酸性神经酰胺酶对组织蛋白酶B→胰酶激活以及NFκB→炎症反应的正向调控影响急性胰腺炎发生发展的作用研究

Trypsinogen premature and NFκB activation which occurred parallely in the pancreatic acinar cells affect the initiation and the development of acute pancreatitis independently. Trypsinogen activated by cathepsin B led to acinar cells injury even cell death while inflammatory cytokines secretion from acinar cells caused by NFκB induced system inflammation finally. Blockage on both of these two pathways could be considered as an efficient treatment for acute pancreatitis. It was found that acid ceramidase could activate cathepsin B. Meanwhile the amount of metabolites of the former like ceramide and sphingosine increased in the serum from acute pancreatitis patients. In our preliminary experiment, the protein level of acid ceramidase was up-regulated in acinar cells under acute pancreatitis induced reagent treatment. Besides cells or mice with inhibitor of acid ceramidase showed less mount of acitve digestive enzyme containing trypsin and TNFα secretion as well as and minor inflammatory cells infiltration in pancreatic tissue. It could be inferred that acid ceramidase induced acute pancreatitis through activating trypsinogen as well as NFκB. In order to prove this hypothesis, pancreatic acinar cells and mice were applied to investigate the effect of acid ceramidase on the cathepsin B/trypsin and NFκB/inflammatory pathway respectively. The aim of this project is to reveal the mechanism underlying acute pancreatitis induced by acid ceramidase. The result will provide new insight into the mechanism underlying acute pancreatitis and will be benefit for new therapy investigation for the illness.

胰腺腺泡细胞中胰酶的活化及NFκB的激活，均可独立发生并促进急性胰腺炎的发生及发展。组织蛋白酶B激活胰酶原导致腺泡细胞损伤甚至死亡，NFκB的激活则促进腺泡细胞分泌相关炎症因子最终引发系统炎症,抑制两条通路能明显抑制急性胰腺炎的发生。酸性神经酰胺酶（AC）可激活组织蛋白酶B，其代谢产物神经鞘氨醇及1-磷酸神经鞘氨醇在急性胰腺炎早期病人的血清中升高，提示AC与急性胰腺炎有关。我们前期发现急性胰腺炎AC的蛋白表达升高，抑制AC可降低胰酶等消化酶的活性、降低TNFα的分泌及胰腺组织炎症细胞的浸润。据此我们假设：AC通过正向调控组织蛋白酶B-胰酶激活通路，以及影响NFκB-炎症通路在急性胰腺炎的发生、发展中起重要作用。本项目拟从细胞水平、以及通过小鼠急性胰腺炎模型研究AC对上述两条通路的影响以及在急性胰腺炎中的作用。本研究的开展，有助于揭示急性胰腺炎的发病机制及发现新的治疗靶点。

急性胰腺炎是一种发生在胰腺的炎症，急性胰腺炎重症可致死，但该疾病无特异性治疗方法存在。胰酶原的提前激活可以引发急性胰腺炎，而不依赖于胰酶原通路，NFkB通路的激活也可引发急性胰腺炎。然而，目前为止，急性胰腺炎的发生机制仍然不完全清楚。在本研究中，我们构建了急性胰腺炎的原代细胞模型及动物模型，在体内外研究神经鞘氨醇生成酶ASAH1在急性胰腺炎中作用。同时，ASAH1的调节蛋白及神经鞘氨醇的靶向蛋白经免疫共沉淀联合质谱的方法进行分析。结果发现，急性胰腺炎发生期间，ASAH1蛋白表达水平增加。ASAH1蛋白的高表达激活胰酶、组织蛋白酶b及NFkB，促进细胞因子的分泌。相反，ASAH1低表达抑制胰酶、组织蛋白酶b及NFkB的激活，抑制细胞因子的分泌。同时，ASAH1通过神经鞘氨醇调节胰酶、组织蛋白酶b、NFkB及细胞因子。除此之外，急性胰腺炎条件下，E3酶MIB1表达降低，从而使得ASAH1与MIB1相互作用减弱。外源性MIB1可以抑制外源性ASAH1对胰酶的激活作用。MIB1对ASAH1泛素化降解被抑制导致ASAH1表达水平的升高。急性胰腺炎条件下，神经鞘氨醇与丙酮酸激酶发生相互作用。丙酮酸激酶可以抑制神经鞘氨醇对胰酶的激活及线粒体过氧化物的产生。总之，急性胰腺炎发生过程中，MIB1下调导致ASAH1表达增加，神经鞘氨醇水平增加，进而胰酶及NFkB通路被激活，而神经鞘氨醇通过丙酮酸激酶调控胰酶激活。

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