线粒体靶向抗氧化肽SS-31对脓毒症急性肾损伤的保护作用  

Protective effect of mitochondria-targeted antioxidant peptide SS-31 in sepsis-induced acute kidney injury

作　　者：孙敏[1] 马建伟[1] 叶继辉[1] 樊恒[1] 乐健伟[1] 朱建华[1] Sun Min;Ma Jianwei;Ye Jihui;Fan Heng;Le Jianwei;Zhu Jianhua(Department of Intensive Care Unit,Ningbo First Hospital,Ningbo 315010,Zhejiang,China)

机构地区：[1]宁波市第一医院重症医学科,浙江宁波315010

出　　处：《中华危重病急救医学》2021年第12期1418-1422,共5页Chinese Critical Care Medicine

基　　金：浙江省宁波市自然科学基金(2018A610296,2019A610310,202003N4256);浙江省医药卫生科技计划项目(2018KY669,2020KY828,2021KY998);浙江省中医药科技计划项目(2019ZA113)。

摘　　要：目的探讨线粒体靶向抗氧化肽SS-31对脓毒症急性肾损伤(AKI)小鼠肾脏的保护作用及其机制。方法选择60只成年雄性C57BL/6小鼠,按随机数字表法分为假手术组(10只)、阳性对照组(10只)、脓毒症模型组(20只)和SS-31肽干预组(20只)。采用盲肠结扎穿孔术(CLP)复制脓毒症AKI模型;假手术组只开腹,不进行CLP。阳性对照组和SS-31肽干预组于术后30 min腹腔注射SS-31肽注射液(5 mg/kg);假手术组和模型组给予等量生理盐水;4组均连续给药7 d。于术后24 h眼眶采血,分离血清检测血肌酐(SCr)、尿素氮(BUN)水平;术后7 d处死小鼠,取肾脏组织行苏木素-伊红(HE)染色,光镜下观察肾脏组织病理学变化,透射电镜下观察肾脏组织线粒体超微结构改变,原位末端缺刻标记试验(TUNEL)检测肾小管上皮细胞凋亡情况,蛋白质免疫印迹试验(Western blotting)检测过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)、腺苷酸活化蛋白激酶(AMPK)、活化的天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的蛋白表达水平。结果与假手术组比较,脓毒症模型组术后24 h SCr和BUN均明显升高〔SCr(μmol/L):93.12±11.80比32.94±3.37,BUN(mmol/L):41.36±6.48比9.49±3.58,均P<0.05〕,肾脏组织AMPK、PGC-1α和活化的caspase-3的蛋白表达水平均明显增加(AMPK/β-actin:0.30±0.02比0.12±0.01,PGC-1α/β-actin:0.38±0.03比0.16±0.02,活化的caspase-3/β-actin:0.20±0.01比0.11±0.02,均P<0.05);光镜下可见炎性细胞浸润,肾小球基底膜暴露,管腔内可见空泡样透明管型;电镜下可见线粒体损伤,出现肿胀、嵴消失、内外膜破裂,凋亡细胞数明显增多〔个:24.00(18.75,31.00)比2.00(0.72,3.25),P<0.05〕。与脓毒症模型组比较,SS-31肽干预组血清SCr、BUN水平及肾脏组织AMPK、PGC-1α、活化的caspase-3的蛋白表达量均明显降低〔SCr(μmol/L):71.33±10.14比93.12±11.80,BUN(mmol/L):27.00±5.52比41.36±6.48,AMPK/β-actin:0.23±0.01比0.30±0.02,PGC-Objective To study the effect and mechanism of mitochondria-targeted antioxidant peptide SS-31 on sepsis-induced acute kidney injury(AKI).Methods Sixty adult male C57BL/6 mice were randomly divided into four groups according to the random number table method:sham group(10 mice),positive control group(10 mice),sepsis model group(20 mice),and SS-31 peptide group(20 mice).The sepsis-induced AKI mouse model was reproduced by cecal ligation and puncture(CLP).The sham group only received laparotomy.SS-31 peptide(5 mg/kg)was intraperitoneally injected in SS-31 peptide group and positive control group 30 minutes after the operation,while an equivalent amount of normal saline was given in sham group and sepsis model group for 7 days.The blood samples were collected 24 hours after the operation from orbit,and the serum was collected to test the serum creatinine(SCr)and blood urea nitrogen(BUN).The mice were sacrificed 7 days after surgery.The kidney tissues were collected to observe the pathologic structure changes under the hematoxylin-eosin(HE)staining by light microscope.And the mitochondrial ultrastructure was checked under the transmission electron microscope.Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method(TUNEL).The expression level of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),adenosine monophosphate-activated protein kinase(AMPK),and cleaved caspase-3 protein were tested by Western blotting.Results Compared with sham group,the levels of SCr and BUN were significantly increased in sepsis model group[SCr(μmol/L):93.12±11.80 vs.32.94±3.37,BUN(mmol/L):41.36±6.48 vs.9.49±3.58,both P<0.05].The expression levels of AMPK,PGC-1αand cleaved caspase-3 protein increased(AMPK/β-actin:0.30±0.02 vs.0.12±0.01,PGC-1α/β-actin:0.38±0.03 vs.0.16±0.02,cleaved caspase-3/β-actin:0.20±0.01 vs.0.11±0.02,all P<0.05).HE staining showed that inflammatory cell was infiltrated,glomerular basement membrane was exposed and vacuole-like transpar

关 键 词：透明管型 肾小球基底膜 器官功能障碍 线粒体结构 干预组 线粒体损伤 细胞肿胀 C57BL/6小鼠 

分 类 号：R459.7[医药卫生—急诊医学] R692[医药卫生—治疗学]