甘珀酸抑制eATP-P2X7R-NLRP3信号通路减轻慢性胰腺炎纤维化的实验研究

摘要：目的　探讨细胞外ATP（eATP）-P2X7R-NLRP3信号通路在慢性胰腺炎（CP）胰腺纤维化中的作用以及ATP抑制剂甘珀酸（CBX）对CP的治疗作用。方法　将6周龄雄性C57BL/6小鼠随机均分为5组：正常组，模型组，CBX低、中、高剂量组（分别为25、50、100 mg/kg）。造模结束后，CBX各剂量组小鼠分别腹腔注射相应剂量的药物。末次给药24 h后将小鼠脱颈处死。HE染色评估胰腺组织病理学改变；ATP化学发光法测定小鼠胰腺组织eATP水平；免疫荧光染色检测P2X7R、NLRP3、半胱氨酸天冬氨酸蛋白酶1（Caspase-1）蛋白表达；实时荧光定量逆转录聚合酶链反应（qPCR）检测P2X7R、NLRP3、Caspase-1、缝隙连接蛋白（PAN-1）mRNA的表达。结果　HE染色，光镜下可见正常组小鼠胰腺细胞分布紧密，排列规则，无纤维化及炎症细胞浸润。模型组细胞间隙增宽、腺泡萎缩、炎症细胞浸润且有大量胶原纤维生成，组织学评分较正常组显著升高（P＜0.01）；与模型组相比，CBX中、高剂量组炎症细胞浸润及胶原纤维生成均减少，组织学评分降低。模型组小鼠造模后eATP水平较正常组显著升高（P＜0.01），CBX中、高剂量组给药2周后，胰腺组织eATP水平均低于模型组。免疫荧光染色法和qPCR检测均显示，与正常组相比，模型组P2X7R、NLRP3、Caspase-1蛋白和mRNA表达升高（P＜0.01）；与模型组相比，CBX中、高剂量组表达均下调（P＜0.05）。此外，qPCR检测结果还显示，模型组小鼠PAN-1 mRNA表达较正常组显著上调；与模型组相比，CBX中、高剂量组表达均下调（P＜0.01）。结论　CP病程中eATP水平显著增加并激活P2X7R，促进NLRP3炎性体组装，加重胰腺纤维化，CBX可下调P2X7R、NLRP3、Caspase-1，减轻胰腺炎症损伤及纤维化，从而发挥治疗作用。

An experimental study on the inhibition of eATP-P2X7R-NLRP3 signaling pathway by carbenoxolone to alleviate fibrosis in chronic pancreatitis

Abstract: Objective　To investigate the effects of extracellular ATP (eATP) -P2X7R -NLRP3 axis on the inflammation and fibrogenesis of chronic pancreatitis (CP), and the therapeutic effect of ATP inhibitor carbenoxolone (CBX) on CP. Methods　C57BL/6 mice (male, 6-week-old) were randomly divided into 5 groups: normal group, model group, CBX low, medium and high-dose groups (25, 50 and 100 mg/kg, respectively). After the mouse models of CP were established, the three CBX groups were intraperitoneally injected with corresponding doses of drugs. The mice were killed by cervical dislocation 24 h after the last drug injection. Pancreatic histopathological changes were evaluated by HE staining. eATP levels were measured by the luminescence ATP detection assay. The protein expressions of P2X7R, NLRP3 and Caspase-1 were measured by immunofluorescence staining, and the expressions of P2X7R, NLRP3, Caspase-1 and Pannexin-1 (PAN-1) mRNA were detected by real-time fluorescent polymerase chain reaction (qPCR). Results　Under light microscope, the normal group showed tightly distributed cells in murine pancreas without fibrosis and inflammatory cell infiltration. Compared with the normal group, the intercellular space of the pancreatic tissue, atrophy of the pancreatic acinus, hyperplasia of the collagen fibers and infiltration of a large number of inflammatory cells among fibers were elevated in model group with an increased histopathological score (P＜0.01). Compared with the model group, the content of collagen and the degree of inflammatory cell infiltration were reduced with a decreased histopathological scores in CBX groups. The eATP level was significantly higher in model group than that of the normal group (P＜0.01). After 2 weeks of treatment, the levels of eATP in murine pancreatic tissue were lower in CBX medium and high-dose groups than that of model group. Compared with the normal group, the protein expressions of P2X7R, NLRP3 and Caspase-1 were significantly increased in the model group (P＜0.01). Compared with the model group, these 3 protein expressions were down-regulated in CBX treatment groups, in which a significant effect was found in the CBX medium and high-dose groups (P＜0.05). Results of qPCR showed that the expression of PAN-1 mRNA was significantly up-regulated in model group compared with that of normal group. Compared with the model group, the levels of PAN-1 mRNA were reduced in CBX medium and high-dose groups (P＜0.01). Conclusion　In the course of CP, the eATP level is significantly increased, and then activates the P2X7R, which can accelerate the NLRP3 inflammasome assembly and aggravates pancreatic fibrosis. CBX can down-regulate the expressions of P2X7R, NLRP3 and Caspase-1, alleviate the injury and fibrosis of pancreatitis and consequently play a therapeutic role.