右美托咪定对严重烫伤大鼠心肌损伤的影响及其机制的研究
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摘要
右美托咪定(dexmedetomidine)是一种高选择性α2-肾上腺素能受体激动剂,具有中枢性抗交感和抗焦虑作用,能产生近似自然睡眠的镇静作用；同时具有一定的镇痛、利尿作用,对呼吸无明显抑制。自1999年美国FDA批准其用于ICU机械通气患者最初24h的镇静以来,已经在临床手术麻醉、围术期及重症监护室镇静镇痛等领域得到广泛的应用。国外有学者报道,右美托咪定可应用于烧伤患者的手术与换药,但其在烧伤领域的应用还不够广泛。右美托咪定对烧伤后机体病理生理过程的影响及相关机制目前也尚未完全阐明。
心脏是机体的循环动力器官。有研究表明,严重烧伤后10min,心肌细胞骨架受损,30min至1h心肌血流量开始明显减少,心功能降低,肌钙蛋白漏出增多,病理形态出现明显变化,心肌细胞凋亡增加。这种即早出现的心肌损害不仅可引起心功能不全,还可能成为严重烧伤休克和全身组织器官缺血缺氧损害的重要启动因素。因此,针对烧伤后心肌损害机制的研究和如何防治严重烧伤后心肌损害一直是国内外学者的研究热点。右美托咪定的主要药理作用是降低交感神经系统的兴奋性,削弱应激反应,减少应激激素的分泌,并预防围术期心肌缺血的发生。国外有研究表明,右美托咪定预处理可减轻心肌缺血再灌注损伤；国内也有学者研究发现,右美托咪定预处理可以减轻缺血再灌注损伤大鼠心肌细胞凋亡。另外,还有大量基础研究表明,右美托咪定可以通过减轻机体炎症反应,产生器官保护作用。右美托咪定对烧伤后心肌损伤的影响及机制如何,目前尚不清楚。由于严重烧伤后心肌损害的机制包括：机体过度的应激反应、心肌细胞缺血缺氧、缺血再灌注损伤、失控性炎症反应、氧利用及能量代谢障碍等,并且心肌细胞出现不同程度的凋亡。因此,我们推测右美托咪定对烧伤后心肌细胞可发挥类似的保护及抗凋亡作用。
目前,已有大量研究证实心肌细胞凋亡是严重烧伤早期心功能损害的原因之一,但具体机制尚未完全阐明。传统的细胞凋亡途径主要包括死亡受体和线粒体途径,两者均通过诱导caspase级联反应激活caspase-3,导致细胞凋亡。近年的研究显示,内质网应激(endoplasmic reticulum stress, ERS)也与细胞凋亡密切相关。在ERS早期,细胞通过一系列蛋白表达调控----未折叠蛋白反应(unfolded protein response, UPR)以恢复内质网稳态；但如果ERS持续而严重,则激活凋亡通路而诱发细胞凋亡。有研究表明,减轻ERS的药物或者措施可以减少缺血再灌注损伤引起的心肌损伤及凋亡。如果右美托咪定能减轻烧伤后心肌损伤,其机制是否与影响心肌细胞凋亡有关?是否有涉及ERS介导的凋亡通路?这些均有待进一步研究探讨。
ERS是真核细胞内自我保护机制的一种反应,对维持细胞内环境稳态具有重要作用,其过程及介导凋亡的通路十分复杂。葡萄糖调节蛋白78(glucose regulated protein78, GRP78)是内质网分子伴侣,是UPR反应的标志分子之一,也是调控ERS的关键分子之一。没有未折叠或错误折叠蛋白质时,GRP78能结合并抑制内质网应激的特殊感受器,一旦出现未折叠蛋白质,GRP78就从感受器上移位到未折叠蛋白上,使感受器得以激活。蛋白激酶R样内质网激酶(PKR-Like ER kinase, PERK)是内质网应激最重要的感受器。ERS时,与PERK结合在一起的GRP78由于位移到大量的未折叠和错误折叠蛋白质上,而使得PERK蛋白暴露,进而发生二聚化和自身磷酸化。磷酸化的PERK又使得内质网外侧的eIF2a发生磷酸化修饰,从而使得蛋白质合成启动过程暂停,缓解了蛋白质的进一步堆积。如果ERS持续存在,磷酸化的PERK则通过ATF4激活下游的相关凋亡通路,诱导细胞凋亡。因此,GRP78与P-PERK表达的上调标志着ERS的发生,并且可以反映ERS激活的程度。C/EBP同源蛋白(C/EBP homologous protein, CHOP)是ERS特异的转录因子,是ERS发挥介导细胞凋亡功能的最主要蛋白。正常情况下,CHOP表达十分低下,当ERS持续剧烈存在时,IRE1、PERK、ATF6的活化均对CHOP产生诱导,促使其激活,并大量表达,从而诱导细胞凋亡,其中PERK通路与CHOP的活化关系最为密切,因为PERK-eIF2a-ATF4是CHOP蛋白表达所必须的复合物。Caspase-12是含半胱氨酸的天冬氨酸蛋白水解酶家族成员之一,位于内质网膜胞质一侧。Caspase-12只有被激活后才发挥介导凋亡的作用。目前相关研究表明,有三种途径可以激活Caspase-12:IRE1途径,m-钙蛋白酶途径和Caspase-7途径,而这三种途径均与ERS有关。烧伤后心肌细胞ERS的具体过程尚未完全阐明,右美托咪定对上述ERS标志性蛋白的影响如何也有待研究探讨。
综上所述,本研究拟以SD大鼠为研究对象,并建立大鼠30%体表面积烧伤模型,应用组织病理学、免疫组织化学、蛋白质电泳等分子生物学手段,比较不同剂量右美托咪定对严重烫伤大鼠心肌损伤及炎症状态的影响,观察严重烫伤大鼠整个休克期ERS相关的标志性蛋白及心肌细胞凋亡水平的变化及趋势,并进一步探讨右美托咪定对严重烫伤大鼠心肌细胞凋亡的影响及其机制,为将来深入阐明右美托咪定对严重烧伤后心肌损伤的影响机制以及右美托咪定在烧伤患者围术期的广泛应用提供重要的理论依据。
第1章右美托咪定对严重烫伤大鼠血清cTnI、CK-MB、TNF-α以及心肌组织TNF-α的影响
目的探讨不同剂量右美托咪定对烫伤大鼠心肌损伤及炎症反应的影响。
方法以SD大鼠为研究对象,将其分为3组(n=32)：烫伤组(A组)、烫伤+右美托咪定10μg/kg组(B组)、烫伤+右美托咪定30μg/kg组(C组)。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液,分别用相应剂量右美托咪定腹腔注射处理,于伤后3、6、12、24h,检测大鼠心肌组织肿瘤坏死因子α(TNF-α)以及血清肌钙蛋白I (cTnI)、肌酸激酶同工酶(CK-MB)、TNF-α水平变化。数据资料以x±s表示,采用SPSS17.0统计软件分析。不同处理组在各时间点测得的相应指标水平比较采用两因素析因设计资料的方差分析,组间比较采用LSD法。P<0.05为差异有统计学意义。
结果A、B、C三组大鼠烫伤后血清cTnI、CK-MB、TNF-α以及心肌组织TNF-α水平逐渐升高,伤后12h达高峰,伤后24h出现下降趋势(P<0.05)。与A组比较,B组、C组大鼠血清cTnI水平下降,且C组下降较B组更显著(P均<0.05)；伤后6、12、24h B组大鼠血清CK-MB水平较A组下降(P均<0.05),伤后3、6、12、24h C组大鼠血清CK-MB水平较A组下降(P均<0.05),伤后3、12、24h C组大鼠血清CK-MB水平显著低于B组(P均<0.05)；伤后12h B组大鼠血清TNF-α水平较A组下降(P=0.019),伤后3、6、12、24h C组大鼠血清TNF-α水平较A组下降(P均<0.05),伤后6、12、24h C组大鼠血清TNF-α水平显著低于B组(P均<0.05)；伤后6、12、24h B组大鼠心肌组织TNF-α水平较A组下降(P<0.05),伤后6、12、24h C组大鼠心肌组织TNF-α水平较A组下降(P均<0.05),伤后12h C组大鼠心肌组织TNF-α水平显著低于B组(P=0.00)
结论严重烫伤后早期大鼠出现心肌损伤,于烫伤后12h血清cTnI、 CK-MB、TNF-α以及心肌组织TNF-α水平达到峰值。右美托咪定可减轻大鼠烫伤后心肌损伤并降低血浆及心脏局部TNF-α水平,且腹腔注射301μg/kg所产生的心肌保护效应较腹腔注射10μg/kg显著。
第2章严重烫伤大鼠早期心肌细胞凋亡以及心肌组织GRP78、P-PERK、 CHOP表达的变化及意义
目的观察大鼠严重烫伤后心肌细胞内质网应激相关蛋白表达的变化,探讨其在心肌细胞凋亡过程中的意义及可能机制。
方法健康成年雄性SD大鼠64只,将其分为8组(n=8)：正常对照组、烫伤后1、3、6、12、24、48、72h组。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液。对照组大鼠于假伤后即刻,其余7组大鼠于相应时间点获取左室心肌组织,采用蛋白印记以及细胞凋亡的原位标记(TUNEL)法,观察大鼠心肌组织GRP78、P-PERK、CHOP表达以及心肌细胞凋亡程度的变化。计量资料以x±s表示,采用SPSS17.0统计软件分析。各指标数据采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果各组大鼠心肌GRP78水平表达有差异(F=133.240,P=0.000),烫伤后大鼠心肌GRP78水平较伤前升高,差异有统计学意义(P<0.01),与伤后1、3、72h相比,伤后6-48h大鼠心肌GRP78进一步升高(P<0.01),从均值上看伤后48h大鼠心肌GRP78表达水平达到峰值,但伤后6-48h之间大鼠心肌GRP78表达水平差异无统计学意义(P>0.05)。各组大鼠心肌P-PERK水平表达有差异(F=154.733,P=0.000),烫伤后3h开始大鼠心肌P-PERK水平较伤前逐渐升高,并于伤后12h-24h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h恢复至伤前水平(P>0.05)。各组大鼠心肌CHOP水平表达有差异(F=223.871,P=0.000),烫伤后6h开始大鼠心肌CHOP水平较伤前逐渐升高,差异有统计学意义(P<0.01),并于伤后12h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h恢复至伤前水平(P>0.05)。各组大鼠心肌细胞凋亡水平有差异(F=322.860,P=0.000),烫伤后1h开始大鼠心肌细胞凋亡水平较伤前逐渐升高,并于伤后12h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h仍高于伤前水平(P<0.05)。
结论大鼠严重烫伤早期心肌细胞存在明显的凋亡,凋亡最高峰在伤后12h。严重烫伤后1h大鼠心肌细胞ERS相关蛋白表达开始逐渐增加,整个烫伤早期阶段心肌细胞内质网应激反应一直持续,并参与了部分的心肌细胞凋亡过程,且CHOP通路所介导的细胞凋亡可能于伤后6h开始,伤后12h达到高峰。
第3章右美托咪定对严重烫伤大鼠心肌细胞凋亡及内质网应激介导凋亡通路相关蛋白的影响
目的右美托咪定对烧伤后心肌细胞凋亡以及内质网应激介导凋亡通路相关蛋白的影响。
方法健康成年雄性SD大鼠24只,将其分为3组(n=8)：对照组(C组)、烫伤组(B组)、烫伤+右美托咪定30μg/kg组(D组)。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液,分别用相应剂量右美托咪定腹腔注射处理,于伤后12h获取心肌组织,通过光学显微镜、电子显微镜以及免疫组织化学、脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)和蛋白印迹等方法观察右美托咪定对严重烫伤大鼠心肌组织病理形态与凋亡以及GRP78、P-PERK、 CHOP、Caspase-12这些内质网应激相关蛋白表达的影响。计量资料以x±s表示,采用SPSS17.0统计软件分析。各指标数据采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果与C组比较,B组和D组大鼠心肌组织GRP78、P-PERK、CHOP、 Caspase-12表达水平以及凋亡指数均升高,差异均有统计学意义(P<0.05)；与B组比较,D组大鼠心肌组织GRP78、P-PERK、CHOP、Caspase-12表达水平以及凋亡指数均减少,差异有统计学意义(P<0.05),光镜下见C组大鼠心肌细胞结构正常,肌丝排列整齐,细胞界限清晰；B组大鼠心肌细胞水肿、间质充血、心肌纤维变性,横纹模糊不清甚至消失,出现广泛的肌纤维波浪状排列；D组大鼠心肌细胞少量水肿,心肌纤维排列基本正常,病变程度较B组有所减轻；电镜下见C组大鼠心肌细胞结构正常,肌丝、肌节排列整齐,线粒体未见肿胀,细胞核正常,核膜完整,间质未见水肿及炎症细胞侵润,毛细血管内皮细胞无肿胀,管腔内未见淤血及血栓形成；B组大鼠心肌细胞肌丝、肌节排列紊乱,结构模糊不清,肌浆网扩张明显,部分肌丝灶性溶解,部分节段细胞膜破坏,可见部分线粒体漏出及固缩,间质毛细血管内皮细胞肿胀；D组大鼠心肌细胞肌丝、肌节排列基本整齐,线粒体轻度肿胀,仅个别线粒体漏出,肌浆网轻度扩张,细胞膜基本完整无断裂,少量间质水肿,毛细血管内皮细胞大致正常。
结论右美托咪定可以减轻严重烫伤大鼠心肌损伤及凋亡,并抑制心肌细胞内质网应激程度以及内质网应激介导的凋亡通路相关蛋白表达,其减少细胞凋亡的机制可能与抑制CHOP和caspase-12这两条凋亡通路有关。
论文主要结论
1、严重烫伤后早期大鼠出现心肌损伤,血浆cTnI及CK-MB水平升高,于烫伤后12h达到峰值。烫伤后大鼠全身及心肌局部出现炎症反应,血浆及心脏局部TNF-α水平升高,烫伤后12h达到峰值。右美托咪定可减轻大鼠烫伤后心肌损伤并降低血浆及心脏局部TNF-α水平,且腹腔注射30μg/kg所产生的心肌保护效应较腹腔注射10μg/kg显著。
2、大鼠严重烫伤早期心肌细胞存在明显的凋亡,凋亡最高峰在伤后12h。严重烫伤后1h大鼠心肌细胞ERS相关蛋白表达开始逐渐增加,整个烫伤早期阶段心肌细胞内质网应激反应一直持续,并参与了部分的心肌细胞凋亡过程,且CHOP通路所介导的细胞凋亡可能于伤后6h开始,伤后12h达到高峰。
3、右美托咪定可以减轻严重烫伤大鼠心肌损伤及凋亡,并抑制心肌细胞内质网应激程度以及内质网应激介导的凋亡通路相关蛋白表达,其减少细胞凋亡的机制可能与抑制CHOP和caspase-12这两条凋亡通路有关。
Dexmedetomidine is one kind of high selectivity α2-adrenergic receptor agonist,which has the effect of central antisympathety and antianxiety.It can produce the sedation like natural sleep. Meanwhile, it has a certain extent of abirritation and diuretic action. It does not inhibit the respiration obviously.In1999,dexmedetomidine was initially approved by FDA for the sedation of patient in ICU who was received mechanical ventilation. Now it is been used widely in the field of clinical anesthesia and the sedation or analgesia of perioperative period and in ICU. Foreign scholars have reported that dexmedetomidine can be used in surgery and treatment of burn patients, but its application in the field of burns is not wide enough. The influence and the related mechanism of dexmedetomidine to the body and pathophysiological process after burns is also has not been fully elucidated.
The heart is the organ that in charge of body's circulation. Research has shown that,the myocardial cytoskeleton had been damaged in10min after severe burns;the myocardial blood flow began to significantly reduce on30min to1h after severe burns,at the same time,cardiac function was reduced,the leaking of troponin was increased,the pathological form of the myocardium changed significantly,the apoptosis of myocardial cell also increased.The myocardial damaging in this early stage can not only cause cardiac insufficiency,but also can become the important factor to star the serious burn shock and body tissues and organs ischemia anoxic damage.Therefore, the study of myocardial injury mechanism after burns and how to prevent and treat the myocardial damage after severe burns has been the research hotspot of scholars both at home and abroad. The main pharmacological effect of dexmedetomidine is to reduce the excitability of sympathetic nervous system, weaken the stress reaction, reduce the secretion of stress hormones, and prevent the occurrence of perioperative myocardial ischemia. Overseas studies have shown that the dexmedetomidine pretreatment can reduce myocardial ischemia-reperfusion injury; domestic scholars study has also found that dexmedetomidine pretreatment can reduce the cell apoptosis of rat myocardial in ischemia reperfusion injury In addition, there are a lot of fundamental research shows that dexmedetomidine can produce organ protection by reduce the body's inflammatory response.The influence and mechanism of dexmedetomidine on myocardial injury after burn is still not clear now.Due to the mechanism of myocardial injury after severe burns includes: ischemia-reperfusion injury, uncontrolled inflammatory response, disorders of oxygen uptake and energy metabolic,and myocardium cells appeared different degree of apoptosis We speculate that dexmedetomidine can exert similar protective effect and anti-apoptosis effect to myocardial injury after severe burns.
At present,a lot of research have confirmed that apoptosis of myocardial cells is one of the reasons of damaging on cardiac function in the early stage after severe burn,but the concrete mechanism has been incompletely recognized.The traditional approaches of cell apoptosis mainly includes death receptors and mitochondrial pathway.These two pathways activate caspase-3by inducing the caspase cascade reaction,then lead to cell apoptosis. Recent studies show that endoplasmic reticulum stress is also closely related with apoptosis. In the early stages of endoplasmic reticulum stress, it through a series of protein expression regulation of cells(unfolded protein response,UPR) to get well the endoplasmic reticulum steady;but if the endoplasmic reticulum stress is sustained and serious, it will active the apoptotic pathways and then induce apoptosis. Studies have shown that the drugs or measures which can alleviate endoplasmic reticulum stress will decrease the myocardial injury and apoptosis caused by ischemia reperfusion injury. If dexmedetomidine can alleviate myocardial injury after burns, is its mechanism related to the influence on myocardial cell apoptosis? If there were any involving the endoplasmic reticulum stress mediated apoptosis pathway? All these needs further research.
Endoplasmic reticulum stress is one kind of reaction about self protection mechanism of a eukaryotic cell.It plays an important role in maintaining cellular homeostasis.It has very complex process and pathway for mediated apoptosis. Glucose regulated protein78(GRP78) is the molecular chaperone in endoplasmic reticulum,and it also is the molecules sign of UPR reaction. And it is one of the key molecules of regulating the endoplasmic reticulum stress. When there are no unfold or misfolded proteins, GRP78can combine and suppress the special receptors of endoplasmic reticulum stress.Once the unfolded proteins appear,GRP78will shift from the special receptors to the unfolded proteins to activate the receptors. PERK is the most important receptor of endoplasmic reticulum stress.When ERS,GRP78which Combine with PERK will move into unfolded and misfolded proteins, and makes the PERK protein exposure. Thus PERK occurs two polymerization and its phosphorylation.Phosphorylation of PERK wiil make the eIF2a that outside of the endoplasmic reticulum added as phosphorylated modification, so the starting process of protein synthesis will be suspended,then eased the further protein accumulation. If ERS persistence for a long time, phosphorylation of the PERK will activate the downstream apoptotic pathway by ATF4.Then induced cell death.Therefore,the raising expression of GRP78and P-PERK not only mark the ERS,but also reflect the extent to which ERS activated. CHOP is the Specific transcription factor in ERS,it is the most important protein which has the function of mediated apoptosis in ERS. Under normal circumstances,the expression of CHOP is very low.When ERS is sustained and severe,the activation of IRE-1,PERK,ATF6will all induce the production of CHOP, prompt it to activate,and expresses abundantly, induced cell death consequently. The relationship between activation of CHOP and PERK pathway is most closely,because the complex of PERK-eIF2a-ATF4are necessary to the expression of CHOP protein. Caspase-12is one of the members of the family that contain cysteine aspartic acid proteolytic enzyme, located in the endoplasmic reticulum retinal cytoplasmic side. Caspase-12wiil play to the role of the mediated for apoptosis only after it be activated.At present, related studies have shown that there are three ways can be activated caspase-12:IRE1pathway,m-calpain activator pathway,caspase-7pathway,and this three ways are all related to the ERS.The concreteness process of ERS in cardiomyocytes after severe burn has not yet been fully elucidated, how dexmedetomidine will affect the ERS iconic protein that above-mentioned is still needed to research.
In conclusion, this study intended to SD rats as the research object,and establish the model rats with30%body surface area burn,using molecular biology method like histopathology, immunohistochemistry, protein electrophoresis,to compare the influence of different dose dexmedetomidine for myocardial injury and the inflammatory state in the rat suffer severe burns,and observe the variation and trend of the trademark protein related to ERS and the level of myocardial apoptosis during the whole shock stage of rat suffer severe burns,and further discuss the effect and its mechanism of dexmedetomidine to the myocardial apoptosis of rat suffer severe burns. To provide important theoretical basis for further clarify of the mechanism and effects about dexmedetomidine acting on myocardial injury after severe burns,and the widespread use of dexmedetomidine of the burn patients in the perioperative period.
Chapter1Effects of dexmedetomidine on plasma levels of cTnI、CK-MB、 TNF-a and myocardial expression of TNF-a after severe scald injury in rats
Objective To investigate the effects of different does of dexmedetomidine on severe scald injury-induced myocardial damage and inflammation in rats.
Methods Ninety-six male SD rats weighing220～280g were used in this study.Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). The animals with thermal injury involving30%of TBS were randomly divided into3 groups(n=32each):group A thermal injury;group B received intraperitoneal dexmedetomidine10μg/kg immediately after thermal injury; group C received intraperitoneal dexmedetomidine30μg/kg immediately after thermal injury.The false injury group and group A received equal volume of normal saline IP instead of dexmedetomidine.The plasma and myocardial tissue samples in each group were harvested at3,6,12,24postburn hours(PBH) for the determination of the contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue. Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The expression of each target of different groups were analyzed by two-way ANOVA, and multiple comparisons tests were performed by LSD.Using Pearson analysis the correlation between the various indicators. A probability value of P<0.05was considered to be statistically significant.
Results The contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue in each group rising after thermal injury,and they peaked at12PBH and decline in the trend at24PBH (P<0.01). The contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue in group B and group C were significantly lower than those in group A (P<0.01),and all the index in group C were more lower than group B significantly (P<0.01)
Conclusion Myocardial injury appear early after severe burns in rats,the peak of the injury was in about12h after severe burns. Dexmedetomidine can alleviate the myocardial injury after severe scald in rats,and reduce the level of TNF-a in plasma and in myocardial tissue. Cardioprotection provided by intraperitoneal dexmedetomidine30μg/kg was more remarkable than10μg/kg.
Chapter2Transformation of cardiomyocytes apoptosis and endoplasmic reticulum stress associated protein(GRP78、P-PERK、CHOP) in cardiomyocytes after severe scald in rat and its possible meaning
Objective To investigate the transformation of endoplasmic reticulum stress associated protein in cardiomyocytes in rat after severe scald.To Discuss the possible meaning and mechanism of endoplasmic reticulum stress during myocardium cell apoptosis in rats after severe scald.
Methods Sixty-four male SD rats weighing220～280g were used in this study.The animals were randomly divided into8group (n=8each):Control group,1PBH(post burn hour) group,3PBH group,6PBH group,12PBH group,24PBH group,48PBH group. Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). Control group were exposed to37℃water for12s to preparation of false injury model.The heart tissues were harvested immediately after false injury in control group,and were harvested at the corresponding time point in each scald group.Myocardium apoptosis was detected with TUNEL assay.The expressions of glucose regulated protein78(GRP78), phosphorylated proteins kinase RNA-like ER kinase(P-PERK),C/EBP-homologous protein(CHOP) in different pathways of ERS were analysed with Westen blot. Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment.Completely random single factor analysis of variance was used to analyse the expression of GRP78,CHOP,P-PERK and cell apoptosis rate at different time points, the comparisons between groups were performed by LSD or Dunnett's T3. A probability value of P<0.05was considered to be statistically significant.
Results The expression levels of myocardial GRP78in the rats were differences between the groups (F=133.240, P=0.000).The expression of GRP78in burn group were significantly higher than those in control group (P<0.01). Compared with1,3,72h after injury, the expression of myocardial GRP78rised further at6-48h (P<0.01). The expression of myocardial GRP78reached the peak at48h after injury when look from the average,but the difference between the expression of myocardial GRP78at6～48h after injury were no statistical significance (P>0.05).The expression levels of myocardial CHOP in the rats were differences between the groups(F=223.871, P=0.000).The expression of CHOP was significantly higher than those in control group from6h after injury (P<0.01). The expression of myocardial CHOP reached the peak at12h after injury (P<0.01),then fall back.lt recovery to pre-injury level at72h after injury (P>0.05).The expression levels of myocardial P-PERK in the rats were differences between the groups (F=154.773, P=0.000).The expression of P-PERK was significantly higher than those in control group from6h after injury (P<0.01). The expression of myocardial P-PERK reached the peak at12h to24h after injury (P<0.01),then fall back.lt recovery to pre-injury level at72h after injury (P>0.05).The apoptosis index of myocardial cell in the rats were differences between the groups (F=322.860, P=0.000).The apoptosis index was significantly higher than those in control group from1h after injury (P<0.01). The apoptosis index reached the peak at12h after injury (P<0.01),then fall back. At72h after injury, it was still higher than pre-injury level (P<0.05)
Conclusion Part of myocardial cell apoptosis in rats after severe scald were mediated by ERS.The apoptosis mediated by ERS arised from6h after severer scald,and reached the peak at12h after injury.
Chapter3Role and mechanism of apoptosis pathway mediated by endoplasmic reticulum stress in attenuation of myocardial injury by dexmedetomidine in rats with severe scald
Objective To investigate the effects of dexmedetomidine on myocardial apoptosis in severe scald rats and its correlative mechanism.
Methods Twenty-four male SD rats weighing220～280g were used in this study.The animals were randomly divided into3group (n=8each):group C false njury; group B thermal injury;group D received intraperitoneal dexmedetomidine30μg/kg immediately after thermal injury.Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). Group C were exposed to37℃water for12s to preparation of false injury model. The false group C and group B received equal volume of normal saline IP instead of dexmedetomidine. The heart tissues were harvested at12h after injury.The morphological change in the myocardial tissue was observed with transmission electronic microscope(TEM) and optical microscope.Myocardium apoptosis was detected with TUNEL assay.The expressions of glucose regulated protein78(GRP78), phosphorylated proteins kinase RNA-like ER kinase(P-PERK),C/EBP-homologous protein(CHOP) in different pathways of ERS were analysed with Westen blot. The expression of caspase-12was analysed with immunohistochemical method.Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment.Completely random single factor analysis of variance was used to analyse the expression of GRP78,CHOP,P-PERK,caspase-12and cell apoptosis rate at different group, the comparisons between groups were performed by LSD or Dunnett's T3. A probability value of P<0.05was considered to be statistically significant.
Results Compared with group C, The expression of GRP78, CHOP, P-PERK, caspase-12and cell apoptosis rate in group B and group D were significantly higher (P<0.05). Compared with group B, The expression of GRP78, CHOP, P-PERK, caspase-12and cell apoptosis rate in group D were significantly lower (P<0.05).Under the light microscope, the myocardial cell structure was normal, myofilament was in alignment, the cell boundaries were clear in group C; in group B,myocardial cell was edema and interstitial congestion, myocardial fibers were degeneration, transverse striation blurred or even disappear and in a wide range of muscle fiber wavy;in group D, myocardial cell was in a small amount of edema, myocardial fibers arranged was normal, the lesion degree was lighter than group B. Under TEM, in group C,myocardial cell structure was normal, myofilament and sarcomere were in alignmen,there was no swelling of mitochondria, the nuclei was normal, nuclear membrane was integrity, interstitial edema and inflammatory cells invasion were no seen, there were no swelling of capillary endothelial cells,and no blood or thrombosis in the lumen;in group B, myofilament and sarcomere were disorganized, the structures of cells become obscure, sarcoplasmic reticulum expansion, part of the myofilament focal dissolved, part of the segmental cell membrane damage, some of the mitochondrial leakage and pyknosis, and interstitial capillary endothelial cells were swelling;in group D, myofilament and sarcomere were in almost alignmen,there were mild swelling of mitochondria and only individual of mitochondrial leak out, there were mild expansion of sarcoplasmic reticulum, the cell membrane almost complete and without fracture, interstitial edema were in a small amount, and the capillary endothelial cells is roughly normal.
Conclusion The myocardial damage and apoptosis in the rats with severe scald can be alleviated by dexmedetomidine.The mechanisms may be associated with decreasing the endoplasmic reticulum stress of the cardiomyocytes and inhibiting the pathways of apoptosis related with endoplasmic reticulum stress.
The main conclusion of the research
1. Myocardial injury appear early after severe burns in rats,the peak of the injury was in about12h after severe burns. Dexmedetomidine can alleviate the myocardial injury after severe scald in rats,and reduce the level of TNF-a in plasma and in myocardial tissue*Cardioprotection provided by intraperitoneal dexmedetomidine30μg/kg was more remarkable than10μg/kg.
2. Part of myocardial cell apoptosis in rats after severe scald were mediated by ERS.The apoptosis mediated by ERS arised from6h after severe scald,and reached the peak at12h after injury.
3. The myocardial damage and apoptosis in the rats with severe scald can be alleviated by dexmedetomidine.The mechanisms may be associated with decreasing the endoplasmic reticulum stress of the cardiomyocytes and inhibiting the pathways of apoptosis related with endoplasmic reticulum stress.
心脏是机体的循环动力器官。有研究表明,严重烧伤后10min,心肌细胞骨架受损,30min至1h心肌血流量开始明显减少,心功能降低,肌钙蛋白漏出增多,病理形态出现明显变化,心肌细胞凋亡增加。这种即早出现的心肌损害不仅可引起心功能不全,还可能成为严重烧伤休克和全身组织器官缺血缺氧损害的重要启动因素。因此,针对烧伤后心肌损害机制的研究和如何防治严重烧伤后心肌损害一直是国内外学者的研究热点。右美托咪定的主要药理作用是降低交感神经系统的兴奋性,削弱应激反应,减少应激激素的分泌,并预防围术期心肌缺血的发生。国外有研究表明,右美托咪定预处理可减轻心肌缺血再灌注损伤；国内也有学者研究发现,右美托咪定预处理可以减轻缺血再灌注损伤大鼠心肌细胞凋亡。另外,还有大量基础研究表明,右美托咪定可以通过减轻机体炎症反应,产生器官保护作用。右美托咪定对烧伤后心肌损伤的影响及机制如何,目前尚不清楚。由于严重烧伤后心肌损害的机制包括：机体过度的应激反应、心肌细胞缺血缺氧、缺血再灌注损伤、失控性炎症反应、氧利用及能量代谢障碍等,并且心肌细胞出现不同程度的凋亡。因此,我们推测右美托咪定对烧伤后心肌细胞可发挥类似的保护及抗凋亡作用。
目前,已有大量研究证实心肌细胞凋亡是严重烧伤早期心功能损害的原因之一,但具体机制尚未完全阐明。传统的细胞凋亡途径主要包括死亡受体和线粒体途径,两者均通过诱导caspase级联反应激活caspase-3,导致细胞凋亡。近年的研究显示,内质网应激(endoplasmic reticulum stress, ERS)也与细胞凋亡密切相关。在ERS早期,细胞通过一系列蛋白表达调控----未折叠蛋白反应(unfolded protein response, UPR)以恢复内质网稳态；但如果ERS持续而严重,则激活凋亡通路而诱发细胞凋亡。有研究表明,减轻ERS的药物或者措施可以减少缺血再灌注损伤引起的心肌损伤及凋亡。如果右美托咪定能减轻烧伤后心肌损伤,其机制是否与影响心肌细胞凋亡有关?是否有涉及ERS介导的凋亡通路?这些均有待进一步研究探讨。
ERS是真核细胞内自我保护机制的一种反应,对维持细胞内环境稳态具有重要作用,其过程及介导凋亡的通路十分复杂。葡萄糖调节蛋白78(glucose regulated protein78, GRP78)是内质网分子伴侣,是UPR反应的标志分子之一,也是调控ERS的关键分子之一。没有未折叠或错误折叠蛋白质时,GRP78能结合并抑制内质网应激的特殊感受器,一旦出现未折叠蛋白质,GRP78就从感受器上移位到未折叠蛋白上,使感受器得以激活。蛋白激酶R样内质网激酶(PKR-Like ER kinase, PERK)是内质网应激最重要的感受器。ERS时,与PERK结合在一起的GRP78由于位移到大量的未折叠和错误折叠蛋白质上,而使得PERK蛋白暴露,进而发生二聚化和自身磷酸化。磷酸化的PERK又使得内质网外侧的eIF2a发生磷酸化修饰,从而使得蛋白质合成启动过程暂停,缓解了蛋白质的进一步堆积。如果ERS持续存在,磷酸化的PERK则通过ATF4激活下游的相关凋亡通路,诱导细胞凋亡。因此,GRP78与P-PERK表达的上调标志着ERS的发生,并且可以反映ERS激活的程度。C/EBP同源蛋白(C/EBP homologous protein, CHOP)是ERS特异的转录因子,是ERS发挥介导细胞凋亡功能的最主要蛋白。正常情况下,CHOP表达十分低下,当ERS持续剧烈存在时,IRE1、PERK、ATF6的活化均对CHOP产生诱导,促使其激活,并大量表达,从而诱导细胞凋亡,其中PERK通路与CHOP的活化关系最为密切,因为PERK-eIF2a-ATF4是CHOP蛋白表达所必须的复合物。Caspase-12是含半胱氨酸的天冬氨酸蛋白水解酶家族成员之一,位于内质网膜胞质一侧。Caspase-12只有被激活后才发挥介导凋亡的作用。目前相关研究表明,有三种途径可以激活Caspase-12:IRE1途径,m-钙蛋白酶途径和Caspase-7途径,而这三种途径均与ERS有关。烧伤后心肌细胞ERS的具体过程尚未完全阐明,右美托咪定对上述ERS标志性蛋白的影响如何也有待研究探讨。
综上所述,本研究拟以SD大鼠为研究对象,并建立大鼠30%体表面积烧伤模型,应用组织病理学、免疫组织化学、蛋白质电泳等分子生物学手段,比较不同剂量右美托咪定对严重烫伤大鼠心肌损伤及炎症状态的影响,观察严重烫伤大鼠整个休克期ERS相关的标志性蛋白及心肌细胞凋亡水平的变化及趋势,并进一步探讨右美托咪定对严重烫伤大鼠心肌细胞凋亡的影响及其机制,为将来深入阐明右美托咪定对严重烧伤后心肌损伤的影响机制以及右美托咪定在烧伤患者围术期的广泛应用提供重要的理论依据。
第1章右美托咪定对严重烫伤大鼠血清cTnI、CK-MB、TNF-α以及心肌组织TNF-α的影响
目的探讨不同剂量右美托咪定对烫伤大鼠心肌损伤及炎症反应的影响。
方法以SD大鼠为研究对象,将其分为3组(n=32)：烫伤组(A组)、烫伤+右美托咪定10μg/kg组(B组)、烫伤+右美托咪定30μg/kg组(C组)。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液,分别用相应剂量右美托咪定腹腔注射处理,于伤后3、6、12、24h,检测大鼠心肌组织肿瘤坏死因子α(TNF-α)以及血清肌钙蛋白I (cTnI)、肌酸激酶同工酶(CK-MB)、TNF-α水平变化。数据资料以x±s表示,采用SPSS17.0统计软件分析。不同处理组在各时间点测得的相应指标水平比较采用两因素析因设计资料的方差分析,组间比较采用LSD法。P<0.05为差异有统计学意义。
结果A、B、C三组大鼠烫伤后血清cTnI、CK-MB、TNF-α以及心肌组织TNF-α水平逐渐升高,伤后12h达高峰,伤后24h出现下降趋势(P<0.05)。与A组比较,B组、C组大鼠血清cTnI水平下降,且C组下降较B组更显著(P均<0.05)；伤后6、12、24h B组大鼠血清CK-MB水平较A组下降(P均<0.05),伤后3、6、12、24h C组大鼠血清CK-MB水平较A组下降(P均<0.05),伤后3、12、24h C组大鼠血清CK-MB水平显著低于B组(P均<0.05)；伤后12h B组大鼠血清TNF-α水平较A组下降(P=0.019),伤后3、6、12、24h C组大鼠血清TNF-α水平较A组下降(P均<0.05),伤后6、12、24h C组大鼠血清TNF-α水平显著低于B组(P均<0.05)；伤后6、12、24h B组大鼠心肌组织TNF-α水平较A组下降(P<0.05),伤后6、12、24h C组大鼠心肌组织TNF-α水平较A组下降(P均<0.05),伤后12h C组大鼠心肌组织TNF-α水平显著低于B组(P=0.00)
结论严重烫伤后早期大鼠出现心肌损伤,于烫伤后12h血清cTnI、 CK-MB、TNF-α以及心肌组织TNF-α水平达到峰值。右美托咪定可减轻大鼠烫伤后心肌损伤并降低血浆及心脏局部TNF-α水平,且腹腔注射301μg/kg所产生的心肌保护效应较腹腔注射10μg/kg显著。
第2章严重烫伤大鼠早期心肌细胞凋亡以及心肌组织GRP78、P-PERK、 CHOP表达的变化及意义
目的观察大鼠严重烫伤后心肌细胞内质网应激相关蛋白表达的变化,探讨其在心肌细胞凋亡过程中的意义及可能机制。
方法健康成年雄性SD大鼠64只,将其分为8组(n=8)：正常对照组、烫伤后1、3、6、12、24、48、72h组。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液。对照组大鼠于假伤后即刻,其余7组大鼠于相应时间点获取左室心肌组织,采用蛋白印记以及细胞凋亡的原位标记(TUNEL)法,观察大鼠心肌组织GRP78、P-PERK、CHOP表达以及心肌细胞凋亡程度的变化。计量资料以x±s表示,采用SPSS17.0统计软件分析。各指标数据采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果各组大鼠心肌GRP78水平表达有差异(F=133.240,P=0.000),烫伤后大鼠心肌GRP78水平较伤前升高,差异有统计学意义(P<0.01),与伤后1、3、72h相比,伤后6-48h大鼠心肌GRP78进一步升高(P<0.01),从均值上看伤后48h大鼠心肌GRP78表达水平达到峰值,但伤后6-48h之间大鼠心肌GRP78表达水平差异无统计学意义(P>0.05)。各组大鼠心肌P-PERK水平表达有差异(F=154.733,P=0.000),烫伤后3h开始大鼠心肌P-PERK水平较伤前逐渐升高,并于伤后12h-24h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h恢复至伤前水平(P>0.05)。各组大鼠心肌CHOP水平表达有差异(F=223.871,P=0.000),烫伤后6h开始大鼠心肌CHOP水平较伤前逐渐升高,差异有统计学意义(P<0.01),并于伤后12h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h恢复至伤前水平(P>0.05)。各组大鼠心肌细胞凋亡水平有差异(F=322.860,P=0.000),烫伤后1h开始大鼠心肌细胞凋亡水平较伤前逐渐升高,并于伤后12h达到峰值水平,差异有统计学意义(P<0.01),而后逐渐回落,伤后72h仍高于伤前水平(P<0.05)。
结论大鼠严重烫伤早期心肌细胞存在明显的凋亡,凋亡最高峰在伤后12h。严重烫伤后1h大鼠心肌细胞ERS相关蛋白表达开始逐渐增加,整个烫伤早期阶段心肌细胞内质网应激反应一直持续,并参与了部分的心肌细胞凋亡过程,且CHOP通路所介导的细胞凋亡可能于伤后6h开始,伤后12h达到高峰。
第3章右美托咪定对严重烫伤大鼠心肌细胞凋亡及内质网应激介导凋亡通路相关蛋白的影响
目的右美托咪定对烧伤后心肌细胞凋亡以及内质网应激介导凋亡通路相关蛋白的影响。
方法健康成年雄性SD大鼠24只,将其分为3组(n=8)：对照组(C组)、烫伤组(B组)、烫伤+右美托咪定30μg/kg组(D组)。建立大鼠30%体表面积烧伤模型后,按Parkland公式补液,分别用相应剂量右美托咪定腹腔注射处理,于伤后12h获取心肌组织,通过光学显微镜、电子显微镜以及免疫组织化学、脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)和蛋白印迹等方法观察右美托咪定对严重烫伤大鼠心肌组织病理形态与凋亡以及GRP78、P-PERK、 CHOP、Caspase-12这些内质网应激相关蛋白表达的影响。计量资料以x±s表示,采用SPSS17.0统计软件分析。各指标数据采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果与C组比较,B组和D组大鼠心肌组织GRP78、P-PERK、CHOP、 Caspase-12表达水平以及凋亡指数均升高,差异均有统计学意义(P<0.05)；与B组比较,D组大鼠心肌组织GRP78、P-PERK、CHOP、Caspase-12表达水平以及凋亡指数均减少,差异有统计学意义(P<0.05),光镜下见C组大鼠心肌细胞结构正常,肌丝排列整齐,细胞界限清晰；B组大鼠心肌细胞水肿、间质充血、心肌纤维变性,横纹模糊不清甚至消失,出现广泛的肌纤维波浪状排列；D组大鼠心肌细胞少量水肿,心肌纤维排列基本正常,病变程度较B组有所减轻；电镜下见C组大鼠心肌细胞结构正常,肌丝、肌节排列整齐,线粒体未见肿胀,细胞核正常,核膜完整,间质未见水肿及炎症细胞侵润,毛细血管内皮细胞无肿胀,管腔内未见淤血及血栓形成；B组大鼠心肌细胞肌丝、肌节排列紊乱,结构模糊不清,肌浆网扩张明显,部分肌丝灶性溶解,部分节段细胞膜破坏,可见部分线粒体漏出及固缩,间质毛细血管内皮细胞肿胀；D组大鼠心肌细胞肌丝、肌节排列基本整齐,线粒体轻度肿胀,仅个别线粒体漏出,肌浆网轻度扩张,细胞膜基本完整无断裂,少量间质水肿,毛细血管内皮细胞大致正常。
结论右美托咪定可以减轻严重烫伤大鼠心肌损伤及凋亡,并抑制心肌细胞内质网应激程度以及内质网应激介导的凋亡通路相关蛋白表达,其减少细胞凋亡的机制可能与抑制CHOP和caspase-12这两条凋亡通路有关。
论文主要结论
1、严重烫伤后早期大鼠出现心肌损伤,血浆cTnI及CK-MB水平升高,于烫伤后12h达到峰值。烫伤后大鼠全身及心肌局部出现炎症反应,血浆及心脏局部TNF-α水平升高,烫伤后12h达到峰值。右美托咪定可减轻大鼠烫伤后心肌损伤并降低血浆及心脏局部TNF-α水平,且腹腔注射30μg/kg所产生的心肌保护效应较腹腔注射10μg/kg显著。
2、大鼠严重烫伤早期心肌细胞存在明显的凋亡,凋亡最高峰在伤后12h。严重烫伤后1h大鼠心肌细胞ERS相关蛋白表达开始逐渐增加,整个烫伤早期阶段心肌细胞内质网应激反应一直持续,并参与了部分的心肌细胞凋亡过程,且CHOP通路所介导的细胞凋亡可能于伤后6h开始,伤后12h达到高峰。
3、右美托咪定可以减轻严重烫伤大鼠心肌损伤及凋亡,并抑制心肌细胞内质网应激程度以及内质网应激介导的凋亡通路相关蛋白表达,其减少细胞凋亡的机制可能与抑制CHOP和caspase-12这两条凋亡通路有关。
Dexmedetomidine is one kind of high selectivity α2-adrenergic receptor agonist,which has the effect of central antisympathety and antianxiety.It can produce the sedation like natural sleep. Meanwhile, it has a certain extent of abirritation and diuretic action. It does not inhibit the respiration obviously.In1999,dexmedetomidine was initially approved by FDA for the sedation of patient in ICU who was received mechanical ventilation. Now it is been used widely in the field of clinical anesthesia and the sedation or analgesia of perioperative period and in ICU. Foreign scholars have reported that dexmedetomidine can be used in surgery and treatment of burn patients, but its application in the field of burns is not wide enough. The influence and the related mechanism of dexmedetomidine to the body and pathophysiological process after burns is also has not been fully elucidated.
The heart is the organ that in charge of body's circulation. Research has shown that,the myocardial cytoskeleton had been damaged in10min after severe burns;the myocardial blood flow began to significantly reduce on30min to1h after severe burns,at the same time,cardiac function was reduced,the leaking of troponin was increased,the pathological form of the myocardium changed significantly,the apoptosis of myocardial cell also increased.The myocardial damaging in this early stage can not only cause cardiac insufficiency,but also can become the important factor to star the serious burn shock and body tissues and organs ischemia anoxic damage.Therefore, the study of myocardial injury mechanism after burns and how to prevent and treat the myocardial damage after severe burns has been the research hotspot of scholars both at home and abroad. The main pharmacological effect of dexmedetomidine is to reduce the excitability of sympathetic nervous system, weaken the stress reaction, reduce the secretion of stress hormones, and prevent the occurrence of perioperative myocardial ischemia. Overseas studies have shown that the dexmedetomidine pretreatment can reduce myocardial ischemia-reperfusion injury; domestic scholars study has also found that dexmedetomidine pretreatment can reduce the cell apoptosis of rat myocardial in ischemia reperfusion injury In addition, there are a lot of fundamental research shows that dexmedetomidine can produce organ protection by reduce the body's inflammatory response.The influence and mechanism of dexmedetomidine on myocardial injury after burn is still not clear now.Due to the mechanism of myocardial injury after severe burns includes: ischemia-reperfusion injury, uncontrolled inflammatory response, disorders of oxygen uptake and energy metabolic,and myocardium cells appeared different degree of apoptosis We speculate that dexmedetomidine can exert similar protective effect and anti-apoptosis effect to myocardial injury after severe burns.
At present,a lot of research have confirmed that apoptosis of myocardial cells is one of the reasons of damaging on cardiac function in the early stage after severe burn,but the concrete mechanism has been incompletely recognized.The traditional approaches of cell apoptosis mainly includes death receptors and mitochondrial pathway.These two pathways activate caspase-3by inducing the caspase cascade reaction,then lead to cell apoptosis. Recent studies show that endoplasmic reticulum stress is also closely related with apoptosis. In the early stages of endoplasmic reticulum stress, it through a series of protein expression regulation of cells(unfolded protein response,UPR) to get well the endoplasmic reticulum steady;but if the endoplasmic reticulum stress is sustained and serious, it will active the apoptotic pathways and then induce apoptosis. Studies have shown that the drugs or measures which can alleviate endoplasmic reticulum stress will decrease the myocardial injury and apoptosis caused by ischemia reperfusion injury. If dexmedetomidine can alleviate myocardial injury after burns, is its mechanism related to the influence on myocardial cell apoptosis? If there were any involving the endoplasmic reticulum stress mediated apoptosis pathway? All these needs further research.
Endoplasmic reticulum stress is one kind of reaction about self protection mechanism of a eukaryotic cell.It plays an important role in maintaining cellular homeostasis.It has very complex process and pathway for mediated apoptosis. Glucose regulated protein78(GRP78) is the molecular chaperone in endoplasmic reticulum,and it also is the molecules sign of UPR reaction. And it is one of the key molecules of regulating the endoplasmic reticulum stress. When there are no unfold or misfolded proteins, GRP78can combine and suppress the special receptors of endoplasmic reticulum stress.Once the unfolded proteins appear,GRP78will shift from the special receptors to the unfolded proteins to activate the receptors. PERK is the most important receptor of endoplasmic reticulum stress.When ERS,GRP78which Combine with PERK will move into unfolded and misfolded proteins, and makes the PERK protein exposure. Thus PERK occurs two polymerization and its phosphorylation.Phosphorylation of PERK wiil make the eIF2a that outside of the endoplasmic reticulum added as phosphorylated modification, so the starting process of protein synthesis will be suspended,then eased the further protein accumulation. If ERS persistence for a long time, phosphorylation of the PERK will activate the downstream apoptotic pathway by ATF4.Then induced cell death.Therefore,the raising expression of GRP78and P-PERK not only mark the ERS,but also reflect the extent to which ERS activated. CHOP is the Specific transcription factor in ERS,it is the most important protein which has the function of mediated apoptosis in ERS. Under normal circumstances,the expression of CHOP is very low.When ERS is sustained and severe,the activation of IRE-1,PERK,ATF6will all induce the production of CHOP, prompt it to activate,and expresses abundantly, induced cell death consequently. The relationship between activation of CHOP and PERK pathway is most closely,because the complex of PERK-eIF2a-ATF4are necessary to the expression of CHOP protein. Caspase-12is one of the members of the family that contain cysteine aspartic acid proteolytic enzyme, located in the endoplasmic reticulum retinal cytoplasmic side. Caspase-12wiil play to the role of the mediated for apoptosis only after it be activated.At present, related studies have shown that there are three ways can be activated caspase-12:IRE1pathway,m-calpain activator pathway,caspase-7pathway,and this three ways are all related to the ERS.The concreteness process of ERS in cardiomyocytes after severe burn has not yet been fully elucidated, how dexmedetomidine will affect the ERS iconic protein that above-mentioned is still needed to research.
In conclusion, this study intended to SD rats as the research object,and establish the model rats with30%body surface area burn,using molecular biology method like histopathology, immunohistochemistry, protein electrophoresis,to compare the influence of different dose dexmedetomidine for myocardial injury and the inflammatory state in the rat suffer severe burns,and observe the variation and trend of the trademark protein related to ERS and the level of myocardial apoptosis during the whole shock stage of rat suffer severe burns,and further discuss the effect and its mechanism of dexmedetomidine to the myocardial apoptosis of rat suffer severe burns. To provide important theoretical basis for further clarify of the mechanism and effects about dexmedetomidine acting on myocardial injury after severe burns,and the widespread use of dexmedetomidine of the burn patients in the perioperative period.
Chapter1Effects of dexmedetomidine on plasma levels of cTnI、CK-MB、 TNF-a and myocardial expression of TNF-a after severe scald injury in rats
Objective To investigate the effects of different does of dexmedetomidine on severe scald injury-induced myocardial damage and inflammation in rats.
Methods Ninety-six male SD rats weighing220～280g were used in this study.Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). The animals with thermal injury involving30%of TBS were randomly divided into3 groups(n=32each):group A thermal injury;group B received intraperitoneal dexmedetomidine10μg/kg immediately after thermal injury; group C received intraperitoneal dexmedetomidine30μg/kg immediately after thermal injury.The false injury group and group A received equal volume of normal saline IP instead of dexmedetomidine.The plasma and myocardial tissue samples in each group were harvested at3,6,12,24postburn hours(PBH) for the determination of the contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue. Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The expression of each target of different groups were analyzed by two-way ANOVA, and multiple comparisons tests were performed by LSD.Using Pearson analysis the correlation between the various indicators. A probability value of P<0.05was considered to be statistically significant.
Results The contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue in each group rising after thermal injury,and they peaked at12PBH and decline in the trend at24PBH (P<0.01). The contents of cardiac troponin I(cTnI),creatine kinase isozyme(CK-MB),tumor necrosis factor-a(TNF-a) in plasma,and the contents of tumor necrosis factor-a(TNF-a) in myocardial tissue in group B and group C were significantly lower than those in group A (P<0.01),and all the index in group C were more lower than group B significantly (P<0.01)
Conclusion Myocardial injury appear early after severe burns in rats,the peak of the injury was in about12h after severe burns. Dexmedetomidine can alleviate the myocardial injury after severe scald in rats,and reduce the level of TNF-a in plasma and in myocardial tissue. Cardioprotection provided by intraperitoneal dexmedetomidine30μg/kg was more remarkable than10μg/kg.
Chapter2Transformation of cardiomyocytes apoptosis and endoplasmic reticulum stress associated protein(GRP78、P-PERK、CHOP) in cardiomyocytes after severe scald in rat and its possible meaning
Objective To investigate the transformation of endoplasmic reticulum stress associated protein in cardiomyocytes in rat after severe scald.To Discuss the possible meaning and mechanism of endoplasmic reticulum stress during myocardium cell apoptosis in rats after severe scald.
Methods Sixty-four male SD rats weighing220～280g were used in this study.The animals were randomly divided into8group (n=8each):Control group,1PBH(post burn hour) group,3PBH group,6PBH group,12PBH group,24PBH group,48PBH group. Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). Control group were exposed to37℃water for12s to preparation of false injury model.The heart tissues were harvested immediately after false injury in control group,and were harvested at the corresponding time point in each scald group.Myocardium apoptosis was detected with TUNEL assay.The expressions of glucose regulated protein78(GRP78), phosphorylated proteins kinase RNA-like ER kinase(P-PERK),C/EBP-homologous protein(CHOP) in different pathways of ERS were analysed with Westen blot. Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment.Completely random single factor analysis of variance was used to analyse the expression of GRP78,CHOP,P-PERK and cell apoptosis rate at different time points, the comparisons between groups were performed by LSD or Dunnett's T3. A probability value of P<0.05was considered to be statistically significant.
Results The expression levels of myocardial GRP78in the rats were differences between the groups (F=133.240, P=0.000).The expression of GRP78in burn group were significantly higher than those in control group (P<0.01). Compared with1,3,72h after injury, the expression of myocardial GRP78rised further at6-48h (P<0.01). The expression of myocardial GRP78reached the peak at48h after injury when look from the average,but the difference between the expression of myocardial GRP78at6～48h after injury were no statistical significance (P>0.05).The expression levels of myocardial CHOP in the rats were differences between the groups(F=223.871, P=0.000).The expression of CHOP was significantly higher than those in control group from6h after injury (P<0.01). The expression of myocardial CHOP reached the peak at12h after injury (P<0.01),then fall back.lt recovery to pre-injury level at72h after injury (P>0.05).The expression levels of myocardial P-PERK in the rats were differences between the groups (F=154.773, P=0.000).The expression of P-PERK was significantly higher than those in control group from6h after injury (P<0.01). The expression of myocardial P-PERK reached the peak at12h to24h after injury (P<0.01),then fall back.lt recovery to pre-injury level at72h after injury (P>0.05).The apoptosis index of myocardial cell in the rats were differences between the groups (F=322.860, P=0.000).The apoptosis index was significantly higher than those in control group from1h after injury (P<0.01). The apoptosis index reached the peak at12h after injury (P<0.01),then fall back. At72h after injury, it was still higher than pre-injury level (P<0.05)
Conclusion Part of myocardial cell apoptosis in rats after severe scald were mediated by ERS.The apoptosis mediated by ERS arised from6h after severer scald,and reached the peak at12h after injury.
Chapter3Role and mechanism of apoptosis pathway mediated by endoplasmic reticulum stress in attenuation of myocardial injury by dexmedetomidine in rats with severe scald
Objective To investigate the effects of dexmedetomidine on myocardial apoptosis in severe scald rats and its correlative mechanism.
Methods Twenty-four male SD rats weighing220～280g were used in this study.The animals were randomly divided into3group (n=8each):group C false njury; group B thermal injury;group D received intraperitoneal dexmedetomidine30μg/kg immediately after thermal injury.Thirty percent of the total body surface(TBS) was shaved and then exposed to94℃water for12s to establish30%TBS burn model(confirmed pathologically). Group C were exposed to37℃water for12s to preparation of false injury model. The false group C and group B received equal volume of normal saline IP instead of dexmedetomidine. The heart tissues were harvested at12h after injury.The morphological change in the myocardial tissue was observed with transmission electronic microscope(TEM) and optical microscope.Myocardium apoptosis was detected with TUNEL assay.The expressions of glucose regulated protein78(GRP78), phosphorylated proteins kinase RNA-like ER kinase(P-PERK),C/EBP-homologous protein(CHOP) in different pathways of ERS were analysed with Westen blot. The expression of caspase-12was analysed with immunohistochemical method.Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment.Completely random single factor analysis of variance was used to analyse the expression of GRP78,CHOP,P-PERK,caspase-12and cell apoptosis rate at different group, the comparisons between groups were performed by LSD or Dunnett's T3. A probability value of P<0.05was considered to be statistically significant.
Results Compared with group C, The expression of GRP78, CHOP, P-PERK, caspase-12and cell apoptosis rate in group B and group D were significantly higher (P<0.05). Compared with group B, The expression of GRP78, CHOP, P-PERK, caspase-12and cell apoptosis rate in group D were significantly lower (P<0.05).Under the light microscope, the myocardial cell structure was normal, myofilament was in alignment, the cell boundaries were clear in group C; in group B,myocardial cell was edema and interstitial congestion, myocardial fibers were degeneration, transverse striation blurred or even disappear and in a wide range of muscle fiber wavy;in group D, myocardial cell was in a small amount of edema, myocardial fibers arranged was normal, the lesion degree was lighter than group B. Under TEM, in group C,myocardial cell structure was normal, myofilament and sarcomere were in alignmen,there was no swelling of mitochondria, the nuclei was normal, nuclear membrane was integrity, interstitial edema and inflammatory cells invasion were no seen, there were no swelling of capillary endothelial cells,and no blood or thrombosis in the lumen;in group B, myofilament and sarcomere were disorganized, the structures of cells become obscure, sarcoplasmic reticulum expansion, part of the myofilament focal dissolved, part of the segmental cell membrane damage, some of the mitochondrial leakage and pyknosis, and interstitial capillary endothelial cells were swelling;in group D, myofilament and sarcomere were in almost alignmen,there were mild swelling of mitochondria and only individual of mitochondrial leak out, there were mild expansion of sarcoplasmic reticulum, the cell membrane almost complete and without fracture, interstitial edema were in a small amount, and the capillary endothelial cells is roughly normal.
Conclusion The myocardial damage and apoptosis in the rats with severe scald can be alleviated by dexmedetomidine.The mechanisms may be associated with decreasing the endoplasmic reticulum stress of the cardiomyocytes and inhibiting the pathways of apoptosis related with endoplasmic reticulum stress.
The main conclusion of the research
1. Myocardial injury appear early after severe burns in rats,the peak of the injury was in about12h after severe burns. Dexmedetomidine can alleviate the myocardial injury after severe scald in rats,and reduce the level of TNF-a in plasma and in myocardial tissue*Cardioprotection provided by intraperitoneal dexmedetomidine30μg/kg was more remarkable than10μg/kg.
2. Part of myocardial cell apoptosis in rats after severe scald were mediated by ERS.The apoptosis mediated by ERS arised from6h after severe scald,and reached the peak at12h after injury.
3. The myocardial damage and apoptosis in the rats with severe scald can be alleviated by dexmedetomidine.The mechanisms may be associated with decreasing the endoplasmic reticulum stress of the cardiomyocytes and inhibiting the pathways of apoptosis related with endoplasmic reticulum stress.
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14. Szabadkai QRizzuto R. Participation of endoplasmic reticulum and mitochondrial calcium handing inapoptosis:More than just neighborhood?Febs Lett, 2004;567:111-115.
15. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death:The calcium-apoptosis link. Nat Rev Mol Cell Biol,2003; 4:552-565.
16. Penzo D, Petronilli V, Angelin A, et al.Arachidonic acid released by phospholipase A2 activation triggers Ca dependent apoptosis through the mitochonodrial pathway. Biol Chem,2004;279:25219-25225.
17. Rao RV, Ellerby HM, Bredesen DE.Coupling endoplasmic reticulum stress to the cell death program.Cell Death Differ,2004;11:372-380.
18. Walter P, Ron D. The unfolded protein response:from stress pathway to homeostatic regulation. Science,2011;334:1081-1086.
19. Ho KY, Yeh TS, Huang HH, et al.Upregulation of phosphorylated HSP27,PRDX2,GRP75,GRP78,GRP94 in acquired middle ear cholesteatoma growth. Int Mol Sci,2013;14:14439-14459.
20. Oommen D, Prise KM. Down-regulation of PERK enhances resistance to ionizing radiation. Biochem Biophys Res Commun,2013;441:31-35.
21.Nishitoh H,Matsuzawa A,Tobiume K.ASK1 is essential for endoplasmic reticulum stress induced neuronal cell death triggered by expanded polyglutamine repeats. Genes,2002;16:1345-1355.
22. Gow A,Wrabetz L.CHOP and the endoplasmic reticulum stress response in myelinating glia. Curr Opin Neurobiol,2009; 19:505-510.
23. Motoyoshi E, Masataka M, Shizuo A, et al. C/EBP homologous protein(CHOP) is crucial for the induction of caspase-11 and the pathogenesis of lipopolysaccharide-induced inflammation. Immunol,2006;176:6245-6253.
24. Di Sano F,Ferraro E,Tufi R,et al.Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase-12 independent mechanism. Biol Chem, 2006;281:2693-2700.
25. Nakagawa T, Zhu H, Morishima N, LI E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature,2000; 403:98-103.
26. Carlson DL, Horton JW. Cardiac molecular signaling after burn trauma. Burn Care Res,2006; 27:669-675.
27. Doroudgar S,Glembotski CC. New concepts of endoplasmic reticulum function in the heart:Programmed to conserve. Mol Cell Cardiol,2012; [Epub ahead of print].
28. Wang J,Wang Y,Shan S,et al.Salusins protect myocardium against ischemic injury by alleviating endoplasmic reticulum stress.Sci China Life Sci,2012; 55:358-366.
29. Chen YH,Wu XD,Yao ST,et al. Calcineurin is involved in cardioprotection induced by ischemic postconditioning through attenuating endoplasmic reticulum stress.China Med J(Engl),2011;124:3334-3340.
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6. Biccard B, Goga S, Beurs J, et al. Dexmedetomidine and cardiac protection for non-cardiac surgery:a meta-analysis of randomised controlled trials. Anaesthesia, 2008; 63:4-14.
7. Yoshitomi O, Cho S, Hara T,et al. Direct protective effects of dexmedetomidine against myocardial ischemia-reperfusion injury in anesthetized pigs. Shock,2012; 38:92-97.
8. Ibacache M, Sanchez G, Pedrozo Z,et al. Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart. Biochimica et Biophysica Acta,2012; 1822:537-545.
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13. Rasheva Ⅵ, Domingos PM. Cellular responses to endoplasmic reticulum stress and apoptosis. Apoptosis,2009; 14:996-1007.
14. Szabadkai QRizzuto R. Participation of endoplasmic reticulum and mitochondrial calcium handing inapoptosis:More than just neighborhood?Febs Lett, 2004;567:111-115.
15. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death:The calcium-apoptosis link. Nat Rev Mol Cell Biol,2003; 4:552-565.
16. Penzo D, Petronilli V, Angelin A, et al.Arachidonic acid released by phospholipase A2 activation triggers Ca dependent apoptosis through the mitochonodrial pathway. Biol Chem,2004;279:25219-25225.
17. Rao RV, Ellerby HM, Bredesen DE.Coupling endoplasmic reticulum stress to the cell death program.Cell Death Differ,2004;11:372-380.
18. Walter P, Ron D. The unfolded protein response:from stress pathway to homeostatic regulation. Science,2011;334:1081-1086.
19. Ho KY, Yeh TS, Huang HH, et al.Upregulation of phosphorylated HSP27,PRDX2,GRP75,GRP78,GRP94 in acquired middle ear cholesteatoma growth. Int Mol Sci,2013;14:14439-14459.
20. Oommen D, Prise KM. Down-regulation of PERK enhances resistance to ionizing radiation. Biochem Biophys Res Commun,2013;441:31-35.
21.Nishitoh H,Matsuzawa A,Tobiume K.ASK1 is essential for endoplasmic reticulum stress induced neuronal cell death triggered by expanded polyglutamine repeats. Genes,2002;16:1345-1355.
22. Gow A,Wrabetz L.CHOP and the endoplasmic reticulum stress response in myelinating glia. Curr Opin Neurobiol,2009; 19:505-510.
23. Motoyoshi E, Masataka M, Shizuo A, et al. C/EBP homologous protein(CHOP) is crucial for the induction of caspase-11 and the pathogenesis of lipopolysaccharide-induced inflammation. Immunol,2006;176:6245-6253.
24. Di Sano F,Ferraro E,Tufi R,et al.Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase-12 independent mechanism. Biol Chem, 2006;281:2693-2700.
25. Nakagawa T, Zhu H, Morishima N, LI E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature,2000; 403:98-103.
26. Carlson DL, Horton JW. Cardiac molecular signaling after burn trauma. Burn Care Res,2006; 27:669-675.
27. Doroudgar S,Glembotski CC. New concepts of endoplasmic reticulum function in the heart:Programmed to conserve. Mol Cell Cardiol,2012; [Epub ahead of print].
28. Wang J,Wang Y,Shan S,et al.Salusins protect myocardium against ischemic injury by alleviating endoplasmic reticulum stress.Sci China Life Sci,2012; 55:358-366.
29. Chen YH,Wu XD,Yao ST,et al. Calcineurin is involved in cardioprotection induced by ischemic postconditioning through attenuating endoplasmic reticulum stress.China Med J(Engl),2011;124:3334-3340.
[1]黄跃生.再论烧伤后“休克心”及其临床意义.中华烧伤杂志,2009,25(3)：161-163.
[2]Xiao R, Lei ZY, Dang YM,et al. Prompt myocardial damage contributes to hepatic, renal, and intestinal injuries soon after a severe burn in rats. Trauma, 2011;71:663-672.
[3]Biccard B, Goga S, Beurs J, et al. Dexmedetomidine and cardiac protection for non-cardiac surgery:a meta-analysis of randomised controlled trials. Anaesthesia, 2008;63:4-14.
[4]Yoshitomi O, Cho S, Hara T,et al. Direct protective effects of dexmedetomidine against myocardial ischemia-reperfusion injury in anesthetized pigs. Shock, 2012;38:92-97.
[5]Ibacache M, Sanchez G, Pedrozo Z,et al. Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart. Biochimica et Biophysica Acta,2012; 1822:537-545.
[6]Peng M, Wang YL, Wang CY, Chen C.Dexmedetomidine attenuates lipopolysaccharide-induced proinflammatory response in primary microglia. Surg Res,2012; [Epub ahead of print].
[7]Benggon M, Chen H, Applegate R,et al. Effect of dexmedetomidine on brain edema and neurological outcomes in surgical brain injury in rats. Anesth Analg, 2012; 115:154-159.
[8]Hanci V, Yurdakan G, Yurtlu S,et al. Sipahi EY Protective effect of dexmedetomidine in a rat model of a-naphthylthiourea-induced acute lung injury. Surg Res,2012;178:424-430
[9]杨宗城.烧伤治疗学.北京人民卫生出版社：91.
[10]Gorbunov BB.Study of the myocardium cell membrane using the Luo-Rudy model. Med Tekh,2012;31:32-34.
[11]肖荣,黄跃生.“休克心”对严重烧伤后早期脏器损害影响的研究进展.中华烧伤杂志,2008,24(2)：157-159.
[12]Biccard B, Goga S, Beurs J, et al. Dexmedetomidine and cardiac protection for non-cardiac surgery:a meta-analysis of randomised controlled trials. Anaesthesia, 2008; 63:4-14.
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[14]Roekaerts PM, Prinzen FW, Lange SD. Beneficial effects of dexmedetomidine on ischaemic myocardium of anaesthetized dogs. Br J Anaesth 1996; 77:427-429.
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