N-乙酰半胱氨酸上调Sirtuin 3蛋白表达对脓毒症小鼠急性肾损伤的保护作用及机制研究  

The protective effect and mechanism of N-acetylcysteine on acute kidney injury by up-regulating Sirtuin3 protein expression in septic mice

作　　者：樊恒[1] 乐健伟[1] 陈国栋[1] 孙敏[1] 朱建华[1] Fan Heng;Le Jianwei;Chen Guodong;Sun Min;Zhu Jianhua(Department of Intensive Care Unit,Ningbo First Hospital,Ningbo 315010,China)

机构地区：[1]宁波市第一医院重症医学科,宁波315010

出　　处：《中华急诊医学杂志》2022年第6期783-788,共6页Chinese Journal of Emergency Medicine

基　　金：浙江省医药卫生科技计划项目(2020KY828,2021KY998)。

摘　　要：目的探讨抗氧化剂N-乙酰半胱氨酸(NAC)调控沉默信息调节因子3(Sirt3)对脓毒症致小鼠急性肾损伤(AKI)的保护作用及其机制。方法将雄性C57BL/6小鼠随机(随机数字法)分为假手术组(Sham)、盲肠结扎和穿孔术组(CLP)CLP+NAC(50 mg/kg)和CLP+NAC(100mg/kg)组,每组10只;CLP造模后24h处死小鼠,留取血液和肾组织样本;采用HE染色法评估各组小鼠肾组织病理损伤;ELISA法检测血清中肌酐(Scr)尿素氮(BUN)、肾损伤分子1(KIM-1)和中性粒细胞明胶酶相关载脂蛋白(NGAL)水平;采用免疫组织化学检测肾组织Sirt3蛋白表达;RT-qPCR法测定Sirt3 mRNA水平;透射电镜下观察肾小管上皮细胞线粒体损伤情况,并计算线粒体密度,同时检测肾皮质中超氧化物歧化酶(SOD)谷胱甘肽过氧化物酶(GSH-Px)过氧化氢酶(CAT)和丙二醛(MDA)水平。结果与Sham组相比,CLP导致肾组织病理损伤明显加重(P<0.001),肾功能指标Scr、BUN、KIM-1和NGAL水平均明显升高(均P<0.001),肾组织Sirt3蛋白和mRNA表达均显著降低(均P<0.001),肾小管上皮细胞线粒体结构破坏增加,线粒体密度的明显减低(P<0.001),肾皮质中抗氧化酶SOD、GSH-Px和CAT水平均显著降低(均P<0.001),同时脂质过氧化物MDA显著升高(P<0.001)。与CLP组相比,虽然50 mg/kg NAC预处理组肾损伤评分、肾功能指标Scr、BUN、KIM-1和NGAL水平均有所降低,肾组织中SOD,GSH-Px和CAT的水平均有所升高,但差异无统计学意义。然而,给予100 mg/kg NAC预处理可显著降低CLP引起的肾组织病理损伤(P<0.001),并且均明显降低Scr、BUN、KIM-1和NGAL水平(均P<0.001),显著升高肾组织Sirt3蛋白[(50.20±2.79)vs.(20.00±0.75),P<0.001]和mRNA[(0.57±0.07)vs.(0.41±0.07),P<0.001]表达水平,肾小管上皮细胞线粒体结构更加稳定,线粒体密度明显增加[(0.60±0.05)vs.(0.43±0.06),P<0.001],均显著升高SOD(U/mg)[(67.37±3.79)vs.(21.09±0.89),P<0.001]、GSH-Px(U/mg)[(265.61±9.61)vs.(180.00±3.31),P<0.001]和CAT(U/mg)[(8.58Objective To explore the protective effect and mechanism of the antioxidant N-acetylcysteine(NAC)regulating silent information regulator 3(Sirt3)on acute kidney injury(AKI)in septic mice.Methods Male C57BL/6 mice were randomly(random number)divided into the sham operation group(sham),cecal ligation and perforation group(CLP),CLP+NAC(50 mg/kg)and CLP+NAC(100 mg/kg)groups,with 10 mice in each group.The mice were sacrificed 24 h after CLP,and blood and kidney tissue samples were collected.HE staining was used to evaluate the pathological damage of the kidney tissue of mice in each group.ELISA was used to detect serum creatinine(Scr),urea nitrogen(BUN),kidney injury molecule 1(KIM-1)and neutrophil gelatinase-associated apolipoprotein(NGAL)levels.Immunohistochemistry was used to detect the expression of Sirt3 protein in kidney tissue.RT-qPCR was used to detect the level of Sirt3 mRNA.Mitochondrial damage of renal tubular epithelial cells was observed under transmission electron microscope,and the mitochondrial density was calculated.Meanwhile,the levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and malondialdehyde(MDA)in the renal cortex were also detected.Results Compared with the sham group,in the CLP group,the pathological damage of renal tissue was significantly aggravated(P<0.001),and the levels of renal function indicators(Scr,BUN,KIM-1 and NGAL)were all increased significantly(all P<0.001).The protein and mRNA expression of Sirt3 were all significantly decreased(all P<0.001),the mitochondrial structure damage of renal tubular epithelial cells was increased,and the mitochondrial density was significantly decreased(P<0.001).The levels of antioxidant enzymes(SOD,GSH-Px and CAT)in the renal cortex were all significantly decreased(all P<0.001),while the lipid peroxide MDA was significantly increased(P<0.001).Compared with the CLP group,the renal injury score and renal function indexes(Scr,BUN,KIM-1 and NGAL levels)in the 50 mg/kg NAC pretreatment group were decreased,and the levels of

关 键 词：N-乙酰半胱氨酸 Sirt3 脓毒症 急性肾损伤 小鼠 线粒体损伤 抗氧化酶 氧化应激 

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