腹腔穿刺引流对重症急性胰腺炎大鼠胰腺细胞焦亡的影响及其意义  

Effect of abdominal paracentesis drainage on pancreatic pyroptosis of rats with severe acute pancreatitis and its significance

作　　者：吴俊[1,2] 卢一琛 黄竹 蒋文 且华吉[1,2] 刘江涛 孙红玉 汤礼军[1,2] Wu Jun;Lu Yi-Chen;Huang Zhu;Jiang Wen;Qie Hua-Ji;Liu Jiang-Tao;Sun Hong-Yu;Tang Li-Jun(College of Medicine,Southwest Jiaotong University,Chengdu,Sichuan 610063,China;PLA Center of General Surgery,Pancreatic Injury and Repair Key Laboratory of Sichuan Province;Basic Medical Laboratory,the General Hospital of Western Theater Command,Chengdu,Sichuan 610083,China)

机构地区：[1]西南交通大学医学院,四川成都610063 [2]西部战区总医院全军普通外科中心/四川省胰腺损伤与修复重点实验室,四川成都610083 [3]西部战区总医院基础医学实验室,四川成都610083

出　　处：《解放军医学杂志》2022年第9期863-870,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81772001);国家临床重点专科项目(41732113);四川省科技计划项目(2019YJ0277)。

摘　　要：目的探讨腹腔穿刺引流(APD)对重症急性胰腺炎(SAP)大鼠caspase-1介导的胰腺细胞焦亡的影响及其意义。方法将48只健康雄性SD大鼠随机分为假手术组、SAP组及APD组,每组16只。采用5%牛磺胆酸钠逆行注入胰胆管构建大鼠SAP模型,APD组在SAP组的基础上于右下腹放置引流管引流。于造模后12 h收集大鼠血清及胰腺组织。采用全自动生化分析仪检测血清脂肪酶及淀粉酶活性,HE染色观察胰腺损伤情况,酶联免疫吸附实验(ELISA)检测血清炎性因子的变化,RT-PCR及Western blotting检测胰腺组织caspase-1介导的焦亡相关基因及蛋白的表达变化,透射电镜观察胰腺腺泡细胞超微结构变化及焦亡的特征性改变。结果与SAP组比较,APD组的胰腺组织病理损伤明显减轻,病理评分明显降低,血清脂肪酶、淀粉酶、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β、IL-18水平明显降低,差异均有统计学意义(P<0.05);SAP组胰腺组织焦亡通路蛋白核苷酸结合寡聚化结构域样受体3(NLRP3)、凋亡相关的斑点样蛋白(ASC)、caspase-1、cleaved-caspase-1、gasdermin家族蛋白D(GSDMD)及cleaved-GSDMD表达水平明显高于假手术组,经APD治疗后,胰腺组织焦亡通路蛋白表达均明显降低,胰腺组织IL-1β及IL-18 mRNA及蛋白表达水平也明显降低,差异均有统计学意义(P<0.05);透射电镜观察显示,SAP组出现明显的内质网及线粒体扩张,核染色质凝聚,以及焦亡的特征性改变即细胞膜上膜孔的形成,而APD组内质网及线粒体扩张明显减轻,核染色质凝聚明显减少。结论Caspase 1介导的焦亡在SAP中被激活;早期实施APD能够减轻SAP的严重程度,并明显抑制caspase-1介导的焦亡激活,从而减轻局部及全身炎症反应。Objective To investigate the role and significance of abdominal paracentesis drainage(APD)to pyroptosis in the pancreas of rats with severe acute pancreatitis(SAP),which is mediated by NLRP3 inflammasome-activated-caspase-1.Methods A total of 48 healthy male SD rats were randomly divided into three groups:sham operation(Sham)group,SAP group,and APD group,with 16 rats in each group.5%sodium sulfonate was retrogradely injected into the pancreaticobiliary duct to establish the SAP model.In the APD group,besides the SAP induction,a drainage tube was placed on the right lower abdomen for drainage.Serum and pancreatic tissue were collected 12 hours after modeling.We examined the activities of serum lipase and amylase using an automatic biochemical analyzer and evaluated pancreatic damage through HE staining.In addition,we quantified the expression of serum inflammatory factors by ELISA and measured the expression level of genes and proteins related to caspase-1 mediated pyroptosis in pancreatic tissue using RT-PCR and Western blotting.Lastly,we observed the structural changes of the subcellular organelles and the characteristic changes of pyrolysis in pancreatic acinar cells using transmission electron microscopy.Results Compared with the SAP group,the pancreas tissue in the ADP group showed alleviated damages with a much lower pathological score;the levels of serum lipase,amylase,tumor necrosis factor(TNF)-α,Interleukin(IL)-6,IL-1β,and IL-18 also showed significant reduction(P<0.05).Compared with the Sham group,the expression levels of NLRP3,ASC,Caspase-1,cleaved caspase-1,GSDMD,and cleaved-GSDMD were all significantly up-regulated in the pancreas in the SAP group.After APD treatment,the expression of these Caspase-1-mediated pyrolysis pathway key genes in pancreatic tissue was significantly down-regulated.In addition,the expression levels of IL-1βand IL-18 mRNA in pancreatic tissue were also significantly reduced(P<0.05).Transmission electron microscopy showed that the SAP group had endoplasmic reticulum and mitoc

关 键 词：重症急性胰腺炎 腹腔穿刺引流 细胞焦亡 CASPASE-1 gasdermin家族蛋白D 

分 类 号：R657.51[医药卫生—外科学]