重症急性胰腺炎大鼠肠源性内毒素、Toll样受体4蛋白与自噬微管相关蛋白1轻链3、p62蛋白、溶酶体相关膜蛋白2的变化及相关性  

Changes and correlation of intestinal endotoxin,toll-like receptor 4 protein and autophagy microtubule-associated protein 1 light chain 3,p62 protein,lysosomal associated membrane protein 2 in rats with severe acute pancreatitis

作　　者：宋剑 段吉明[1] 岳浩浩 朱燕琼 李文星[1] SONG Jian;DUAN Jiming;YUE Haohao;ZHU Yanqiong;LI Wenxing(Department of General Surgery,Second Hospital of Shanxi Medical University,Taiyuan,Shanxi 030000,Chinav)

机构地区：[1]山西医科大学第二医院普外科,山西太原030000

出　　处：《安徽医药》2024年第10期1956-1960,I0005,共6页Anhui Medical and Pharmaceutical Journal

基　　金：山西省自然科学研究面上项目(20210302123261)。

摘　　要：目的探讨重症急性胰腺炎(SAP)大鼠肠源性内毒素、Toll样受体4(TLR4)蛋白与自噬微管相关蛋白1轻链3(LC3)、p62蛋白(p62)、溶酶体相关膜蛋白2(LAMP-2)的变化及相关性。方法2022年7月至2023年3月进行该研究。选取SD大鼠36只,随机数字表法分为假手术(SO)组和SAP组,两组以术后时间点3 h、6 h、12 h各自再平均分成三组。采用胰胆管逆行注射5%牛磺胆酸钠(0.1 mL/100 g)制备SAP组模型,SO组找到胰腺后关腹。酶联免疫吸附测定(ELISA)检测大鼠血清中α-淀粉酶(AMS)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和脂多糖(LPS)的含量。苏木精-伊红染色法对比各组胰腺、小肠病理学评分变化。免疫组织化学法比较各组LC3、p62和LAMP-2的表达。蛋白质印迹法检测TLR4蛋白表达。结果SAP组大鼠血清AMS、TNF-α、IL-1β、LPS,胰腺、小肠病理学评分,胰腺LC3、p62阳性细胞总积分,胰腺组织TLR4蛋白表达量与SO组相比均明显升高(均P<0.05),SAP组各项指标随术后时间延长不断升高。SAP组胰腺LAMP-2阳性细胞总积分[3 h:(4.23±0.29)分,6 h:(0.33±0.16)分,12 h:(0.20±0.13)分]与SO组[3 h:(6.27±0.10)分,6 h:(6.23±0.34)分,12 h:(6.30±0.28)分]相比明显降低(P<0.05)。SAP组中,大鼠血清LPS与血清TNF-α、IL-1β,胰腺LC3、p62、TLR4表达变化呈正相关(LPS比TNF-α,r=0.92;LPS比IL-1β,r=0.99;LPS比LC3,r=0.98;LPS比p62,r=0.99;LPS比TLR4,r=0.96;均P<0.001);大鼠血清LPS与胰腺LAMP-2表达变化呈负相关(r=−0.98,P<0.001)。结论SAP时LPS突破肠道屏障移位至胰腺,激活TLR4通路表达大量炎症因子促进胰腺炎症反应,使细胞自噬蛋白LC3与p62表达上调,但LAMP-2表达下降致自噬通量受损,肠源性内毒素移位与细胞自噬受损可共促进SAP病程发展。Objective To investigate the changes and correlation of intestinal endotoxin,Toll-like receptor 4(TLR4)protein and autophagy microtubule-associated protein 1 light chain 3(LC3),p62 protein(p62)and lysosome associated membrane protein 2(LAMP-2)in rats with severe acute pancreatitis(SAP).Methods The study will be conducted from July 2022 to March 2023.Thirty-six SD rats were randomly assigned into sham operation(SO)group and SAP group.The two groups were further assigned into three groups at 3 h,6 h and 12 h after surgery.The SAP group model was prepared by retrograde injection of 5%sodium taurocholate(0.1 mL/100 g)into the pancreaticobiliary duct.The abdomen of the SO group was closed after the pancreas was found.The levels ofα-amylase(AMS),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and lipopolysaccharide(LPS)in the serum of rats were detected by enzyme-linked immunosorbent assay(ELISA).The changes of pancreatic and small intestinal pathological scores among the groups were compared by hematoxylin-eosin staining.The expressions of LC3,p62 and LAMP-2 among the groups were compared by immunohistochemistry.The expression of TLR4 protein was detected by western blot detection.Results Serum AMS,TNF-α,IL-1β,LPS,pathological score of pancreas and small intestine,total score of LC3 and p62 positive cells in pancreas,and the expression of TLR4 protein of pancreatic tissue in SAP group were significantly higher than those in SO group(P<0.05),all the indexes in SAP group increased with time.The total score of LAMP-2 positive cells in pancreas in SAP group[3 h:(4.23±0.29)points,6 h:(0.33±0.16)points,12 h:(0.20±0.13)points]was significantly lower than that in SO group[3 h:(6.27±0.10)points,6 h:(6.23±0.34)points,12 h:(6.30±0.28)points](P<0.05).In the SAP group,serum LPS of rats was positively correlated with the changes in serum TNF-α,IL-1β,and the expressions of pancreatic LC3,p62,and TLR4(LPS vs.TNF-α,r=0.92,P<0.001;LPS vs.IL-1β,r=0.99,P<0.001;LPS vs.LC3,r=0.98,P<0.001;LPS vs.p62,r=0.99,P<0.001;LPS v

关 键 词：重症急性胰腺炎 内毒素 细胞自噬 炎症因子 肠道屏障 大鼠 Sprague-Dawley 

分 类 号：R576[医药卫生—消化系统]