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OBJECTIVE@#To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine.@*METHODS@#Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting.@*RESULTS@#After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05].@*CONCLUSIONS@#TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.
Assuntos
Masculino , Animais , Suínos , Traumatismos Abdominais , Apoptose , Reanimação Cardiopulmonar , NefropatiasRESUMO
Objective To investigate the effects of therapeutic hypothermia (TH) on myocardial Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and cell autophagy after cardiopulmonary resuscitation (CPR) in swine.Methods Twenty healthy male domestic swine weighing 33-40 kg were randomly (random number) divided into 3 groups:sham group (n=4),CPR group (n=8) and TH group (n=8).Sham animals only underwent general preparation without experiencing cardiac arrest and resuscitation.The animal model was established by 8 min of electrically induced ventricular fibrillation and then 5 min CPR in the CPR and TH groups.Successful resuscitation was regarded as an organized rhythm with a mean arterial pressure of greater than 50 mmHg for 5 min or more.After successful resuscitation,body temperature was decreased to 33 ℃ by a cooling blanket and then maintained until 24 h post-resuscitation,and followed by a rewarming at a rate of 1 ℃/h for 5 h in the TH group.A normal temperature was maintained by the blanket throughout the experiment in the sham and CPR groups.At 6,12,24 and 30 h after resuscitation,the values of stroke volume (SV) and global ejection fraction (GEF) were measured by PiCCO,and meanwhile the serum concentrations of cardiac troponin Ⅰ (cTnI) were measured by ELISA assay and the serum activities of creatine kinase-MB (CK-MB) were evaluated by an automatic biochemical analyzer.At 30 h after resuscitation,the animals were sacrificed and left ventricular myocardium was obtained for the determination ofCaMK Ⅱ,microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ) and p62 expressions by Western blot.The variables were compared with One way analysis of variance and then the Bonferroni test among the three groups.Results Compared with the sham group,myocardial dysfunction and injury after resuscitation were observed in the CPR and TH groups,which were indicated by decreased SV and GEF and also increased cTnI concentration and CK-MB activity in serum (all P＜0.05).Compared with the CPR group,the values of SV and GEF were significantly increased at 6 h after resuscitation,and serum cTnI concentration and CK-MB activity were significantly decreased starting 12 h after resuscitation in the TH group [SV (mL):25.0±6.9 vs 31.9±3.3 at 6 h,26.7±5.1 vs 34.6±3.7 at 12 h,28.8±3.3 vs 35.7±3.2 at 24 h,29.2±5.2 vs 36.7±3.3 at 30 h;GEF (％):17.1±2.7 vs 19.9±1.8 at 6 h,18.7±1.9 vs 21.6±1.8 at 12 h,19.3±2.3 vs 23.0±2.4 at 24 h,21.0±1.7 vs 23.7±1.7 at 30 h;cTnI (pg/mL):564±51 vs 466±56 at 12 h,534±38 vs 427±60 at 24 h,476±55 vs 375±46 at 30 h;CK-MB (U/L):803±164 vs 652±76 at 12 h,693±96 vs 557±54 at 24 h,633±91 vs 480±77 at 30 h,all P＜0.05].Tissue detection indicated that the expression of CaMK Ⅱ and LC3 Ⅱ were increased while the expression of p62 was decreased in post-resuscitation myocardium in the CPR and TH groups compared with the sham group (all P＜0.05).However,the expression of CaMK Ⅱ and LC3 Ⅱ were decreased and the expression of p62 was increased in postresuscitation myocardium in the TH group compared to the CPR group (CaMK Ⅱ:0.73±0.06 vs 0.58±0.05;LC3 Ⅱ:0.69±0.09 vs 0.50±0.07;p62:0.40±0.07 vs 0.68±0.14,all P＜0.05).Conclusion The mechanism of TH alleviating post-resuscitation myocardial dysfunction and injury may be related to the inhibition of CaMK Ⅱ expression and cell autophagy.
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Objective To evaluate the effects of hypothermia on Ca2+∕calmodulin-dependent pro-tein kinase Ⅱ ( CaMKⅡ) and cell autophagy in brain tissues after cardiac arrest and cardiopulmonary re-suscitation ( CA-CPR) in swine. Methods Twenty-one healthy male white swine, weighing 33-40 kg, were divided into 3 groups using a random number table method: sham operation group ( group S, n=5) , CA-CPR group ( n=8) and hypothermia group ( group H, n=8) . The experimental model of CA-CPR was established in CA-CPR and H groups. The Swan-Ganz catheters were placed in the right femoral artery and vein to monitor the pressure of thoracic aorta and right atrium and body temperature and to collect blood sam-ples. A pacing catheter was advanced from the right external jugular vein into the right ventricle. Ventricu-lar fibrillation was induced by using a 1 mA alternating current through the pacing catheter. Once ventricular fibrillation was successfully induced, mechanical ventilation was discontinued for 8 min, and then CPR was initiated. Epinephrine 20 μg∕kg was intravenously injected at 2. 5 min of CPR, followed by repetition once every 3 min. Defibrillation was delivered at 5 min of CPR, and then spontaneous circulation was evaluated. If the spontaneous circulation was not restored, CPR was immediately resumed for 2 min, and then defibril-lation was delivered again. Mechanical ventilation was continued for 30 h after successful CPR. At 5 min af-ter successful resuscitation, body temperature was decreased to 33 ℃ by using a cooling blanket, then maintained at 33 ℃ until 24 h after resuscitation, and finally increased at a rate of 1℃∕h for 5 h in group H. The temperature was maintained at a normal level of 37. 5-38. 5 ℃ with the aid of a cooling blanket in S and CA-CPR groups. At 1, 6, 12, 24 and 30 h after resuscitation (T1-5), blood samples were collected from the femoral vein for measurement of the concentration of neuron specific enolase ( NSE) and S100βprotein in serum by enzyme-linked immunosorbent assay. Five animals in each group were then sacrificed, and brains were removed to determine the expression of CaMKⅡ, microtubule-associated protein 1 light chain 3 Ⅱ( LC3Ⅱ) and p62 in cerebral cortex by Western blot. Neurological deficit score was evaluated in the remaining three swine at 48, 72 and 96 h after resuscitation (T6-8) in CA-CPR and H groups. Results Compared with group S, the concentrations of NSE and S100β protein in serum were significantly in-creased at T1-5 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was up-regulated, and the expres-sion of p62 in cerebral cortex was down-regulated in CA-CPR and H groups (P<0. 05). Compared with group CA-CPR, the concentrations of NSE and S100βprotein in serum were significantly decreased at T3-5, the neurological deficit score was decreased at T6-8 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was down-regulated, and the expression of p62 in cerebral cortex was up-regulated in group H ( P<0. 05) . Conclusion The mechanism by which hypothermia alleviates brain injury after CA-CPR may be related to inhibiting CaMKⅡ activation and reducing cell autophagy in brain tissues of swine.
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Objective To investigate the effects of rapid hypothermia induced via esophagus on intestinal mucous injury in early stage after cardiopulmonary resuscitation (CPR) in a swine model of cardiac arrest.Methods Twenty-seven male domestic pigs weighing (36±2)kg were utilized.The animals were randomly crandom number divided into 3 groups (n=9 in each):normothermia group (NT group),surface cooling group (SC group),and esophageal cooling group (EC group).The pig model was established by 8 mins of untreated ventricular fibrillation and then 5 mins of CPR.At 5 mins after restoration of spontaneous circulation (ROSC),therapeutic hypothermia was applied by either an esophageal cooling device in the EC group or a surface cooling blanket in the SC group to reach a targeted temperature of 33 ℃ maintained for 24 h after ROSC,and then followed by warming up in a rate of 1 ℃ / hr for 5 hrs.A normal temperature of (38.0±0.5)℃ was maintained throughout the experiment in the NT group.The core temperature was continuously monitored during a period of 30 h after ROSC.At 3 h,6 h,12 h,24 h and 30 h after ROSC,intestinal fatty acid binding protein (IFABP) content and diamine oxidase (DAO) activity in serum were measured by ELISA.At 30 h after ROSC,the pigs were sacrificed,and then intestinal tissue was rapidly obtained for the determination of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) contents by ELISA,cell apoptosis by TUNEL,and caspase-3 expression by immunohistochemistry.Results The rate of temperature decrease was 2.8 ℃/h and the time required for target temperature was 102 min in the EC group,while the rate of temperature decrease was 1.5 /h and the time consumed for target temperature was 185 mins in the SC group,which suggested the efficacy of cooling was significantly better in the EC group than that in the SC group (both P＜0.05).Compared with the NT group,serum IFABP content and DAO activity were significantly decreased at 3 hrs after ROSC in the EC group and at 6 hrs after ROSC in the SC group.Compared with the SC group,serum IFABP content at 6 hrs after ROSC and DAO activity at 12 h after ROSC were significantly decreased in the EC group IFABP (pg/mL):(710±32) vs.(777±52) at 6 h,(870±49) vs.(960±64) at 12 h,(1 022±65)vs.(1 143±63) at 24 h,(882±71) vs.(1 006±45) at 30 h DAO (U/mL):(39.9±1.9) vs.(43.4±3.2) at 12 h,(30.6±2.4) vs.(34.0±3.1) at 24 h,(26.1±2.7) vs.(29.4±2.2) at 30 h,all P＜0.05.In the intestinal tissue,TNF-α and IL-6 contents were significantly reduced,and cell apoptosis index and caspase-3 expression were significantly decreased in the SC and EC groups compared with the NT group.Additionally,inflammatory response and cell apoptosis in intestinal tissue were further significantly lesser in the EC group compared with the SC group TNF-α (pg/mL):(721±94) vs.(922±125);IL-6(pg/mL):(454±69) vs.(697±132);Apoptotic index(％):(6.2±2.6)vs.(12.8±3.0);caspase-3 expression (IOD):(8.9±1.6) vs.(15.9±1.9),all P＜0.05.Conclusions In a swine model of cardiac arrest,rapid hypothermia could be successfully induced via esophagus and consequently produced a greater protective effect on post-resuscitation intestinal injury compared with the conventional surface cooling.The protective mechanisms are associated with the inhibition of inflammatory response and cell apoptosis.
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Objective To evaluate the effect of mild hypothermia on inositol-requiring enzyme 1 (IRE1) signaling pathway during myocardial injury after cardiac arrest and resuscitation in swine.Methods Twenty-one healthy male white swine,weighing 33-41 kg,were divided into 3 groups using a random number table method:sham operation group (group S,n =5),cardiac arrest-cardiopulmonary resuscitation group (group CA-CPR,n=8),and mild hypothermia group (group MH,n=8).The model of cardiac arrest and resuscitation was established based on the previously reported method.The catheters placed in the left femoral artery and right internal jugular vein were connected to the PiCCO Monitor system,and another pacing catheter was advanced from the right external jugular vein into the right ventricle.Ventricular fibrillation was induced by using a 1 mA alternating current through the pacing catheter.Once ventricular fibrillation was successfully induced,mechanical ventilation was discontinued for 8 min,and then cardiopulmonary resuscitation was initiated.Epinephrine 20 μg/kg was administered at 2.5 min of resuscitation followed by repetition every 3 min.Defibrillation was delivered at 5 min of resuscitation,and then spontaneous circulation was evaluated.If return of spontaneous circulation was not achieved,cardiopulmonary resuscitation was immediately resumed for 2 min and then defibrillation was delivered again.Mechanical ventilation was continued for 30 h after successful resuscitation.Animals in group S only underwent surgical preparation without experiencing cardiac arrest and resuscitation.At 5 min after successful resuscitation,body temperature was cooled down to 33 ℃ by using a cooling blanket,and then maintained at this level until 24 h after resuscitation,followed by 5 h of re-warming at a rate of 1 ℃/h in group MH.The temperature was maintained at 37.5-38.5 ℃ with the aid of surface cooling blanket in the other two groups.At 1,6,12,24 and 30 h after resuscitation (T1-5),the values of stroke volume (SV) and global ejection fraction (GEF) were recorded,and meanwhile 2 ml of blood samples was obtained via the femoral vein to measure the concentration of serum cardiac troponin Ⅰ (cTnI) (by enzyme-linked immunosorbent assay) and activity of serum creatine kinase-MB (CK-MB) (by immunosuppression).The swine were sacrificed at 30 h after resuscitation,and then myocardial specimens from the left ventricle were obtained for determination of the expression of caspase-3 (by immunohistochemistry),cell apoptosis (by TUNEL),and expression of IRE1 and casepase-12 (by Western blot).Apoptosis index was calculated.Results Compared with group S,SV and GEF were significantly decreased and the serum CK-MB activity was increased at T1-5,the concentration of serum cTnI was increased at T2-5,the expression of IRE1,caspase-12 and caspase-3 in myocardium was up-regulated,and apoptosis index was increased in CA-CPR and MH groups (P＜0.05).Compared with group CA-CPR,the SV and GEF were significantly increased and the concentration of serum cTnI was decreased at T2-5,the activity of serum CK-MB was decreased at T3-5,the expression of IRE1,caspase-12 and caspase-3 in myocardium was down-regulated,and apoptosis index was decreased in group MH (P＜0.05).Conclusion The mechanism by which mild hypothermia mitigates myocardial injury after cardiac arrest and resuscitation may be related to inhibiting IRE1 signaling pathway in swine.