自噬通过降低肠道黏膜氧化应激减少细菌移位发生  

Autophagy reduces bacterial translocation by regulating intestinal mucosal oxidative stress

作　　者：鹿兴[1] 尹承芬[1] 苏亚肖 高心晶[1] 王凤梅[3] 徐磊[1,4] Lu Xing;Yin Chengfen;Su Yaxiao;Gao Xinjing;Wang Fengmei;Xu Lei(Department of Critical Care Medicine,Third Central Hospital,Tianjin 300170,China;The Third Central Clinical College,Tianjin Medical University,Tianjin 300170,China;Institute of Hepatobiliary Disease,Third Central Hospital,Tianjin 300170,China;Key Laboratory of Extracorporeal Life Support for Critical Diseases,Third Central Hospital,Tianjin 300170,China)

机构地区：[1]天津市第三中心医院重症医学科,天津300170 [2]天津医科大学三中心临床学院,天津300170 [3]天津市第三中心医院肝胆疾病研究所,天津300170 [4]天津市第三中心医院重症疾病体外生命支持重点实验室,天津300170

出　　处：《中华危重病急救医学》2025年第2期153-159,共7页Chinese Critical Care Medicine

基　　金：天津市自然科学基金(21JCYBJC01200);天津市卫生健康委员会中医中西医结合科研课题(2023220,2023221)。

摘　　要：目的研究自噬在肠道高毒力肺炎克雷伯菌(hvKp)移位感染中的作用机制,探讨减少肠源性细菌移位感染的方法。方法以50只C57BL/6J小鼠为研究对象,随机分为灌胃组(n=40)和对照组(CO组,n=10)。灌胃组用200μL/d的hvKp(菌落数为10^(9) CFU/mL)连续灌胃5 d,建立hvKp肠道感染模型;CO组使用等量无菌生理盐水灌胃。实验结束后,用异氟醚麻醉小鼠实施颈椎脱位安乐死。取小鼠外周静脉血,用16S rDNA测序检测是否发生细菌移位,并分为移位组(BT+组)和未移位组(BT-组)。用苏木素-伊红(HE)染色评估肠道黏膜形态学改变;电镜下观察肠道黏膜超微结构改变;测定肠道氧化应激指标超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GPx)的水平;用原位杂交检测hvKp是否发生细菌转移;用蛋白质免疫印迹试验(Western blotting)检测紧密连接蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和自噬相关蛋白Beclin-1的表达;用反转录-聚合酶链反应(RT-PCR)检测连接蛋白ZO-1和Claudin-2的mRNA表达;用免疫荧光法观察自噬蛋白及连接蛋白的分布表达情况。结果灌胃组40只小鼠中有2只发生吸入性肺炎后死亡;CO组小鼠全部存活。16S rDNA测序结果显示,CO组外周血标本未检出细菌;灌胃组有18只小鼠外周血中检出细菌,细菌移位率为47.4%。BT-组和BT+组出现肠黏膜组织损伤,其中BT+组损伤严重。与CO组比较,BT-组和BT+组MDA水平显著升高,SOD和GPx活性显著降低。与BT-组比较,BT+组MDA水平进一步升高,SOD和GPx活性进一步降低〔MDA(mmol/mg):2.98±0.11比2.48±0.11,SOD(U/mg):62.40±5.45比73.40±4.08,GPx(U/mg):254.72±10.80比303.55±8.57,均P<0.01〕。原位杂交检测结果显示,BT+组连续灌胃5 d后在肠黏膜固有层和肝脏组织中检测到移位的hvKp。与CO组相比,BT-组和BT+组LC3-Ⅱ、Beclin-1蛋白表达明显增加;而BT+组LC3-Ⅱ、Beclin-1蛋白表达显著低于BT-组(LC3-Ⅱ/β-actin:0.38±0.04比0.70±0.09,BObjective To investigate the mechanism of autophagy in regulating bacterial translocation in intestinal infection caused by hypervirulent Klebsiella pneumonia(hvKp)and explore the method of reducing translocation infection of intestinal bacteria.Methods Fifty C57BL/6J mice were divided into gavage group(n=40)and control group(CO group,n=10).The gavage group was orally administered with 200μL/d of hvKp(colony count of 10^(9) CFU/mL)continuously for 5 days to establish a hvKp intestinal infection model.CO group was given an equal amount of normal saline.After the experiment,the mice were anesthetized with lsofluraneand euthanized with cervical dislocation under anesthesia.Peripheral venous blood of mice was collected to detect bacterial translocation by 16S rDNA sequencing,then divided into translocation group(BT+group)and non-translocation group(BT-group).Hematoxylin-eosin(HE)staining was used to evaluate intestinal morphology.The ultrastructural changes of intestinal tissues were observed by electron microscope.The levels of intestinal oxidative stress indicators such as superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GPx)were measured.Translocation was detected by in situ hybridization.The expression of tight junction protein microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ)and autophagy protein Beclin-1 were measured by Western blotting.The mRNA expression of tight junction proteins ZO-1 and Claudin-2 were detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of autophagy protein and tight junction protein were observed by immunofluorescence.Results Two out of 40 mice in the gavage group died after developing aspiration pneumonia.All mice in the CO group survived.The 16S rDNA sequencing results showed that no bacteria were detected in the peripheral blood of the CO group,but bacteria were detected in the peripheral blood of 18 mice in the gavage group,with a bacterial translocation rate of 47.4%.The BT-and BT+groups showed intestinal mucosal tissu

关 键 词：氧化应激 自噬 细菌移位 肺炎克雷伯菌 肝脓肿 

分 类 号：R563.1[医药卫生—呼吸系统] R563[医药卫生—内科学]