早期脓毒症对延髓内脏带的病理损害及胆碱能抗炎通路的干预效应  

Pathological damage of medullary visceral zone induced by early sepsis and intervention effect of the cholinergic anti-inflammatory pathway

作　　者：李楚娟 李红兵 史兰[2] 柳仙[2] 王颖 郑权 Li Chujuan;Li Hongbing;Shi Lan;Liu Xian;Wang Ying;Zheng Quan(Department of Gynecological Tumors,Hubei Cancer Hospital;Department of General Medicine,The First People’s Hospital of Guiyang)

机构地区：[1]湖北省肿瘤医院妇科肿瘤科,武汉430060 [2]贵阳市第一人民医院全科医学科,贵阳550002

出　　处：《重庆医科大学学报》2024年第9期1095-1104,共10页Journal of Chongqing Medical University

基　　金：贵州省自然科学基金重点课题资助项目(编号:黔科合基础-ZK[2023]重点001);贵州省科技厅科研课题资助项目(编号:黔科合基础[2019]1005);贵州省卫健委科学技术基金资助项目(编号:gzwkj2021-001)。

摘　　要：目的:探索脓毒症对延髓内脏带(medullaryvisceralzone,MVZ)的影响及胆碱能抗炎通路(cholinergicantiinflammatory pathway,CAP)的干预效应。方法:64只成年SPF级雄性Sprague-Dawley大鼠纳入本研究,随机分为对照组8只:正常饲养;假手术组8只:大鼠剖腹,游离盲肠后缝合腹腔,并予哌拉西林(50 mg/kg,腹腔注射,1次/d,连续3 d)预防感染及生理盐水(1 mL/100 g,腹腔注射,1次/d,连续3 d);脓毒症组共48只,采用盲肠结扎穿刺(cecal ligation and puncture,CLP)制备脓毒症模型后随机分为:模型组16只:CLP后,余处理同假手术组;GTS-21[α7-烟碱样乙酰胆碱受体(nicotinic acetylcholine receptors,nAChRs)激动剂,用于激活CAP]组16只,哌拉西林使用同脓毒症组,并以GTS-21(4 mg/kg,腹腔注射,1次/d,连续3 d)进行干预;甲基牛扁亭(methyllycaconitine,MLA)强效选择性α7nAChRs拮抗剂,用于阻断CAP组16只:哌拉西林使用同脓毒症组。干预3 d后,收集每组大鼠延髓样本,用于延髓内脏带的病理观察、TdT介导的dUTP缺口末端标记(TdT mediated dUTP Nick End Labeling,TUNEL)检测凋亡、酪氨酸羟化酶(tyrosine hydroxylase,TH)/Caspase 3或胆碱乙酰转移酶(choline acetyltransferase,CHAT)/Caspase 3的双重免疫荧光标记检测两种神经元的凋亡情况,PCR检测关键基因如生长相关蛋白43(grouth associated protein-43,GAP-43)mRNA,少突胶质细胞转录因子2(oligodendrocyte transcription factor 2,Olig-2)mRNA,血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA,胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)mRNA,和基质金属蛋白酶(matrix metalloprotein,MMP)-9 mRNA的表达。结果:模型组MVZ凋亡指数明显高于对照组(P<0.05),GTS-21具有降低脓毒病导致的凋亡的趋势(P>0.05),MLA较GTS-21明显增加凋亡(P<0.05);脓毒症诱导MVZ儿茶酚能神经元和胆碱能神经元较对照组Caspase 3表达明显升高P<0.05),TH明显降低(P<0.05),但CHAT仅有下降的趋势(P>0.05);GTS-21具有降低脓毒症�Objective:To investigate the influence of sepsis on medullary visceral zone(MVZ)and the intervention effect of the cholin⁃ergic anti-inflammatory pathway(CAP).Methods:A total of 64 spe⁃cific pathogen-free adult male Sprague-Dawley rats were randomly divided into control group with 8 rats(normal feeding);shamoperation group with 8 rats undergoing laparotomy and suture of the abdominal cavity after isolation of the cecum,followed by piperacil⁃lin(50 mg/kg,intraperitoneal injection,once a day for 3 consecutive days)to prevent infection and normal saline(1 mL/100 g,intraperi⁃toneal injection,once a day for 3 consecutive days);sepsis group with 48 rats undergoing cecal ligation and puncture(CLP)to establish a models of sepsis,then they were randomly divided into model group(given the same treatment as the sham-operation group),GTS-21(a selectiveα7 nicotinic acetylcholine receptor agonist used to activate the cholinergic anti-inflammatory pathway[CAP])group with 16 rats treated with piperacillin(the same treatment as the sepsis group)and intervened with GTS-21(4 mg/kg,intraperitoneal injection,once a day for 3 consecutive days),and the methyllycaconitine(MLA,a potent and selective nicotine acetylcholine receptor antagonist used to block CAP)group with 16 rats given the same piper⁃acillin treatment as the sepsis group and intervened with MLA(4.8 mg/kg,intraperitoneal injection,once a day for 3 consecutive days).After 3 days of intervention,medulla oblongata samples were collected from each group for pathological observation;TdT-mediated dUTP nick end labeling(TUNEL)was used to measure apoptosis;double immunofluorescence labeling for tyrosine hydroxylase(TH)/Caspase 3 or choline acetyltransferase(CHAT)/Caspase 3 was used to observe the apoptosis of neurons;PCR was used to measure the mRNA expression levels of the key genes such as growth-associated protein-43(GAP-43),oligodendrocyte transcription factor 2(Olig-2),vascular endothelial growth factor(VEGF),glial fibrillary acidic protein(GFAP),and matrix metallop

关 键 词：脓毒症 延髓内脏带 胆碱能抗炎通路 神经炎症 

分 类 号：R114[医药卫生—卫生毒理学]