P2X7R拮抗剂对小鼠慢性胰腺炎的作用及机制研究

Protective effects of P2X7R antagonist on chronic pancreatitis in mice

目的探讨嘌呤能P2X7受体(P2X7R)拮抗剂——氧化三磷酸腺苷(OxATP)和亮蓝G(BBG)在抑制下游靶蛋白——核苷酸结合寡聚化结构域样受体3(NLRP3)炎性体活化和慢性胰腺炎(CP)胰腺纤维化进程中的作用及可能机制。方法 40只C57BL/6小鼠随机均分为正常对照组、CP模型组、Ox ATP组和BBG组。除正常对照组腹腔注射与处理组等体积的生理盐水外,其余组均通过腹腔注射雨蛙素法制作CP小鼠模型(6周)。造模结束后,CP模型组、Ox ATP组和BBG组分别予以腹腔注射生理盐水、Ox ATP(20μL,300μmol/L)和BBG(20μL,10μmol/L)处理,连续注射2周。然后处死各组小鼠并取胰腺组织,行病理学检查,以HE染色对胰腺病理组织学(炎性细胞浸润、腺泡萎缩及纤维化程度)进行评分,天狼星红染色和α-平滑肌肌动蛋白(α-SMA)免疫组化染色分别对胰腺纤维化程度进行评估;免疫组化法检测胰腺P2X7R、NLRP3和半胱氨酸天冬氨酸蛋白酶1(Caspase-1)中的累积光密度(IOD)值。结果与正常对照组比,CP模型组炎症损伤组织学评分增加,胰腺纤维化程度显著增加,P2X7R、NLRP3和Caspase-1免疫组化IOD值显著升高(P<0.05);与CP模型组相比,Ox ATP和BBG组炎症损伤组织学评分降低,HE染色纤维化评分、天狼星红染色和α-SMA免疫组化染色均显著减轻(P<0.05),胰腺P2X7R、NLRP3和Caspase-1 IOD值均明显降低(P<0.05)。结论 P2X7R拮抗剂Ox ATP和BBG能够显著减轻CP小鼠模型的慢性炎症和纤维化程度,阻断P2X7R-NLRP3炎性体信号通路,这有望成为治疗CP及其纤维化进程的一种潜在新策略。

Objective To investigate the role of purinergic 2X7 receptor(P2X7R) and its downstream target- NLRP3 inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis(CP).Methods Forty C57BL/6 mice were randomly divided into normal control group,CP group,P2X7 R antagonist oxidized ATP(OxATP) group and brilliant blue G(BBG) group.The chronic pancreatitis model was induced by repeated intraperitoneal injection of the cholecystokinin analogue caerulein with the dose of 50 μg/kg for six weeks.Normal saline,OxATP(20 μL,300 μmol/L) or BBG(20 μL,10 μmol/L) were administered for CP group,OxATP group and BBG group for two weeks after the last caerulein injection.Then all mice were sacrificed and the histopathological changes of the pancreas,especially the fibrotic degrees were evaluated by HE stain,fibrosis score,Sirius red staining and α- SMA immunohistochemical stain.The pancreatic P2X7 R,NLRP3 and Caspase-1 expressions were detected by immunohistochemistry respectively to compare the changes between the groups,and explore the role of P2X7R-NLRP3 signaling pathway in pancreatic fibrosis.Results Compared with the normal control group,the scores of pancreatic fibrosis and the expressions of P2X7 R,NLRP3 and Caspase- 1 in pancreas were significantly increased in CP model group(P<0.05).Compare to CP group,the pancreatic chronic inflammation and the fibrosis indices such as HE fibrosis score,Sirius red staining and α- SMA immunohistochemical stain were ameliorated obviously in OxATP and BBG groups(P<0.05).And expressions of P2X7 R,NLRP3 and Caspase-1 in the pancreas were all reduced greatly in both OxATP and BBG groups(P<0.05).Conclusion P2X7 R antagonist OxATP and BBG can significantly decrease pancreatic chronic inflammation and flbrosis in the mouse model of CP,which suggests that the blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.