研究目的

第一部分：对感染性休克患者宏观循环复苏后的能量代谢障碍进行分类，并与包括心脏在内的器官功能障碍关系进行研究。

第二部分：研究脓毒症时大鼠心肌线粒体分裂融合的变化情况，并探讨相关分子机制。

第三部分：研究调节心肌线粒体分裂融合失衡后心肌能量代谢以及功能变化。

研究方法

第一部分：前瞻性收集2016年10月—2017年1月重症医学科连续收治的完成宏观循环复苏并行机械通气的感染性休克患者，行10 min-OCT检查，抽取动静脉血气计算Ratio。根据10 min-OCT与Ratio水平将患者分为三组：A组（10 min-OCT>66 mmHg，并且Ratio≤1.23）；B组（10 min-OCT≤66 mmHg）；C组（10 min-OCT >66 mmHg，并且Ratio>1.23）。记录三组患者一般资料，完成宏观循环复苏时、宏观循环复苏24h、48h后MAP、Lac、ScvO₂、累计液体平衡等血流动力学参数资料以及强心药物使用情况、KIDGO评分、SOFA评分等器官功能障碍评价指标。采用SPSS 19.0软件进行统计学分析。

第二部分：32只SD大鼠随机分为4组（每组8只大鼠）包括：假手术组（SAM组）、脓毒症2h组（CLP_2h组）、脓毒症10h组（CLP_10h组）、脓毒症18h组（CLP_18h组）。SAM组于术后18h，CLP_2h组、CLP_10h组、CLP_18h组分别于2h、10h、18h进行开胸取心脏标本，RT-PCR检测Drp1、Mfn2、Opa1 mRNA水平，Western Blot检测Drp1蛋白水平，电镜观察线粒体形态，评估心肌线粒体分裂融合状态。

第三部分，32只SD大鼠随机分为4组（每组8只大鼠）包括：假手术组（SAM组）、假手术加Mdivi-1组（SAM+Mdi组）、脓毒症组（CLP组）、脓毒症加Mdivi-1组（CLP+Mdi组）。四组大鼠手术18h后再次麻醉行心脏超声评估心脏功能后，开胸取心脏标本。电镜观察心脏线粒体形态，评估心肌线粒体分裂融合状态。检测心肌线粒体呼吸链复合物 IV活性以及ATP水平。

研究结果

第一部分：共49例患者入选研究，A、B、C组分别为19、21、9例。强心药物使用率B组与C组在各时点均高于A组（P<0.05），C组强心药物使用率高于B组，但差异无统计学意义（P>0.05）。AKI发生率在T0时点三组差异无统计学意义（P=0.114），T24 h、T48 h时点C组AKI发生率高于A组（P=0.038），AKI加重率C组高于A组与B组（P<0.05）。SOFA评分C组各时点均高于A组（P<0.05），C组ΔSOFA评分在T24 h时点高于A组与B组（P<0.05）。在Kaplan-Meier生存分析中，C组死亡率高于A组与B组（P<0.05）。Ratio预测复苏后24 h SOFA评分增加≥1的ROC曲线AUC面积为0.766。

第二部分：CLP_2h组心肌线粒体形态与SAM组比较无明显变化，CLP_10h组、CLP_18h组长管状线粒体明显减少，圆球状线粒体明显增加，出现分裂融合失衡，形态构成比与SAM组比较，差异具有统计学意义（P<0.05）。CLP_10h组、CLP_18h组分裂蛋白Drp1 mRNA水平高于SAM组（P<0.05）；CLP_2h组、CLP_10h组、CLP_18h组融合蛋白Mfn2与OPA1 mRNA水平高于SAM组（P<0.05）。CLP_2h组Drp1蛋白水平与SAM组相比，差异无统计学意义（P=0.543）；CLP_10h组、CLP_18h组高于SAM组，差异具有统计学意义（P<0.05）。

第三部分：CLP组线粒体形态构成比与SAM组比较差异具有统计学意义（P<0.05），CLP+Mdi组线粒体形态构成比与CLP组比较差异具有统计学意义（P<0.05），CLP组复合体IV活性与ATP水平低于SAM组（P<0.05），CLP+Mdi组复合体IV活性与ATP水平高于CLP组（P<0.05）。心脏超声结果显示，CLP组LVEF、SV低SAM组（P<0.05），CLP+Mdi组LVEF、SV高于CLP组（P<0.05）。

结论

第一部分：1.10 min-OCT联合Ratio应用可以对感染性休克患者复苏后组织细胞能量代谢障碍进行分类。2.感染性休克患者宏观与微循环灌注恢复后部分患者仍存在无氧代谢，并且提示器官功能障碍进行性加重，预后不良。3.感染性休克患者合并细胞氧利用障碍，心、肾较呼吸功能障碍表现更重，提示等线粒体含量丰富器官功能障碍与组织细胞氧利用异常相关。4.Ratio对于感染性休克患者复苏后器官功能障碍进展具有较好预测价值。

第二部分：1.持续存在的过度分裂是CLP脓毒症大鼠心肌线粒体分裂融合失衡的特征性改变。2. Drp1蛋白表达增加与脓毒症心肌线粒体持续过度分裂相关。

第三部分：1.持续存在的线粒体过度分裂是与氧化呼吸功能异常相关，是脓毒症心肌抑制发生、发展的重要机制。2.避免线粒体过度分裂是脓毒症心肌抑制干预治疗的新靶点。3. Mdivi-1可以纠正CLP脓毒症大鼠心肌线粒体过度分裂，保护心脏功能。

Objective

(1) To investigate the corrrelation between metabolic energy dysfunction and organs dysfunction in septic shock patients

(2) To investigate the charatertiscs of myocardial mitochondrial fission and fusion, and related molecular mechanisms in sepsis rat

(3) To investigate the effect of the regulation of mitochondrial fission and fusion imbalance on myocardial energy metabolism and function

Methods

（1）The septic shock patients were prospectively observed, who with mechanical ventilation from October 2016 to January 2017. All the patients received transcutaneous oxygen challenge test （10 min-OCT  inspired 1.0 FiO2 for 10-min）, simultaneous arterial and central venous blood gas were obtained to calculate Ratio. patients were divided into three groups based on level of 10 min-OCT and Ratio: Group A （10 min-OCT>66mmHg and Ratio≤1.23）；Group B（10 min-OCT≤66mmHg）Group C （10 min-OCT >66mmHg and Ratio>1.23）. All the patients’ general information,T0, T24, T48 haemodynamics（MAP Lac ScvO₂）and organs dysfunction data（KIDGO score SOFA score）were collected.

    （2）SD rats were divided into four groups randomly（8 in each group）: Group SAM；Group CLP_2h；Group CLP_10h；GroupCLP_18h. RT-PCR technology were applied to assess the level of Drp1、Mfn2、Opa1 mRNA，protein level of Drp1 were assessed by Western Blot technology. Myocardial mitochondrial fission and fusion were assessed by transmission electron microscope.

（3）SD rats were divided into four groups randomly（8 in each group）: Group SAM；Group SAM+Mdi；Group CLP；Group CLP+Mdi. Cardiac function of rats in each group were assessed by echocardiography 18h after operations. Myocardial mitochondrial fission and fusion were assessed by transmission electron microscope，and the level of complex IV and ATP in myocardium were measured.

Result

     （1）49 patients were included in the study, The utilization rate of inotropes in Group B and Group C were significantly higher in Group A（P<0.05）, but no significant difference between Group B and Group C（P>0.05）. There was no significant difference in the incidence of AKI among three groups at T0（P=0.114）, but Group C had a significantly higher incidence of AKI than Group A at both T24 and T48（P=0.038）. Group C had a significantly higher aggravating AKI rate than both Group A and Group B（P=0.015）. Group C had a significantly higher SOFA score than Group A（P<0.05）. Group C had a significantly higher Mortality than Group A（P=0.01）. The ratio predicted in 24hours after resuscitation the area of which the SOFA score under the ROC curves was 0.766.

    （2）There were no difference in myocardial mitochondrial morphology between Group SAM and Group CLP_2h, The Group CLP_10h and Group CLP_18h had myocardial mitochondrial morphology imbalance compared with Group SAM. Group SAM had a significantly lower Drp1 mRNA and protein level than CLP10h and Group CLP18h（P<0.05）; Group SAM had a significantly lower Mfn2 and OPA1 mRNA level in Group CLP2h, Group CLP10h and Group CLP18h（P<0.05）.

（3）The Group CLP had significantly myocardial mitochondrial morphology difference compared with Group SAM, and Group CLP+Mdi had significantly better myocardial mitochondrial morphology than Group CLP. Group CLP had significantly higher complex IV and ATP level than Group SAM（P<0.05）, and CLP+Mdi Group had significantly higher complex IV and ATP level than Group CLP（P<0.05）Group CLP had significantly lower LVEF、SV than Group SAM, but Group CLP+Mdi had a significantly higher LVEF、SV than Group CLP.

Conclusion

Part one：（1）Apply of 10 min-OCT and Ratio is helpful to determine oxygen utilization abnormability in septic shock patients.（2）If anaerobic metabolism still persists exit after circulation recovery, the septic shock patients will have a worse organs dysfunction and prognosis （3）Organs dysfunction in organs with abundant mitochondria like heart and kidney are likely to be in progress when septic shock patients combine tissue oxygen utilization abnormal.（4）Ratio showned a good value for prediction of the progression of organ dysfunction in septic shock patients.

Part two：（1）The CLP sepsis rats myocardium had a excessive fission mitochondria , which is related to the sepsis-induced myocardial dysfunction.（2）Drp1 protein expression was associated with excessively mitochondrial fission in septic myocardial.

Part three：（1）Excessive fission mitochondria is associated with oxidation respiratory abnormal，and excessive fission mitochondria palys an important role in the sepsis-induced myocardial dysfunction.（2）Reduction of excessive fission mitochondria might be a new therapeutic target for sepsis-induced myocardial dysfunction.（3）Mdivi-1 intraperitoneal could correct excessively mitochondrial fission and improve the cardiac function in septic shock.