自噬和线粒体辅酶Q对急性脓毒症大鼠胰腺外分泌功能的影响
자서화선립체보매Q대급성농독증대서이선외분비공능적영향
Effect of autophagy and mitochondrial coenzyme Q on exocrine function of pancreas in rats with acute sepsis
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2015年 2期 86-91 ,共6页

    陈胜利%黄锦达%曾其毅%贾玉娥%王金华

    진성리%황금체%증기의%가옥아%왕금화

    自噬%线粒体辅酶Q%胰腺外分泌%脓毒症%大鼠 自噬%線粒體輔酶Q%胰腺外分泌%膿毒癥%大鼠
    자서%선립체보매Q%이선외분비%농독증%대서
    Autophagy%Mitochondrial coenzyme Q%Exocrine pancreas%Sepsis%Rat
    目的：探讨自噬对急性脓毒症大鼠胰腺外分泌功能的影响，以及线粒体辅酶Q（Mito Q）是否通过自噬对急性脓毒症大鼠胰腺外分泌功能障碍起保护作用。方法实验1：将30只雄性SD大鼠按随机数字表法分为3组，每组10只。所有大鼠腹腔注射脂多糖（LPS）10 mg/kg，1 h后分别经尾静脉注射自噬特异性抑制剂渥曼青霉素（Wortmannin）2 mg/kg（LPS+ Wortmannin组）、 Mito Q 6.5μmol/kg（LPS+ Mito Q组）或等量生理盐水（LPS组）。观察LPS后12 h内动物存活情况。实验2：将100只雄性SD大鼠按随机数字表法分为10组：对照4 h、6 h、12 h组，LPS 4 h、6 h、12 h组，LPS+ Wortmannin 4 h组，Wortmannin 4 h组，LPS+ Mito Q 6 h组， Mito Q 6 h组，每组10只，制模及给药方法同实验1。在相应时间点留取大鼠血标本，用速率法测定血清淀粉酶含量；取胰腺组织，用酶联免疫吸附试验（ELISA）检测活性氧（ROS）含量，蛋白质免疫印迹试验（Western Blot）检测自噬相关基因LC3蛋白表达，镜下观察胰腺组织病理学改变。结果① LPS+ Wortmannin组大鼠存活时间明显短于LPS组（h：7.50±0.64比11.90±0.13，χ2＝19.847，P＝0.001）；LPS+ Mito Q组大鼠存活时间与LPS组无差异（h：11.60±0.24比11.90±0.13，χ2＝1.055，P＝0.137）。② LPS 6 h组、LPS+ Wortmannin 4 h组、LPS+ Mito Q 6 h组血淀粉酶含量明显高于同时间点对照组（U/L：2881.00±550.12比2099.20±249.57，3672.00±779.24比2081.36±245.18，2975.20±687.03比2099.20±249.57，均P＜0.05），LPS 12 h组血淀粉酶含量明显低于同时间点对照组（U/L：794.00±218.71比2086.80±261.75，P＜0.01）；LPS 6 h和12 h组、LPS+Wortmannin 4 h组、 LPS+ Mito Q 6 h组胰腺组织ROS含量均明显高于同时间点对照组（kU/L：3.18±1.06比1.78±0.37，3.63±1.08比1.85±0.41，3.14±0.98比1.65±0.34，3.17±1.03比1.78±0.37，均P＜0.05）。LPS+Wortmannin 4 h组血淀粉酶和胰腺组织ROS含量均明显高于同时间点LPS组（U/L：3672.00±779.24比2432.20±442.85， kU/L：3.14±0.98比1.87±0.42，均P＜0.05），而LPS+ Mito Q 6 h组血淀粉酶和胰腺组织ROS含量与同时间点LPS组无差异（U/L：2975.20±687.03比2881.00±550.12，kU/L：3.17±1.03比3.18±1.06，均P＞0.05）。光镜下观察，LPS 6 h和12 h组、 LPS+ Wortmannin 4 h组、LPS+ Mito Q 6 h组均可见明显胰腺病理改变；电镜下观察，LPS 6 h后自噬体增多，Mito Q干预后自噬体数量与相应时间点LPS组无差异，而给予Wortmannin后未见自噬体。Western Blot检测显示，LPS 6 h和12 h组、 LPS+ Mito Q 6 h组LC3蛋白表达均明显高于同时间点对照组（A值：0.34±0.02比0.17±0.02，0.37±0.03比0.18±0.04，0.36±0.02比0.17±0.02，均P＜0.05），但LPS 12 h组和LPS+ Mito Q 6 h组LC3蛋白表达与LPS 6 h组比较均无差异（均P＞0.05）。结论自噬对脓毒症大鼠胰腺外分泌功能障碍起保护作用，且自噬能力或自噬体形成速率可能是胰腺是否出现外分泌功能障碍的机制之一；而线粒体靶向抗氧化剂Mito Q对脓毒症大鼠胰腺外分泌功能障碍无保护作用。
    목적：탐토자서대급성농독증대서이선외분비공능적영향，이급선립체보매Q（Mito Q）시부통과자서대급성농독증대서이선외분비공능장애기보호작용。방법실험1：장30지웅성SD대서안수궤수자표법분위3조，매조10지。소유대서복강주사지다당（LPS）10 mg/kg，1 h후분별경미정맥주사자서특이성억제제악만청매소（Wortmannin）2 mg/kg（LPS+ Wortmannin조）、 Mito Q 6.5μmol/kg（LPS+ Mito Q조）혹등량생리염수（LPS조）。관찰LPS후12 h내동물존활정황。실험2：장100지웅성SD대서안수궤수자표법분위10조：대조4 h、6 h、12 h조，LPS 4 h、6 h、12 h조，LPS+ Wortmannin 4 h조，Wortmannin 4 h조，LPS+ Mito Q 6 h조， Mito Q 6 h조，매조10지，제모급급약방법동실험1。재상응시간점류취대서혈표본，용속솔법측정혈청정분매함량；취이선조직，용매련면역흡부시험（ELISA）검측활성양（ROS）함량，단백질면역인적시험（Western Blot）검측자서상관기인LC3단백표체，경하관찰이선조직병이학개변。결과① LPS+ Wortmannin조대서존활시간명현단우LPS조（h：7.50±0.64비11.90±0.13，χ2＝19.847，P＝0.001）；LPS+ Mito Q조대서존활시간여LPS조무차이（h：11.60±0.24비11.90±0.13，χ2＝1.055，P＝0.137）。② LPS 6 h조、LPS+ Wortmannin 4 h조、LPS+ Mito Q 6 h조혈정분매함량명현고우동시간점대조조（U/L：2881.00±550.12비2099.20±249.57，3672.00±779.24비2081.36±245.18，2975.20±687.03비2099.20±249.57，균P＜0.05），LPS 12 h조혈정분매함량명현저우동시간점대조조（U/L：794.00±218.71비2086.80±261.75，P＜0.01）；LPS 6 h화12 h조、LPS+Wortmannin 4 h조、 LPS+ Mito Q 6 h조이선조직ROS함량균명현고우동시간점대조조（kU/L：3.18±1.06비1.78±0.37，3.63±1.08비1.85±0.41，3.14±0.98비1.65±0.34，3.17±1.03비1.78±0.37，균P＜0.05）。LPS+Wortmannin 4 h조혈정분매화이선조직ROS함량균명현고우동시간점LPS조（U/L：3672.00±779.24비2432.20±442.85， kU/L：3.14±0.98비1.87±0.42，균P＜0.05），이LPS+ Mito Q 6 h조혈정분매화이선조직ROS함량여동시간점LPS조무차이（U/L：2975.20±687.03비2881.00±550.12，kU/L：3.17±1.03비3.18±1.06，균P＞0.05）。광경하관찰，LPS 6 h화12 h조、 LPS+ Wortmannin 4 h조、LPS+ Mito Q 6 h조균가견명현이선병리개변；전경하관찰，LPS 6 h후자서체증다，Mito Q간예후자서체수량여상응시간점LPS조무차이，이급여Wortmannin후미견자서체。Western Blot검측현시，LPS 6 h화12 h조、 LPS+ Mito Q 6 h조LC3단백표체균명현고우동시간점대조조（A치：0.34±0.02비0.17±0.02，0.37±0.03비0.18±0.04，0.36±0.02비0.17±0.02，균P＜0.05），단LPS 12 h조화LPS+ Mito Q 6 h조LC3단백표체여LPS 6 h조비교균무차이（균P＞0.05）。결론자서대농독증대서이선외분비공능장애기보호작용，차자서능력혹자서체형성속솔가능시이선시부출현외분비공능장애적궤제지일；이선립체파향항양화제Mito Q대농독증대서이선외분비공능장애무보호작용。
    ObjectiveTo investigate the effects of autophagy on exocrine function of pancreas in rats with acute sepsis, and to determine whether the mitochondrial coenzyme Q (Mito Q) can prevent exocrine dysfunction of pancreas mediated by autophagy.Methods ExperimentⅠ: 30 Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group. All the rats were given lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally, and Wortmannin (2 mg/kg), the specific inhibitor of autophagy (LPS+ Wortmannin group), Mito Q (6.5μmol/kg, LPS+Mito Q group), or the same volume of normal saline (LPS group) was respectively injected via the tail vein 1 hour later.&nbsp;Survival rate was assessed within 12 hours after LPS injection. ExperimentⅡ: another 100 male SD rats were randomly divided into ten groups with 10 rats in each group: namely control 4, 6 and 12 hours groups, LPS 4, 6 and 12 hours groups, and LPS+ Wortmannin 4 hours group, Wortmannin 4 hours group, LPS+ Mito Q 6 hours group, and Mito Q 6 hours group. The protocols of model reproduction and drug administration were the same as in the experimentⅠ. Blood samples were collected at each time point, and the amylase content was determined with the velocity method. The levels of reactive oxygen species (ROS) in the pancreases were measured with enzyme-linked immunosorbent assay (ELISA). The expression of the autophagy-related protein LC3 was determined with Western Blot. The pathological changes in the pancreas were observed with microscopy.Results① The survival time in the LPS+ Wortmannin group was significantly shorter than that in the LPS group (hours: 7.50±0.64 vs. 11.90±0.13,χ2= 19.847,P= 0.001). There was no significant difference in the survival time between LPS+ Mito Q and LPS groups (hours: 11.60±0.24 vs. 11.90±0.13,χ2= 1.055,P= 0.137).② The serum amylase in the LPS 6 hours, LPS+ Wortmannin 4 hours, and LPS+ Mito Q 6 hours groups were significantly higher than those in the control group at the same time points (U/L:2 881.00±550.12 vs. 2 099.20±249.57, 3 672.00±779.24 vs. 2 081.36±245.18, 2 975.20±687.03 vs. 2 099.20± 249.57, allP< 0.05), and were significantly lowered in LPS 12 hours group (U/L: 794.00±218.71 vs. 2 086.80±261.75, P< 0.01). The pancreatic ROS in the LPS 6 hours and 12 hours groups, LPS+ Wortmannin 4 hours group, and LPS+Mito Q 6 hours group were significantly higher than those of the control group at the same time points (kU/L: 3.18±1.06 vs. 1.78±0.37, 3.63±1.08 vs. 1.85±0.41, 3.14±0.98 vs. 1.65±0.34, 3.17±1.03 vs. 1.78±0.37, allP< 0.05). The serum amylase and pancreatic ROS in LPS+ Wortmannin 4 hours group were significantly higher than those of the LPS group at the same time points (U/L: 3 672.00±779.24 vs. 2 432.20±442.85, kU/L: 3.14±0.98 vs. 1.87±0.42, both P< 0.05), but there were no differences in above two parameters between LPS+ Mito Q 6 hours group and LPS group (U/L: 2 975.20±687.03 vs. 2 881.00±550.12, kU/L: 3.17±1.03 vs. 3.18±1.06, bothP> 0.05). Light microscopy showed that obvious pathological changes were found in the pancreas in the LPS 6 hours and 12 hours groups, LPS+Wortmannin 4 hours group, and LPS+ Mito Q 6 hours group. Electron microscopy showed that the number of autophagic vacuoles increased 6 hours after LPS administration. There was no difference at any time point in the number of autophagic vacuoles between LPS+ Mito Q 6 hours group and LPS 6 hours group, and the autophagic vacuoles were not found after Wortmannin intervention. It was demonstrated by Western Blot that the levels of LC3 protein in the LPS 6 hours and 12 hours groups, and LPS+ Mito Q 6 hours group were significantly higher than those of the control group at the same time points (A value: 0.34±0.02 vs. 0.17±0.02, 0.37±0.03 vs. 0.18±0.04, 0.36±0.02 vs. 0.17±0.02, allP< 0.05), but there were no differences between LPS 12 hours group or LPS+ Mito Q 6 hours group and LPS 6 hours group (bothP> 0.05).Conclusions Autophagy prevents exocrine dysfunction of pancreas in septic rats, and the autophagic capacity or autophagosome-formation rate may determine the development of exocrine pancreatic dysfunction. The mitochondria-targeted antioxidant Mito Q does not prevent exocrine dysfunction of pancreas.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문