并发多器官功能障碍的病理过程密切相关，在本团队既往的研究中，我们发现，阻断PARP活性，能够改善SAP大鼠肾上腺的病理和功能损伤，本项目中我们继续深入探索阻断PARP对SAP大鼠肾上腺损伤的具体机制。本课题组通过建立牛黄胆酸钠诱导的SAP大鼠模型，采用PARP抑制剂PJ34进行干预，利用血清酶学、免疫组化、免疫荧光、TUNEL、ELISA、western blot等实验技术，观察并评价了SAP胰腺炎的严重指标，肾上腺组织功能与结构变化，超微病理改变，PI3K/Akt信号通路相关蛋白的表达水平，了解了PARP活化对PI3K/Akt信号通路蛋白及下游因子GSK3β、Caspase3、Caspase9 等的表达变化，证实PARP抑制剂PJ34有助于减轻SAP病情，并能减轻大鼠肾上腺结构和功能损伤，进一步活化PI3K/Akt通路，减少肾上腺组织内炎症反应和细胞凋亡水平。通过双氧水刺激肾上腺皮质癌细胞（NCI-H295R）模拟SAP时肾上腺的损伤，并应用PARP抑制剂PJ34和PI3K抑制剂LY294002单独和联合干预，进一步证实，PJ34能够通过抑制PARP，进一步活化PI3K/Akt通路，改善双氧水诱导的H295R细胞的炎症反应和细胞凋亡水平，为SAP合并肾上腺损伤的发病机制及防治提供理论基础和实验依据。