高迁移率族蛋白1在小鼠热射病相关急性肾损伤发病机制中的意义

Role of high mobility group box-1 protein in pathogenesis of acute kidney injury induced by heat stroke in mice

目的观察高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)在热射病(heat stroke)小鼠动物模型肾组织中的表达,探讨HMGB1在热射病相关急性肾损伤发病机制中的意义。方法采用健康雄性C57BL/6J小鼠20只,按照随机数字表随机分为正常对照组(n=10)及热射病组(n=10),热射病组小鼠进入生物模拟仓(温度41℃,湿度70%)2 h,以肛温大于40℃为成模标准,采用18F脱氧葡萄糖标记小型正电子发射断层成像/X射线断层成像(micro-PET/CT)核素显像技术行活体显像。采集血标本检测血肌酐水平;HE染色观察肾组织病理改变;透射电镜检测线粒体形态改变;免疫组化法检测HMGB1及线粒体凋亡诱导因子2(apoptosis inducing factor mitochondria associated 2,Aifm2)定位表达情况;Western印迹法检测HMGB1、糖基化终末产物受体(advanced glycosylation end product-specific receptor,RAGE)表达情况;原位末端转移酶标记技术(TUNEL)检测肾组织细胞凋亡水平;液相色谱-质谱联用仪(LC-MS)检测小鼠肾组织代谢组学,并通过KEEG数据库进行通路富集分析。结果(1)热射病组小鼠造模后45 min体温显著高于正常对照组(P<0.05);热射病组血肌酐水平显著高于正常对照组(P<0.05);micro-PET/CT显示热射病组小鼠骨骼肌、内脏出现18F脱氧葡萄糖广泛浓聚。(2)HE染色显示热射病组小鼠肾组织出现集合管出血、小管内皮细胞肿胀,在透射电镜下可见线粒体肿胀变形。(3)免疫组化显示Aifm2在肾小管上皮细胞广泛表达,热射病组表达水平显著高于正常对照组(P<0.05);HMGB1呈现细胞核阳性表达,在热射病组阳性细胞比例显著高于正常对照组(P<0.05)。(4)Western印迹结果显示热射病组肾组织HMGB1、RAGE表达水平显著上升(P<0.05)。(5)TUNEL检测结果显示热射病组凋亡细胞水平显著高于正常对照组(P<0.05)。(6)正常对照组和热射病组肾组织间检出136种差异代谢物,经过KEGG数据库分析,不饱和脂肪酸代谢紊乱等代谢通路存在显著性差异,肾上腺素酸(adrenic acid)等在内的多种差异代谢物可能是发病机制中的重要调节点。结论急性肾损伤是热射病常见并发症,可能原因为全身高分解代谢导致的肾脏线粒体功能障碍及凋亡通路激活,与不饱和脂肪酸代谢紊乱及HMGB1活化可能存在相关性。部分差异代谢物可能在热射病相关急性肾损伤研究中存在较高的研究价值。

Objective To observe the differential expression of high mobility group box-1 protein(HMGB1)in renal tissues of heat stroke mice models,and to explore its role in the pathogenesis of heat stroke associated acute kidney injury(HS-associated AKI).Methods According to random number table,20 healthy male C57BL/6J mice were randomly divided into 2 groups,including normal control(n=10)and heat stroke group(n=10).The mice in heat stroke group were given with a 2-hour-exposure in biological simulation chamber(temperature 41℃,humidity 70%).Heat stroke was defined as anal temperature lasting more than 40 degrees Celsius.A 18F-deoxyglucose nuclide labeled vivo imaging was conducted with micro-positron emission tomography(PET)/computer tomography(CT).Serum creatinine was examined with blood example.In order to evaluate the pathological changes,HE stain was conducted with kidney tissue,and mitochondrial morphological changes in kidney tissue were observed by transmission electron microscopy.The expressions of HMGB1 and apoptosis inducing factor mitochondria associated 2(Aifm2)were examined by immunohistochemical method,and the levels of HMGB1 and RAGE were examined by Western blotting.The cell apoptosis of renal tissue was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL).The metabolomics of kidney tissue in mice were detected by liquid chromatography-mass spectrometry(LC-MS),and the pathway enrichment analysis was carried out by KEEG database.Results(1)The body temperature of the mice in heat shock group was significantly higher than that in normal control group 45 min after model establishment(P<0.05).The level of serum creatinine in heat shock group was significantly higher than that in normal control group(P<0.05),and the levels of 18F-deoxyglucose increased in skeletal muscle and visceral tissue of the mice in heat-shock group.(2)HE staining showed hemorrhage in collecting duct and tubular endothelial cell swelling,and mitochondrial swelling and deformation were observed by transmission electron microscopy in kidney tissue of the heat shock group.(3)Immunohistochemical method showed that the levels of Aifm2 and HMGB1 in heat shock group were higher(P<0.05).(4)Western blotting showed that the levels of HMGB1 and RAGE in heat shock group were higher than those in normal control group(P<0.05).(5)TUNEL showed that the number of cells with positive stain in kidney tissue of the heat shock group was higher than that in normal control group(P<0.05).(6)Between normal control group and heat shock group,136 differential metabolites were detected in kidney tissues.After analysis by KEGG database,pathway abnormalities such as unsaturated fatty acid metabolism disorder may be associated with HS-associated AKI,and many differential metabolites such as adrenic acid may be important regulatory points in the pathogenesis.Conclusion Acute kidney injury is a common complication of heat shock.It may be related to the dysfunction of renal mitochondria and activation of apoptotic pathway caused by systemic hypercatabolism,which may be related to the disorder of unsaturated fatty acid metabolism and activation of HMGB1.Some differential metabolites may be of high value in HS-associated AKI studies.