摘要
研究背景
非致命性创伤(如交通事故引起的机械损伤)一直是医学界广泛关注的重要课题。随着医疗护理水平的提高,由创伤本身所引起的直接损伤危害逐年减少,而其继发的创伤后脏器功能衰竭正日益受到重视。近年来多项临床研究显示,非致命性创伤后的患者在未发生冠脉夹层和直接心肌损伤的情况下,其心肌梗死(MI)和继发性心功能不全的发病率均明显增加,但其机制尚不清楚。
在对小鼠建立创伤性休克模型的研究中我们发现,可导致大鼠死亡的创伤强度不能使小鼠出现全身脏器损伤。小鼠在创伤完全恢复后(创伤7天后)若发生心肌缺血,其损伤程度远重于未经受创伤的动物。既往研究表明,创伤使全身循环中肿瘤坏死因子α(TNF-α)以及其他炎性因子升高,但创伤后炎性因子的升高是一过性的,因此用TNF-α等炎性因子直接加重在创伤后远期出现的心肌缺血性损伤解释不通,提示最大的可能性为:创伤后TNF-α等炎性因子升高诱发了机体的某种继发反应,而这种继发反应可能是导致创伤远期心肌出现缺血性损伤时损伤程度加重的直接原因。但TNF-α等诱发了何种继发性反应因而加重缺血性损伤,目前仍是未解之谜。
脂联素是脂肪细胞分泌的细胞因子。研究表明,血浆脂联素水平与缺血性心肌病发病呈高度负相关,脂联素可通过减轻氧化/硝化应激等机制对心肌缺血/再灌注(MI/R)损伤具有明确的保护作用。基础和临床研究均已证实,TNF-α等炎性因子与脂联素在一系列病理生理活动中具有相互拮抗关系。炎性因子的升高可抑制脂联素分泌,降低血浆脂联素水平。因此我们设想:非致命性创伤所导致的TNF-α等一过性升高所致的继发性脂联素水平下降可能是创伤远期心肌缺血性损伤加重的重要原因。本研究拟对该假设进行验证,并进而阐明非致命创伤后心肌缺血性损伤加重的分子机制,为深入认识创伤后所发生的病理改变、进一步揭示创伤继发的心肌缺血性损伤机制提供实验依据。
研究目的
(1)观察非致命性创伤对MI/R损伤严重程度(梗死面积、心功能和心肌细胞凋亡)的影响。
(2)明确创伤后血浆TNF-α的时相变化及其对血浆脂联素水平的调节关系。
(3)观察脂联素水平变化对心肌氧化/硝化应激及MI/R损伤严重程度的影响,进一步阐明脂联素水平变化在小鼠非致命性创伤后MI/R损伤加重中的作用及其分子机制。
实验方法
(1)小鼠创伤模型:用戊巴比妥钠(40 mg/kg, i.p.)麻醉雄性C57B16/J小鼠或脂联素基因敲除小鼠(Adp-/-小鼠,20 - 25g)。将麻醉完全的小鼠置于Noble-Collip鼓中,以40 rpm的速率共200转诱导全身非致命性创伤模型。假创伤小鼠采用同样的转速和转数,但小鼠用胶带粘于鼓的内壁上,因此避免了创伤。
(2)小鼠MI/R模型:用6-0丝线在小鼠冠脉左前降支中点处打一活结造成心肌缺血,30 min后,将活结打开行再灌注。再灌注3 h后进行心肌细胞凋亡及氧化/硝化应激指标检测,再灌注24 h后检测心梗面积及心功能。假手术组采用同样的方法处理,只是LAD不行结扎。
(3)心室插管检测小鼠心功能:经左侧颈动脉将1.4F Millar-tip导管换能器插入左室腔。用计算机算法和交互式视频图像程序(Po-Ne-Mah Physiology Platform P3 Plus, Gould Instrument Systems, Inc.)得出心率、左室舒张末压(LVEDP)、左室压最大上升速率的正值和负值( +dP/dtmax和-dP/dtmax)。
(4)心梗面积测定:伊文氏蓝/三苯四唑氯盐(TTC)双染色法。
(5)心肌细胞凋亡检测:①末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法;②Caspase-3活性测定。
(6)血浆TNF-α及脂联素水平:采用ELISA检测。
(7)诱导型一氧化氮合酶(iNOS)表达:采用Western Blot检测。
(8)一氧化氮(NO)及其在体代谢产物(NO2和NO3),统称为NOx,采用硝酸还原酶法检测。
(9) ?O2?生成采用荧光素增强的化学发光法和原位二氢乙啶(DHE)染色法检测。
(10)心肌ONOO-水平:采用竞争ELISA法检测及免疫组织化学方法检测。
实验结果
(1)与假创伤+ MI/R组(假创伤7 d后行MI/R)相比,创伤+ MI/R组(创伤7 d后行MI/R)小鼠的心梗面积增加(P < 0.05)、LVEDP进一步增大(P < 0.01)而±dP/dtmax进一步减小(P < 0.05)、心肌细胞凋亡指数(AI)及caspase-3活性进一步增加(P < 0.01)。
(2)创伤后3 h血浆TNF-α达到峰值(P < 0.01 vs. 0时间点组),此后迅速降低,未再出现第二个峰值。创伤后血浆脂联素水平逐渐下降,至创伤后3 d达到谷值(P < 0.01 vs. 0时间点组),之后较快恢复到正常水平。在创伤前给予TNF-α抑制剂etanercep(t创伤前16 h和1 h,8 mg/kg×2)可阻断脂联素水平的时相变化。
(3)用etanercept抑制TNF-α可减轻创伤7 d后MI/R所造成的心肌梗死、心功能下降及心肌细胞凋亡。再灌注前10 min给予小鼠腹腔注射脂联素球状片段(gAd,0.5 mg/kg)或在创伤3 d后给予gAd也可明显抑制MI/R所造成的心肌损伤。
(4)创伤可使MI/R后心肌?O2?生成增加~1.6倍(P < 0.01 vs.假创伤)。在创伤前用etanercept抑制TNF-α、或再灌注前10 min给予gAd,或在创伤3 d后给予gAd均可使MI/R后心肌?O2￣生成显著减少(P < 0.01~0.05 vs.假创伤)。
(5)创伤可使MI/R后心肌iNOS表达增强,NOx的生成增加(~1.5倍,P < 0.05 vs.假创伤)。在创伤前用etanercept抑制TNF-α、创伤3 d后或创伤7 d后再灌注前10 min给小鼠腹腔注射gAd均可使MI/R后心肌NOx生成显著减少(P < 0.01~0.05 vs.假创伤)。
(6)创伤+ MI/R组小鼠心肌过氧化亚硝酸盐免疫组化信号增强,ONOO-生成显著增加(~ 5倍,P < 0.05 vs.假创伤+ MI/R组)。在创伤前用etanercept抑制TNF-α、创伤3 d后或创伤7 d后再灌注前10 min给小鼠腹腔注射gAd均可使MI/R后心肌过氧化亚硝酸盐免疫组化信号减弱及ONOO-生成明显减少(P < 0.01~0.05 vs.假创伤)。
(7)在再灌注前10 min给予Mn(III)TBAP(?O2￣清除剂,10 mg/kg)或EUK134(ONOO-清除剂,5 mg/kg)可缩小创伤+ MI/R后的心梗面积、改善心功能,同时抑制心肌细胞凋亡及caspase-3激活。
(8) Etanercept仅可轻度缩小创伤+ MI/R诱导的Adp-/-小鼠心梗面积(12.4%,P = 0.046 vs. vehicle),而补充外源性gAd可缩小创伤+ MI/R诱导的心梗面积达37.8%(P < 0.01 vs. vehicle)。但etanercept不能改善创伤+ MI/R组Adp-/-小鼠的±dp/dtmax降低(P >0.05 vs. vehicle),只有补充外源性gAd对其具有改善作用(P < 0.01 vs. vehicle)。
结论
(1)非致命性创伤可使小鼠MI/R损伤加重,创伤可能是心肌缺血性损伤加重的“隐性杀手”。
(2)创伤后血浆TNF-α升高可继发性抑制脂联素水平。抑制TNF-α或直接补充脂联素(gAd)可减轻创伤对MI/R损伤的增敏作用,提示TNF-α诱导的脂联素减少很有可能参与创伤后MI/R损伤加重。
(3)创伤后TNF-α升高可通过降低脂联素水平导致MI/R后心肌氧化/硝化应激增加,进而增加心肌对I/R损伤的敏感性。
(4)补充外源性脂联素(而非给予etanercept)可明显改善Adp-/-小鼠创伤后MI/R诱导的心肌梗死和心功能下降,证实TNF-α诱导的脂联素水平下降是创伤后MI/R损伤加重的重要因素。
Background
Non-lethal trauma (e.g. traffic accidents-induced mechanical trauma) has always been one of the most concerned issues in the medical world. With the development of medical care provisions, direct hazards rendered by trauma per se have been substantially reduced, whereas secondary organ failure induced by trauma is increasingly recognised. Recent multi-hospital evidence showed that the prevalence of myocardial infarction (MI) and secondary heart failure was significantly increased in post-traumatic patients even in the absence of coronary artery dissection and direct myocardial injury. However, the mechanisms remain elusive.
In a recent study employing a mouse traumatic shock model we found that the intensity of traumatic injury leathal to rats failed to induce multi-organ injury in mice. Mice fully recovered from trauma (7 days after trauma) sustained far more severe myocardial injury when subjected to myocardial ischemia. Studies have shown that trauma can induce the elevation of plasma tumor necrosis factorα(TNF-α) and other inflammatory cytokines. However, the elevation of these inflammatory cytokines are only transient and are unlikely to directly enhance myocardial ischemic injury long after trauma. This prompted us to speculate that a secondary change might be induced by these cytokines, which can serve as a more direct injury-inducing factor that enhances post-traumatic myocardial ischemic injury. However, it still remains an enigma as to the validity and nature of this secondary change induced by inflammatory cytokines that increases myocardial ischemic injury.
Adiponectin is a cytokine secreted by adipocytes and is present in high abundance in the circulation. Studies showed that plasma adiponectin is conversely related to the morbidity of ischemic cardiac injury, and adiponectin can protect myocardium against ischemia/reperfusion injury (MI/R) by attenuating oxidative/nitrative stress. Basic and clinical studied both confirmed that reciprocal regulations exist between adiponectin and TNF-α(and possibly several other inflammatory cytokines). The elevation of inflammatory cytokines can inhibit the secretion of adiponectin and decrease its plasma level. Therefore, we hypothesized that the decrease in the plasma adiponectin level secondary to the temporary elevation of TNF-αand other inflammatory cytokines induced by non-lethal trauma might be an important reason that sensitizes post-traumatic myocardium to ischemic injury. In the present study, we aimed to test this hypothesis and further to elucidate the molecular mechanisms responsible for the aggravation of post-traumatic myocardial ischemia injury. Our study might shed light to trauma-induced pathological changes and further provide experimental basis for the mechanisms underlying trauma-induced secondary ischemic cardiac injury.
Objectives
(1) To investigate the effects of non-lethal trauma on the the serevity of MI/R injury (myocardial infarction, cardiac function and cardiomyocyte apoptosis).
(2) To ascertain the time-course changes of plasma TNF-αafter trauma and to verify the possible existance of a modulatory effect between TNF-αand adiponectin.
(3) To observe the effects of the changes in plasma adiponectin level on myocardial oxidative/nitrative stress and the serevity of MI/R injury. Meanwhile, to further define the role of the changes in plasma adiponectin level in the aggravation of post-traumatic MI/R injury and to elucidate the underlying mechanisms.
Methods
(1) Mice traumatic injury model: Male C57B16/J mice or adiponectin knockout mice (Adp-/-, 20 - 25g) were fully anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and placed in a Noble-Collip drum apparatus. Whole-body non-lethal trauma was induced by a total of 200 revolutions at a rate of 40 rpm. Sham traumatized mice were subjected to the same revolution but the animals were taped on the inner wall of drum, thus avoiding traumatic injury.
(2) Mice MI/R model: A slipknot was placed at the halfway point of the left anterior descending artery (LAD) with 6-0 silk sutures and ischemia was induced by ligation of the slipknot. Reperfusion was performed by releasing the slipknot 30 min after the ligation. Cardiomyocyte apoptosis and oxidative/nitrative stress were determined at 3 h, and myocardial infarction and cardiac function determined at 24 h, after the conclusion of the reperfusion. Sham operation was performed in a same manner except that the LAD will be left unligated.
(3) Determination of cardiac function by intraventricular catheterization: A 1.4F Millar-tip catheter transducer was inserted to the left ventricular cavity through the left carotid artery. Heart rate, left ventricular end diastolic pressure (LVEDP), maximal positive and negative values of the instantaneous first derivative of LVP (+dP/dtmax and -dP/dtmax) were obtained using computer algorithms and an interactive videographics program (Po-Ne-Mah Physiology Platform P3 Plus, Gould Instrument Systems, Inc.).
(4) Determination of myocardial infarction: Myocardial infarct size was determined by Evans blue/2,3,5-triphenyl tetrazolium chloride (TTC) double staining.
(5) Determination of myocardial apoptosis: Myocardial apoptosis was determined by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining (TUNEL) and caspase-3 activity assay.
(6) Plasma TNF-αand adiponectin assays: Plasma TNF-αand adiponectin levels were determined by enzyme-linked-immunosorbent-assay (ELISA).
(7) Expression of inducible nitric oxide synthase (iNOS): Expression of iNOS was determined by Western blotting.
(8) Contents of nitric oxide (NO) and its in vivo metabolites NO2 and NO3 (collectively termed as NOx) were determined using nitrate reductase method.
(9) ?O2? production was determined by lucigenin-enhanced luminescence and in situ dihydroethidium (DHE) staining.
(10) Myocardial ONOO- formation was determined by competitive ELISA and immunohistochemistry.
Results
(1) Mice in the trauma + MI/R group (MI/R induced 7 d after trauma) showed increased myocardial infarct size as compared to those in sham truam + MI/R group (MI/R induced 7 d after sham trauma) (P < 0.05). Meanwhile, mice in the former group had increased LVEDP (P < 0.01) and decreased±dP/dtmax (P < 0.05). Cardiomyocyte apoptosis index (AI) and caspase-3 activity were also enhanced in mice subjected to trauma + MI/R (P < 0.01 vs. sham truam + MI/R).
(2) Plasma TNF-αlevel peaked 3 h after trauma (P < 0.01 vs. time point 0) and swiftly declined thereafter, without showing a second peak. Plasma adiponectin level gradually decreased and reached the lowest level 3 d after trauma (P < 0.01 vs. time point 0) and was quickly restored to basal level. The time-course change of adiponectin was abolished by the administration of TNF-αinhibitor etanercept (16 h and 1 h before trauma, 8 mg/kg×2).
(3) Blockade of TNF-αby etanercept alleviated myocardial infarction, cardiac dysfunction and cardiomyocyte apoptosis induced by MI/R 7 d post-truama. Globular domain of adiponectin (gAd, 0.5 mg/kg) intraperitoneally administered at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma markedly alleviated MI/R injury.
(4) Myocardial ?O2? production was significantly enchanced in mice subjected to MI/R injury after trauma (P < 0.01 vs. sham trauma). Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial ?O2? production (P < 0.01~0.05 vs. sham trauma).
(5) Myocardial iNOS expression was enhanced and NOx production increased (~ 1.5 fold, P < 0.05 vs. sham trauma) in mice subjected to MI/R injury after trauma. Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial iNOS expression and NOx production (P < 0.01~0.05 vs. sham trauma).
(6) Myocardial histochemistry signal of nitrotyrosine was enhanced and ONOO- formation increased (~ 5 fold, P < 0.05 vs. sham trauma) in mice subjected to MI/R injury after trauma. Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial histochemistry signal of nitrotyrosine and ONOO- formation (P < 0.01~0.05 vs. sham trauma).
(7) Intraperitoneal administration of Mn(III)TBAP (?O2? scavenger, 10 mg/kg) or EUK134(ONOO- scavenger, 5 mg/kg)attenuated myocardial infarction, cardiac dysfunction and cardiomyocyte apoptosis and caspase-3 activity induced by MI/R at the 7th day post-truama.
(8) The replenishment of exogenous gAd reduced myocardial infarct size by 37.8% in Adp-/- mice subjected to MI/R injury 7 days after trauma (P < 0.01 vs. vehicle), whereas etanercept only mildly reduced the infarct size by (12.4%, P = 0.046 vs. vehicle). Meanwhile, gAd significantly alleviate the decline in±dp/dtmax in such an animal model (P < 0.01 vs. vehicle), but etanercept failed to alleviate such a decline (P > 0.05 vs. vehicle).
Conclusions
(1) Non-lethal trauma can aggravate MI/R injury in mice and may serve as a‘silent killer’that renders myocardium more susceptible to ischemic injury.
(2) Elevation in plasma TNF-αcan induce a secondary decline in adiponectin concentraion. Blockade of TNF-αor direct replenishment of exogenous gAd can reduce the susceptibility of myocardium to post-traumatic MI/R injury, indicating that the decrease in plasma adiponectin level induced by elevated TNF-αmight play a role in the aggravation of MI/R injury after trauma.
(3) Decreased plasma adiponectin induced by elevated TNF-αcan enhance myocardial oxidative/nitrative stress after MI/R and, therefore, increase the susceptibility of myocardium to I/R injury.
(4) Replenishment of exogenous gAd, but not the administration of etanercept, can attenuate myocardial infarction and cardiac dysfunction induced by post-traumatic MI/R injury in Adp-/- mice, thus implicating the decrease in plasma adiponectin level secondary to elevated TNF-αas an important factor in the aggravation of post-traumatic MI/R injury.
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