STIM1/Orai1-SOCE通路调控急性胰腺炎肺微血管内皮细胞损伤机制及清胰汤干预作用

Acute pancreatitis associated lung injury (APALI) is the most common serious complication of early stage for severe acute pancreatitis.Pulmonary microvascular endothelial cells (PMVEC) injury of APALI is one of the key factors which mechanism is unclear.The traditional Chinese medicine Qingyi decoction is effective in the clinical treatment of APALI, its mechanism is a hot spot in current research.Our previous research found that the intracellular calcium overload participated in the PMVEC apoptosis regulation, preliminary experiment found that store-operated calcium entry (SOCE) associated proteins STIM1 and Orai1 was related to the changes of calcium ion concentration,and was closely related to the cell damage.According to that, we get the hypothesis that STIM1 / Orai1 - SOCE pathway may participate the regulation of PMVEC apoptosis in the pathogenesis of APALI. This research project proposed by rat model and invitro cell experiment, application of confocal microscope,flow cytometry, gene transfection, liquid gene chip and immunocytochemical method, to detect the effect of SOCE pathway proteins STIM1 and Orai1 on intracellular calcium ion concentration and cell damage, analysis the change of nuclear transcription factor activity. Through these studies, trying to clarify the role of STIM1 / Orai1-SOCE pathway in the pathogenesis of the damage of lung microvascular endothelial cells in APALI and treatment mechanism of Qingyi decoction, and to establish the new theoretical basis for further popularization use of Qingyi decoction in ALI.

急性胰腺炎相关性肺损伤（APALI）是重症急性胰腺炎早期最常见的严重并发症，肺微血管内皮细胞（PMVEC）损伤是APALI发病的关键因素之一，但其机制尚不明确。通里攻下中药清胰汤是临床治疗APALI的有效方剂，其作用机制是当前研究热点。申请人前期研究发现细胞内钙超载参与PMVEC凋亡调节，预实验发现钙库操控钙内流（SOCE）相关蛋白STIM1和Orai1同细胞内钙离子浓度变化相关。据此提出STIM1/Orai1—SOCE通路参与PMVEC凋亡调控的科研假说。本课题拟通过大鼠模型和体外细胞学实验，应用共聚焦显微镜、流式细胞术、基因转染、液相基因芯片、免疫细胞化学等方法，检测双向调节SOCE通路蛋白对细胞内钙离子浓度与细胞损伤的影响，检测分析细胞核转录因子活性变化。以阐明STIM1/Orai1—SOCE通路调控PMVEC损伤机制及清胰汤干预机制，为探索APALI发病机制和治疗措施提供新的思路。

目的：探讨SOCE相关蛋白在重症急性胰腺炎肺损伤发病机制中的作用及清胰汤、清胰颗粒干预的可能机制，ENO2表达水平与SAP-ALI肺组织损伤严重程度的相关性，sST2 对SAP大鼠心肌损伤的影响及机制，为临床治疗和预防重症急性胰腺炎肺损伤找到新的靶点。.方法：制备大鼠胰腺炎模型，测定肺组织中 Orai1、STIM1 mRNA、蛋白水平。检测肺微血管上皮细胞Orai1、STIM1蛋白的表达；检测微血管上皮细胞凋亡的情况；观察内皮细胞超微结构变化；检测肺组织中Bax、caspase3 mRNA 的水平。分离外周血PMN，流式细胞术检测外周血PMN凋亡率；免疫组化检测肺组织PBEF的表达水平，检测PMN凋亡率，检测BALF和血清中ENO1、ENO2的表达变化；检测肺组织中ENO1、ENO2 mRNA 和蛋白表达变化。检测肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白T（cTnT）、乳酸脱氢酶（LDH）的表达及Na+-K+-ATPase 活性，检测血清中细胞因子和sST2 以及IL-33 的表达；检测心肌细胞凋亡情况， 检测心肌细胞Bax、cleaved caspase-3、Bcl-2 蛋白表达情况。设计、合成针对大鼠STIM1、Orail基因的小干扰RNA序列和扩增质粒，转染大鼠肺微血管内皮细胞及清胰颗粒干预治疗。检测细胞凋亡比率；检测细胞STIM1、 Orai1蛋白水平及STIM1、 Orai1、NFATc3、Bax和caspase3 mRNA的水平。培养细胞系H9C2 细胞，LPS（10μg/mL）构建心肌细胞损伤模型流式细胞术检测心肌细胞凋亡情况，检测心肌细胞Bcl-2、Bax、cleaved-caspase3 以及ST2 表达。.结论：SOCE 通路对急性胰腺炎肺微血管内皮细胞损伤的调控作用。中药清胰汤及中药制剂清胰颗粒可能通过SOCE通路参与PMVECs凋亡途径的调控，进而调节了SAP-ALI的发病过程。清胰汤可能通过下调PBEF表达，进而促进PMN凋亡，对SAP-ALI起到保护作用。清胰汤有效成分大黄素可能通过影响PMN细胞凋亡的死亡受体途径和线粒体途径中相关蛋白的表达促进细胞凋亡。ENO2可能成为预测SAP-ALI大鼠肺组织损伤严重程度的新靶点。sST2可通过竞争性抑制膜受体的IL-33/ST2 信号通路，进而抑制心肌细胞凋亡途径的激活，缓解胰腺炎所致心肌损伤。

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