摘要
目的探讨参附注射液对脓毒症大鼠心肌线粒体分裂融合失衡和自噬的影响。方法将46只健康雄性SD大鼠按随机数字表法分为对照组(n=6)、模型组(n=20)、参附注射液组(n=20)。采用腹腔注射脂多糖(LPS)20 mg/kg的方法复制脓毒症大鼠模型;制模后6 h参附注射液组大鼠腹腔注射参附注射液;模型组和对照给予10 mL/kg生理盐水干预。于基础状态和制模后6 h、12 h行心脏二维斑点追踪显像(2D-STI)评估心功能;制模后12 h取大鼠尾静脉血检测血清心肌钙蛋白I(cTnI);每组取6只大鼠处死留取心脏组织,其余继续饲养观察48 h累积生存率。取大鼠心脏组织,电镜下观察心肌线粒体形态;采用酶联免疫吸附试验(ELISA)检测心肌线粒体三磷酸腺苷(ATP)含量;采用2’,7’-二氯荧光素二乙酸酯(DCFH-DA)荧光探针检测心肌细胞活性氧(ROS)含量;采用蛋白质免疫印迹试验(Western blotting)检测心肌动力相关蛋白1(Drp1)、视神经融合蛋白1(Opa1)、线粒体融合蛋白(Mfn1,Mfn2)以及自噬蛋白轻链3(LC3)的蛋白表达水平。结果与对照组比较,模型组心肌线粒体数目明显减少,体积变小且伴有明显破损;血清cTnI含量明显升高(μg/L:1.51±0.14比0.51±0.06),制模后6 h左室收缩期环向应变(GCS)和纵向应变(GLS)明显下降(GCS:13.80±2.45比18.10±2.58,GLS:20.30±3.12比26.50±2.40,均P<0.05),制模后12 h进一步降低(GCS:9.31±2.12比18.90±3.11,GLS:16.30±2.84比24.40±3.11,均P<0.05),心肌ATP含量亦明显降低(μmol/g:8.42±1.67比14.50±2.23,P<0.05),心肌细胞内ROS含量和心肌线粒体分裂蛋白Drp1的蛋白表达水平及自噬蛋白LC3-Ⅱ/Ⅰ比值均明显增加〔ROS:(24.30±1.42)%比(11.78±1.17)%,Drp1/GAPDH:0.51±0.04比0.22±0.05,LC3-Ⅱ/Ⅰ比值:0.80±0.08比0.38±0.07,均P<0.05〕,融合蛋白Opa1和Mfn1、Mfn2的蛋白表达水平明显减少(Opa1/GAPDH:0.27±0.03比0.49±0.05,Mfn1/GAPDH:0.36±0.05比0.54±0.04,Mfn2/GAPDH:0.28±0.05比0.45±0.04,均P<0.05);与模型组比较,参附注射液能够改善心肌线粒体功能,增加心肌线粒体中ATP含量(μmol/g:10.97±2.13比8.42±1.67,P<0.05)和心功能指标GCS及GLS水平(GCS:11.70±2.25比9.31±2.12,GLS:18.80±2.17比16.30±2.84,均P<0.05),拮抗LPS诱导的cTnI升高(μg/L:1.30±0.22比1.51±0.14,P<0.05),减少ROS的产生〔(22.20±0.86)%比(24.30±1.42)%,P<0.05〕,降低分裂蛋白Drp1的蛋白表达水平和LC3-Ⅱ/Ⅰ比值(Drp1/GAPDH:0.43±0.05比0.51±0.04,LC3-Ⅱ/Ⅰ比值:0.68±0.07比0.80±0.08,均P<0.05),提高融合蛋白Opa1和Mfn1、Mfn2的蛋白表达水平(Opa1/GAPDH:0.34±0.02比0.27±0.03,Mfn1/GAPDH:0.41±0.04比0.36±0.05,Mfn2/GAPDH:0.34±0.02比0.28±0.05,均P<0.05),并能提高大鼠48 h累积生存率(Log-Rank检验:χ^(2)=4.094,P=0.043)。结论参附注射液可通过调控脓毒症大鼠心肌线粒体分裂融合失衡,减少线粒体自噬,改善脓毒症大鼠心功能障碍。
Objective To investigate the effects of Shenfu injection(SFI)on myocardial mitochondrial fission/fusion imbalance and autophagy in septic rats.Methods Forty-six healthy male Sprague-Dawley(SD)rats were randomly divided into three groups:control(n=6),model(n=20)and SFI(n=20)groups;the septic rat models were replicated by intra-peritoneal injection of lipopolysaccharide(LPS,20 mg/kg);Six hours after modeling,the rats were intra-peritoneally injected with SFI in SFI group and with normal saline 10 mL/kg in the control group and model group for intervention.Cardiac function was evaluated by Two Dimensional Speckle Tracking Imaging(2D-SFI)at baseline,6 hours and 12 hours after modeling;12 hours after modeling,the blood was taken from the caudal vein for detection of serum cardiac troponin I(cTnI)levels in various groups;6 rats from each group were sacrificed to retain heart tissue,the rest were continuously fed and the cumulative survival rates were observed within 48 hours.Rats'heart tissues were taken to observe the myocardial mitochondrial morphology under electron microscope;the content of adenosine triphosphate(ATP)in myocardial mitochondria was determined by enzyme linked immunosorbent assay(ELISA);the myocyte reactive oxygen species(ROS)content was detected by using 2',7'-dichlorodihydrofluorescin diacetate(DCFH-DA)as fluorescence probe;The expressions of myocardial mitochondria danamin-related protein 1(Drp1),optic atrophy 1(Opa1),mitofusin 1(Mfn1),mitofusin 2(Mfn2)as well as autophagy protein light chain 3(LC3)were detected by Western blotting.Results Compared with the control group,the number of myocardial mitochondria in model group was significantly reduced and their sizes were smaller with obvious damage;the level of serum cTnI was significantly increased(μg/L:1.51±0.14 vs.0.51±0.06),left ventricle global circumferential strain(GCS)and global longitudinal strain(GLS)were decreased remarkably 6 hours after modeling(GCS:13.80±2.45 vs.18.10±2.58,GLS:20.30±3.12 vs.26.50±2.44,both P<0.05)and further decline occurred 12 hours after modeling(GCS:9.31±2.12 vs.18.90±3.11,GLS:16.30±2.84 vs.24.40±3.11,both P<0.05);the myocardial ATP content was obviously decreased(μmol/g:8.42±1.67 vs.14.50±2.23,P<0.05),the ROS content in myocytes and the protein expression of Drp1 and the ratio of LC3-Ⅱ/Ⅰin model group were increased significantly[ROS:(24.30±1.42)%vs.(11.78±1.17)%,Drp1/GAPDH:0.51±0.04 vs.0.22±0.05,LC3-Ⅱ/Ⅰratio:0.80±0.08 vs.0.38±0.07,both P<0.05];the fusion protein expressions of Opa1,Mfn1 and Mfn2 in model group were reduced(Opa1/GAPDH:0.27±0.03 vs.0.49±0.05,Mfn1/GAPDH:0.36±0.05 vs.0.54±0.04,Mfn2/GAPDH:0.28±0.05 vs.0.45±0.04,all P<0.05);compared with the model group,SFI improved myocardial mitochondrial function and the ATP production(μmol/g:10.97±2.13 vs.8.42±1.67,P<0.05)as well as cardiac parameters GCS and GLS(GCS:11.70±2.25 vs.9.31±2.12,GLS:18.80±2.17 vs.16.30±2.84,both P<0.05);SFI antagonized LPS-induced increase in cTnI(μg/L:1.30±0.22 vs.1.51±0.14,P<0.05)and reduced ROS production[(22.20±0.86)%vs.(24.30±1.42)%,P<0.05]and the protein expression of Drp1 and the ratio of LC3-Ⅱ/Ⅰ(Drp1/GAPDH:0.43±0.05 vs.0.51±0.04,LC3-Ⅱ/Ⅰratio:0.68±0.07 vs.0.80±0.08,both P<0.05);SFI increased the protein expression levels of fusion protein Opa1,Mfn1 and Mfn2(Opa1/GAPDH:0.34±0.02 vs.0.27±0.03,Mfn1/GAPDH:0.41±0.04 vs.0.36±0.05,Mfn2/GAPDH:0.34±0.02 vs.0.28±0.05,all P<0.05)and elevate the cumulative survival rates within 48 hours(Log-Rank:χ^(2)=4.094,P=0.043).Conclusion SFI may improve the cardiac dysfunction of septic rats by regulating myocardial mitochondrial fission/fusion imbalance and decreasing mitochondrial autophagy.
作者
陈瑞娟
何安霞
赵熙璇
芮庆林
Chen Ruijuan;He Anxia;Zhao Xixuan;Rui Qinglin(Department of Emergency,the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine/Jiangsu Provincial Hospital of Traditional Chinese Medicine,Nanjing 210029,Jiangsu,China;Department of Functional Examination,the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine/Jiangsu Provincial Hospital of Traditional Chinese Medicine,Nanjing 210029,Jiangsu,China)
出处
《中国中西医结合急救杂志》
CAS
CSCD
北大核心
2022年第6期657-662,共6页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
江苏省“十二五”中医药重点学科建设项目(js1303)
关键词
脓毒症心功能障碍
线粒体分裂融合
线粒体自噬
Sepsis induced myocardial dysfunction
Mitochondrial fission and fusion
Mitochondrial autophagy
作者简介
通信作者:芮庆林,Email:rql1964@njucm.edu.cn
