Research On Effects Of Dexmedetomidine On Myocardial Damage After Severe Scald Injury And Its Endoplasmic Reticulum Stress Related Mechanism In Rats

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 ratsObjective 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 meaningObjective 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 scaldObjective 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 research1. 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.