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Objective To investigate the role of reactive oxygen species ( ROS ) in emulsified isoflurane postconditioning?induced promotion of nuclear factor?E2 related factor 2 ( Nrf2 )∕antioxidant re?sponse element ( ARE) signaling pathway activation in rats with myocardial injury. Methods Healthy male Sprague?Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized. Their hearts were excised and perfused in a Langendorff apparatus with K?H solution. Eighty isolated rat hearts were divided into 5 groups ( n=16 each) using a random number table: control group ( group C);ischemia?reperfusion (I∕R) group; emulsified isoflurane postconditioning group (group EIP); fat emulsion postconditioning group (group FEP); N?(2?mercaptopropionyl)?glycine (a ROS scavenger) plus emulsi?fied isoflurane postconditioning group ( group N+EIP ) . Group C was perfused with K?H solution for 100 min, and the other 4 groups were subjected to 40 min of ischemia followed by 60 min of reperfusion. EIP and FEP groups were perfused for 2 min with K?H solution containing 1?68 mmol∕L emulsified isoflurane and 712 mg∕L intralipid, respectively, starting from the onset of reperfusion, and then continuously per?fused with K?H solution for 58 min. Group N+EIP was perfused for 3 min with K?H solution containing N?(2?mercaptopropionyl)?glycine 2 mmol∕L, and then emulsified isoflurane postconditioning was performed. Heart rate ( HR ) , left ventricular developed pressure ( LVDP ) , left ventricular end?diastolic pressure (LVEDP) and the maximum rate of increase of left ventricular pressure (+dp∕dtmax) were recorded at the end of equilibration and of reperfusion. At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained for determination of ROS content. At the end of reperfusion, myocardial specimens were obtained for examination of the ultrastructure of myocardial cells and for determination of the expression of Nrf2, heme oxygenase?1 ( HO?1 ) , quinone oxidoreductase 1 ( NQO1 ) and superoxide dismutase?1 ( SOD?1) protein and mRNA in myocardial tissues. Mitochondrial injury scores ( Flameng scores) were e?valuated. Results Compared with group C, HR, +dp∕dtmax and LVDP were significantly decreased, LV?EDP , mitochondrial Flameng scores and ROS contents were increased, and the expression of Nrf2, HO?1, NQO1 and SOD?1 protein and mRNA was down?regulated at the end of reperfusion in I∕R group ( P0?05) . Compared with group EIP, HR, +dp∕dtmax and LVDP were significantly decreased, LVEDP and mitochondrial Flameng scores were increased, ROS contents at 5 min of reperfusion were decreased, ROS contents at the end of reperfu?sion were increased, and the expression of Nrf2, HO?1, NQO1 and SOD?1 protein and mRNA was down?regulated in group N+EIP ( P<0?05) . The degree of myocardial injury was reduced in group EIP as com?pared with group I∕R. There was no significant difference in the degree of myocardial injury between group N+EIP and group I∕R. Conclusion The mechanism by which emulsified isoflurane postconditioning pro?motes Nrf2?ARE signaling pathway activation is totally related to ROS in rats with myocardial injury.
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Astrocytes were traditionally be deemed as supportive cells in the central nervous system.However,recent researches proved astrocytes exerted more important neurophysiological functions,such as regulation of synaptic transmission and integration of neural information.This paper summarized the researches on the functions of astrocytes related to synaptic transmission and analyzed the new features of astrocyte excitability,the communication between neurons and astrocytes and the effect of general anesthetics on astrocytes.Based on these new findings,this paper also suggested the underlying relationship between astrocytes and the loss of consciousness during general anesthesia.
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Objective To observe the activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts.Methods The hearts of adult male Sprague Dawley rats were established ischemia-reperfusion injury model,and devided into six groups(n =8,each group),i.e.Normal group(Group N),ischemiareperfusion group (Group Con,I/R),ischemic postconditioning group (Group IPO),pinacidil postconditioning group (Group P50),N-(2-mercaptopropionyl)-glycine(MPG,2mmol/L) + IPO group(Group M + IPO),MPG + P50 group(Group M + P50).Rat hearts were perfused with Krebs-Henseleit(K-H) buffer for 20 minutes for equilibration.Subsequently,Group N was perfused with K-H buffer for 100 minutes after equilibration,Group Con was perfused with 4℃ ST.Thomas solution to stop the heart beating after equilibration,then the hearts were underwent 40 minutes global ischemia under 32℃,and followed by the K-H solution for 60 minutes.Group IPO after global ischemia period,the hearts were subjected to six 10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion,then were reperfused for 58 minutes.Group P50 after global ischemia,rat hearts were perfused with K-H buffer containing pinacidil(50.μmol/L) for 2 minutes before reperfusion.Group M + IPO after global ischemia,the hearts were subjected to perfuse with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then underwent six 10-seconds cycles of ischemia/reperfusion before reperfusion.Group M + P50 after global ischemia,the hearts were perfused with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then subjected to perfuse with K-H buffer containing pinacidil(50 μmol/L) for 2 minutes before reperfusion.Cardiac function indexes(such as HR,LVDP,LVEDP,and the Max dp/dt) at the end point of equilibration and repeffusion were observed and recorded.The ultrastructure of myocardial tissue was observed by electron microscopy and the mitochondrial Flameng score was calculated.RT-PCR and western-blot were applied to detect the gene transcription and protein expression of HO-1,NQO1,SOD1,and Nrf2 in left ventricular myocardial tissue after reperfusion.Results The HR,LVDP and + dp/dtmax at the end of reperfusion:the cardiac function indexes are lower among each group compared with group N,group 1PO and group P50 are better than group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group group P50,but group M + IPO is obviously decreased(P ＜ 0.05).Compared with group P50,group M + P50 index is decreased significantly(P ＜ 0.05).The LVEDP at the end of reperfusion is lower than that among each group as compared with group Con,which is significantly increased in group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group P50,but group M + IPO is significantly increased(P ＜ 0.05).Compared with group P50,the group M + P50 is obviously decreased(P ＜ 0.05).The ultrastructure of myocardial tissue in group N is mostly normal,group Con presence serious damage.The ultrastructure damage of myocardial tissue is improved in group IPO and group P50 as compared with that in group Con,while group M + IPO is more serious than group IPO,group M + P50 is more serious group P50.The mitochondrial Flameng score is higher among each group as compared with group N (P ＜ 0.05),the score is lower in group IPO and group P50 as compared with group Con and corresponding nonblocking group (M + IPO,M + P50,P ＜0.05).The mRNA and the protein expressions of HO-1,NQO1,SOD1 and Nrf2 among each group are lower as compared with group N(P ＜0.05).Compared with those in group Con,the mRNA and the protein expressions in group IPO and group P50 are obviously increased(P ＜ 0.05),group IPO and group P50 are higher than those in group adding active oxygen scavenger(MPG) (P ＜ 0.05).Conclusion Ischemic postconditioning and pinacidil postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury,while improve the cardiac function index.The cardiac protective effect of Ischemic and Pinacidil postconditioning methods may be involved the ROS in early reperfusion,which activate the Nrf2-ARE pathway,and up-regulate the expression downstream antioxidant protein and phase Ⅱ detoxifying enzyme,ultimately improve the cardiac function index during the reperfusion period.
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Objective To investigate the relationship between the mechanism of ischemic postconditioning-induced activation of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion (I/R) and reactive oxygen species (ROS).Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized with intraperitoneal 1％ pentobarbital sodium 40 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution.Thirty-two isolated rat hearts were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C) , group I/R,ischemic postconditioning group (group IPO) , and N-(2-mercaptopropionyl)-glycine (a ROS scavenger) + IPO group (group M + IPO).After 20 min of equilibration, group C was continuously perfused with K-H solution for 100 min, and the isolated hearts received the drugs via the perfusion system in the other groups.Group I/R was perfused with cardioplegic solution 4 ℃ St.Thomas, and then was subjected to 40 min of ischemia at 32 ℃ followed by 60 min of reperfusion.In group IPO, ischemic postconditioning was induced by 6 cycles of 10 s reperfusion followed by 10 s limb ischemia starting from the onset of reperfusion, and the hearts were then perfused for 58 min.In group M + IPO, the hearts were perfused with K-H solution containing N-(2-mercaptopropionyl)-glycine 2 m mol/L for 3 min starting from the onset of reperfusion,underwent 2 min of ischemic postconditioning, and then was perfused for 55 min.Heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP),and positive maximal pressure of left ventricular increase (+dp/dtmax) were recorded at the end of equilibration and of reperfusion.At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained from the left ventricle for determination of ROS content by enzyme-linked immunosorbent assay.At the end of reperfusion, myocardial specimens were obtained from the left ventricle for examination of the ultrastructure of myocardial cells and for determination of Nrf2, heme oxygenase-1 (HO-1) , quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1 (SOD1) mRNA and protein expression (by using Western blot and real-time polymerase chain reaction).The damage to myocardial mitochondria was assessed using Flameng scoring.Results Compared with group C, HR, +dp/dtmax and LVDP were significantly decreased, and LVEDP was increased at the end of reperfusion in I/R and M+IPO groups, HR and LVDP were decreased, LVEDP was increased, and no significant changes were found in +dp/dtmax at the end of reperfusion in IPO group, Flameng score was increased in I/R, IPO and M+IPO groups , the ROS content was increased at the end of reperfusion in I/R, IPO and M+IPO groups, and Nrf2, HO-1,NQO1 and SOD1 mRNA and protein expression was down-regulated at the end of reperfusion in I/R, IPO and M+IPO groups.Compared with group I/R, HR, +dp/dtmax and LVDP were significantly increased, and LVEDP and ROS content were decreased at the end of reperfusion, Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was up-regulated at the end of reperfusion in IPO and M+IPO groups, Flameng score was decreased in IPO group, there was no significant change in Flameng score in M+IPO group.Compared with group IPO, HR, +dp/dtmax and LVDP were significantly decreased, LVEDP and ROS content were increased at the end of reperfusion, Flameng score was increased, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated in M+IPO group.Conclusion Ischemic postconditioning can regulate ROS level and activate Nrf2-ARE signaling pathway, thus attenuating myocardial I/R injury in rats.
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Objective To evaluate the effects of ischemic postconditioning on mitochondrial cardiolipin synthesis during myocardial ischemia-reperfusion (Ⅰ/R) in rats in vitro.Methods Healthy male Sprague-Dawley rats,aged 16-20 weeks,weighing 250-350 g,were used in the study.The animals were anesthetized with intraperitoneal pentobarbital sodium 40 mg/kg and received intraperitoneal heparin 250 U/kg.Their hearts were excised and retrogradely perfused in a Langendorff apparatus.Sixty-four isolated rat hearts were randomly divided into 4 groups (n =16 each) using a random number table:control group (group C),Ⅰ/R group,ischemic postconditioning group (group IPO) and 5-hydroxydecanoate (5-HD) plus ischemic postconditioning group (group 5-HD + IPO).After 20 min of equilibration,the hearts were continuously perfused with K-H solution for 70 min in group C.In Ⅰ/R group,after 20 min of equilibration,the hearts were continuously perfused with 4 ℃ ST.Thomas cardioplegic solution 10 ml/kg,and exposed to 40 min of ischemia followed by reperfusion with oxygenated K-H solution at 37 ℃ for 30 min.In group IPO,after 20 min of equilibration,the hearts were subjected to 6 cycles of 10 s reperfusion followed by 10 s ischemia starting from 40 min of ischemia,and then were reperfused with oxygenated K-H solution at 37 ℃ for 28 min.In group 5-HD + IPO,after 20 min of equilibration,the hearts were perfused with K-H solution containing 100 μmol/L 5-HD (mitochondrial ATP-sensitive potassium channel blocker) for 5 min starting from 40 min of ischemia,and then the other procedures were similar to those previously described in group IPO.At 20 min of equilibration (T1) and 30 min of reperfusion (T2),HR,left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP) and coronary flow (CF) were recorded.The coronary effluent 2 ml was collected for detection of lactic dehydrogenase (LDH) and creatine kinase (CK) activities.The mitochondria were extracted for determination of cardiolipin content.Results HR,LVDP,and CF were significantly lower,LVEDP was higher,and the LDH and CK activities in coronary effluent were higher at T2 than at T1 in the four groups.Compared with group C,HR,LVDP and CF were significantly decreased,LVEDP was increased,and the LDH and CK activities in coronary effluent were increased at T2 in the other three groups.Compared with Ⅰ/R group,HR,LVDP and CF were significantly increased,LVEDP was decreased,and the LDH and CK activities in coronary effluent were decreased at T2 in IPO group.Compared with IPO group,HR,LVDP and CF were significantly decreased,LVEDP was increased,and the LDH and CK activities in coronary effluent were increased at T2 in 5-HD+IPO group.Conclusion The mechanism by which ischemic postconditioning reduces myocardial Ⅰ/R injury is related to opening of mitochondrial ATP sensitive potassium channels and increasing mitochondrial cardiolipin synthesis in rats.
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AIM:To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics .METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury .Sprague-Dawley rats were randomly divided into 2 groups:pinacidil post-conditioning group (Pina group) and ischemia/reperfusion injury group (I/R group).After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group.In Pina group at the end of 40 min global ischemia , the isolated hearts were perfused with K-H solution containing pinacidil ( 50μmol/L) for 2 min followed 58-min perfusion with regular K-H solution.Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE).The differentially expressed protein spots over 2 times were evaluated by a software .Then they were subjected to in-gel digestion , and analyzed by spectrometry .RESULTS:The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH 1 were decreased in Pina group compared with I/R group.Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ.The ex-pression levels of one spot was elevated , while the other was decreased .CONCLUSION:Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA 10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning .
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AIM: To investigate the effect of diazoxide (D) postconditioning on Cardiac function and mito-chondrial cardiolipin in isolated rat heart and to explore the protective effect of ATP sensitive potassium channel on diazo-xide postconditioning myocardium.METHODS: The myocardial ischemia/reperfusion injury model in isolated rat hearts was established by Langendorff apparatus.The isolated rat hearts were randomized into 4 groups ( n=8): control group ( control) , myocardial ischemia/reperfusion injury group ( I/R) , diazoxide postconditioning group ( I/R+D) , 5-hydroxy decanoic acid (5-HD) plus diazoxide postconditioning group (I/R+5-HD+D).The hearts in each group were started with 20 min perfusion for equilibration.The hearts in control group perfused for 70 min;The hearts in I/R group was global ischemia for 40 min after ischemia reperfusion at 4℃ST.Thomas cardioplegia, then reperfusion for 30 min;The hearts in I/R+D group were treated with diazoxide (50μmol/L) in K-H perfusion for 5 min after global ischemia for 40 min, then reperfusion for 25 min;The hearts in I/R+5-HD+D group were treated with 5-HD (100μmol/L) in K-H perfusion for 5 min before diazoxide postconditioning, then reperfusion for 20 min.The heart rate, coronary outflow volume, heart func-tion, myocardial enzymes and myocardial mitochondrial cardiolipin at the end of perfusion in each group were determined. RESULTS:Compared with control group and I/R+D group, the heart rate, the concentration of heart phospholipid and the coronary outflow volume were reduced, the heart function was significantly impaired the contents of myocardial enzymes were increased in I/R group.However, no significant difference between I/R group and I/R+5-HD+D group was ob-served.CONCLUSION:The diazoxide postconditioning protects the myocardium by increasing mitochondrial cardiolipin content, reducing the release of myocardial enzymes, improving heart function and reducing myocardial reperfusion injury. The myocardial protective effect of diazoxide is completely blocked by 5-hydroxy decanoic acid.
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Objective To evaluate the role of phosphoinositide 3 kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/Akt/GSK-3β) signaling pathway in mitigation of ischemia-reperfusion (I/R) injury by diazoxide postconditioning in isolated rat hearts.Methods Pathogen-free Sprague-Dawley rats were used in the study.Thirty hearts were excised and passively perfused in a Langendorff apparatus with oxygenated K-H solution at 37 ℃.The hearts were randomly divided into 5 groups (n =6 each) using a random number table:control group (group C),I/R group,diazoxide postconditioning group (group DZ),PI3K inhibitor LY294002 group (group LY),and diazoxide postconditioning + LY294002 group (group DZ + LY).In group C,the hearts were continuously perfused with K-H solution for 70 min.In group I/R,the hearts were perfused with cardioplegic solution 4 ℃ ST-Thomas 10 ml/kg,the perfusion pump was then stopped to induce global ischemia,and 40 min later the hearts were perfused with K-H solution for 30 min.In DZ group,5 min of retrograde perfusion with diazoxide 50μmol/L was performed through the aorta starting from the onset of reperfusion.In LY group,5 min of retrograde perfusion with LY294002 15 μnol/L was performed through the aorta starting from the onset of reperfusion.In LY + DZ group,5 min of retrograde perfusion with LY294002 15 μmol/L was performed through the aorta starting from the onset of reperfusion,followed by 5 min of retrograde perfusion with diazoxide 50 μmol/L.At 20 min of stabilization (T1) and 30 min of reperfusion (T2),heart rate (HR),coronary flow (CF),left ventricular developed pressure (LVDP),left ventricular developed pressure (LVEDP) and ± dp/dtmax were measured.The expression of total Akt (t-Akt) and total GSK-3β (t-GSK-3β) and phosphorylation of Akt and GSK-3β in myocardial tissues were determined by Western blot.Results Compared with C group,HR,LVDP and ± dp/dtmax were significantly decreased,and LVEDP was increased at T2 in the other four groups,CF was decreased in I/R,LY and DZ + LY groups,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group.Compared with I/R group,HR,CF,LVDP and ± dp/dtmax were significantly increased,and LVEDP was decreased at T2,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group,and no significant changes were found in the phosphorylation of Akt and GSK-3 β in LY and DZ + LY groups.Compared with DZ group,HR,CF,LVDP and ± dp/dtmax were significantly decreased,LVEDP was increased,and the phosphorylation of Akt and GSK-3β in myocardial tissues was decreased in LY and DZ + LY groups.Conclusion PI3K/Akt/GSK-3β signaling pathway is involved in the mechanism by which diazoxide postconditioning mitigates I/R injury in isolated rat hearts.
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Aim To discuss whether specific mitochon-drial ATP-sensitive potassium channel opener diazoxide ( DZ ) postconditioning activates RISK signaling path-way to protect isolated rat hearts against ischemica reperfusion injury ( IRI ) . Methods Langendorff de-vice was used to establish rat in vitro model of myocar-dial ischemia reperfusion. SD rats were randomly di-vided into normal group ( NOR ) , control group ( CON ) , diazoxide after treatment group ( DZ ) , and LY294002 antagonistic nitrogen Triazine group ( DZ +LY) , with 8 cases in each. The following was com-pared:①whether heart function of each group changed at the end of equilibration and reperfusion; ② at the end of myocardial perfusion and separation, protein was extracted, and protein kinase B ( PKB / Akt ) , P70S6 kinase (P70S6K), endothelial nitric oxide syn-thase ( eNOS) phosphorylation level of expression were analysed by Western blot. Results ① Indicators of changes in heart function: for DZ group at the end of reperfusion , HR , CF , LVDP , LVEDP , +d p/d tmax and -dp/dtmax were significantly better than those in CON group and DZ + LY group ( P 0.05 ) . Conclusion Diazoxide postconditioning through the activation of RISK signa-ling pathway can protect isolated rat hearts against is-chemia reperfusion injury.
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The competency oriented management theory and method,which is proved to be an effective management measure,have been widely used in China's health field. This essay probed into the possibility of introducing the competency oriented management theory into the education of targeted medical students in the rural. Combining the features of clinical medicine with training requirements of general medicine, this essay elaborated on the main contents of competency for targeted medical students in the rural including the ability of the community-levelled medical health service, practical ability, general medical treatment capacity, traditional medicine knowledge and skill and humanistic quality. Meanwhile, this paper summarized the practical experiences in training such elite students.
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Objective To evaluate the role of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in cardio-protection by ischemic or pinacidil postconditioning ( IP,PP) against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Fifty-six male SD rats of both sexes weighing 200-250 g were anesthetized with intraperitoneal amobarbital sodium.The isolated rat hearts were perfused in a Langendorff apparatus with Krebs-Hensleit buffer (K-H).Fifty-six isolated rat hearts with I/R injury were randomly divided into 7 groups ( n =8 each):normal control group (group C) ; group I/R; group IP and group PP1-4 postconditioning with 4 different concentrations of pinacidil.After 20 min of equilibration,the perfusion was suspended for 40 min (global ischemia) followed by 60 min of reperfusion in group I/R.In group IP after 40 min of global ischemia,the isolated hearts underwent 6 cycles of 10 s reperfusion and 10 s ischemia followed by 58 min of reperfusion.In group PP1-4 at the end of 40 min of global ischemia,the isolated hearts were perfused with K-H containing pinacidil 5,10,30 and 50μmol/L for 5 min respectively followed by 55 min reperfusion with regular K-H.Left ventricular developed pressure (LVDP) and LVEDP were measured immediately before global ischemia and at the end of 60 min reperfusion.Myocardial specimens were obtained at the end of reperfusion for detection of Nrf2,quinopeoxidoreductase (NQO1),HO-1 and SOD1 mRNA (by RT-PCR) and protein (by Western blot) expression.Results I/R significantly up-regulated Nrf2,NQO1,HO-1 and SODI mRNA and protein expression,decreased LVDP and increased LVEDP in group I/R as compared with group C.IP and 30,50 μmol/L pinacidil postconditioning further significantly increased Nrf2,NQO1,HO-1 and SOD1 mRNA and protein expression and IP,5,10,30,50 μmol/L pinacidil postconditioning significantly increased LVDP and decreased LVEDP as compared with group I/R.Conclusion Ischemic or pinacidil postconditioning can attenuate I/R injury by activating Nrf2-ARE pathway in isolated rat hearts.
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ObjectiveTo investigate the effect ofpinacidil hyperpolarizaed arrest on p38 mitogen-activited protein kinase (p38MAPK) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts.MethodsFortyeight male SD rats weighting 250-300 g were randomly divided into 6 groups( n =8 each): natural arrest group (group A) ; St.Thomas group (group B) ; pinacidil hyperpolarization arrest group (group C) ;5-hydroxydecanoate (5-HD) group (group D);HMR-1098 group(group E) and 5-HD + HMR-1098 group(group F).Langendorff reperfusion model was established and K-H solution was retrogradely perfused for 15 min.In group A the hearts were arrested naturally afar perfusion was stopped; in group B St.Thomas solution was perfused; in group C pinacidil hyperpolarization solution was perfused; in the other three groups,K-H solution was perfused to isolated rat hearts for 10 min followed by 5 min 5-HD (group D) or HMR-1098(group E) or 5-HD and HMR-1098(group F) perfusion,then hyperpolarization arrest solution was given in each group.Each hearts suffered 60 min ischemia after arrest followed by 30 min K-H solution reperfusion.Coronary flow(CF),HR,left ventricular developed pressure( LVDP),left ventricular systolic pressure(LVSP) and the maximum rate of pressure rise (dp/dtmax) were measured at the end of 15 min K-H solution perfusion and at 20 min of reperfusion.Myocardial phosphatic and nonphosphatic p38MAPK expression was determined by Western blot at 30 min of reperfusion.ResultsCompared with group C,CF,HR,LVDP,LVSP and dp/dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups A,B,D,E and F (P ＜ 0.05).Compared with group E,CF,HR,LVDP,LVSP and dp/ dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups D and F ( P ＜0.05).ConclusionHyperpolarized arrest induced by pinacidil can improve cardiac function following myocardial I/R injury by up-regulating phosphatic p38MAPK expression,down-regulating non-phosphatic p38 MAPK expression and mitochondrial potassium channel is more important than membranous one during the regulation of phosphatic p38MAPK expression.
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Objective To investigate the effect of heart preservation solution containing pinacidil on mitochondrial function in isolated rat hearts. Methods One hundred and twenty pathogen-free SD rats of both sexes weighing 250-350 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital 65 mg/kg. Their hearts were immediately removed and perfused in a Langendorff apparatus. Left ventricular enddiastolic pressure was measured from a fluid-filled latex balloon inserted in the left ventricle. The isolated hearts were randomized into 5 groups (n = 24 each):group Ⅰ was perfused with cardioplegic solution HTK; group Ⅱ with HTK containing pinacidil (a non-specific sarcKATP and mitoKATP channel opener) 0.5 mmol/L; group Ⅲ with HTK containing pinacidil 0.5 mmol/L + 5-HD (a selective mitoKATP channel blocker) 100 μmol/L; group Ⅳ with HTK containing pinacidil 0.5 mmol/L + HMR-1098 100 μmol/L (a selective sarcKATP channel blocker) and group Ⅴ with HTK containing pinacidil 0.5 mmol/L + 5-HD 100 μmol/L + HMR-1098 100μmol/L. The isolated hearts were perfused with simple HTK or HTK containing pinacidil or pinacidil + 5-HD and/or HMR 20 ml/kg at 10 ml/min and then removed from Langendorff apparatus and dipped into the same HTK solution for 8 h at 4 ℃followed by 60 min reperfusion. The respiratory function of mitochondria (respiratory control rate (RCR), the rate of oxygen consumption in state 3/state 4 and P/O) was measured at the end of equilibration (T1) after 8 hpreservation (T2) and at the end of 60 min reperfusion (T3). The CK-MB and LDH activities and cTnI expression in myocardium was detected at T1 and T3. The ultrastructure of myocardium was examined at T3. Results Perfusion suspension-reperfusion (PS/R) significantly decreased mitochondrial respiratory function (RCR, P/O and rate of O2 consumption in state 3) and increased myocardial cTnI concentration and CK-MB and LDH activities at T3 compared with baseline at T1 in group Ⅰ. Pinacidil significantly increased mitochondrial respiratory function (RCR, P/O and rate of O2 consumption in state 3) and decreased myocardial cTnI concentration and CK-MB and LDH activities in group Ⅱ as compared with group Ⅰ-indicative of protective effect of pinacidil on mitochondria against PS/R injury. The protective effect of pinacidil against PS/R injury was attenuated by 5-HD and/or HMR1098. The myocardial damage was slightest in group Ⅱ . Conclusion Both sarcolemmal and mitochondrial KATPchannel are involved in the protective effect of pinacidil against PS/R-induced myocardial damage during heart preservation.
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Objective To investigate the effect of hypoxic postconditioning and diazoxide postconditioning on the calreticulin expression during hypoxia/reoxygenation (H/R) in rat cardiomyocytes. Methods Primary cultured male SD rat cardiomyocytes were randomly divided into 6 groups (n = 8 each): control group (group C),H/R group; hypoxic postconditioning group (group HP) and diazoxide postconditioning group (group DP). Group C were cultured continuously for 2 h. Group H/R, Hp and DP were exposed to 45 min hypoxia (95% N2-5% CO2)followed by 1 h reoxygenation. In group HP, the cells were subjected to three cycles of 3 min hypoxia at 3 min intervals at the end of 45 min hypoxia before 1 h reoxygenation. In group DP, diazoxide 50 μmol/L was giyen at the end of hypoxia. Caspase-3 activity, calreticulin expression and intracellular free calcium ion concentrations were determined. Results Compared with group C, caspase-3 activity was significantly increased in the other groups,the calreticulin expression was up-regulated in group HP and DP, and the free calcium ion concentrations were increased in group H/R (P ＜ 0.05 or 0.01). Compared with group H/R, caspase-3 activity was significantly decreased in HP and DP, the calreticulin expression was up-regulated and the free calcium ion concentrations were decreased in group HP and DP (P ＜ 0.01). Conclusion Hypoxic postconditioning and diazoxide postconditioning attenuate H/R injury in rat cardiomyocytes through up-regulating the expression of calreticulin and reducing intracellular calcium overload.
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Objective To investigate the effects of diazoxide postconditioning on myocardial ischemiareperfusion (I/R) injury in isolated rat hearts. Methods Male SD rats weighing 250-300 g were anesthetized with intraperitoneal pentobarbital 40 mg/kg. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃. Sixty-four isolated rat hearts were randomized into 4 groups after 20 min of equilibration (n = 16 each): group control (group C), group I/R, group diazoxide postconditioning (group D) and group mito-KATP channel blocker 5-hydroxydecanoate (5-HD) + diazoxide postconditioning (group 5-HD + D). Group C received continuous perfusion for another 70 min. In group I/R, D and 5-HD + D, the hearts underwent 40 min of iscbemia followed by 30 min of reperfusion. 4 ℃ ST. Thomas cardioplegic solution was administered prior to ischemia in group I/R. Group D received 5 min of perfusion with K-H solution containing 50μ mol/L diazoxide at 5 min of reperfusion. Group 5-HD + D received 5 min of perfusion with K-H solution containing 50 μmol/L 5-HD before reperfusion with diazoxide. Eight hearts were taken at the end of equilibration and reperfusion and the indexes of cardiac function were recorded. Then the mitochondria were extracted for determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) release, and respiratory function indexes. Results Compared with group C, MMP was significantly decreased, ROS release was significantly increased, mitochondrial respiratory function and cardiac function declined at the end of reperfusion in the other three groups ( P ＜ 0.05 or 0.01 ). MMP was significantly increased, ROS release was significantly decreased, mitochondrial respiratory function and cardiac function were improved in group D compared with group I/R and 5-HD + D.Conclusion Diazoxide postconditioning can reduce myocardial I/R injury in rats via the opening of mito-KATPchannels and improving the mitochondrial function.
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Objective To investigate the effect of hydrogen sulfide postconditioning on the systolic function of left ventricle during myocardial ischemia-reperfusion (IR) in rats. Methods Part Ⅰ Adult male SD rats weighing 200-250 g were anesthetized with pentobarbital 40 mg/kg and heparin 250 U/kg. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃. Forty isolated rat hearts were randomly divided into 5 groups ( n = 8 each) after 20 min of equilibration: control group (group C); IR group; sodium hydrosulfide 1,10, 100 μmol/L postconditioning group (group SP1, SP10, SP100 ).In group Cthe hearts were perfused continuously for another 100 min. In group IR, the hearts were reperfused for 60 min after 40 min ischemia induced by 10 ml/kg ST. Thomas solution. In group SP1 , SP10 and SP100 the hearts were perfused with K-H solution containing sodium hydrosulfide 1, 10, 100 μmol/L for 2 min before reperfusion.LVDP and ± dp/dtmax were recorded at the end of equilibration and reperfusion. Part Ⅱ Cardiomyocytes were isolated from the male SD rats (weighing 200-250 g) and then cultured in CO2 incubator for 4 h. Sixty-four dishes of cultured myocytes were randomly divided into 4 groups( n = 16 each): control group (group C), hypoxia/reoxygenation group (group HR), hydrogen sulfide postconditioning group (group SP) and hypoxia postconditioning group (group HP). Group C were cultured continuously for 2 h. Group HR, SP and HP were exposed to 1 h hypoxia (95%N2-5%CO2 ) followed by 1 h reoxygenation. In group SP 10 μmol/L sodium hydrosulfide was added and the myocytes were then incubated for 2 min before reoxygenation. In group HP the cultured myocytes were expased to 3 min reoxygenation followed by 3 min hypoxia for 3 times before the 1 h reoxygenation. Mitochondrial membrane potential and F-actin expression were determined. Results Part Ⅰ Compared with group C, LVDP and ± dp/dtmax were significantly decreased at the end of reperfusion in group IR (P ＜ 0.05), while no significant difference was found in group SP1 , SP10 and SP100(P ＞0.05). Compared with group IR, LVDP and ± dp/dtmax were significantly increased in group SP ( P ＜ 0.05). There was no significant difference in LVDP and ± dp/dtmax among group SP1, SP10 and SP100(P ＞0.05). Part H Compared with group C, the mitochondrial membrane potential was significantly decreased in group HR and HP, and the expression of F-actin was significantly up-regulated in group HR, SP and HP ( P ＜ 0.05). Compared with group HR, the mitochondrial membrane potential was significantly increased and the expression of F-actin up-regulated in group SP and HP ( P ＜ 0.05 ). There were no significant difference in the mitochondrial membrane potential and expression of F-actin between group SP and HP ( P ＞0.05).Conclusion Hydrogen sulfide postconditioning can improve left ventricular systolic function during IR in rats by stabilizing mitochondrial membrane potential and promoting aggregation of F-actin.
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AIM: To investigate the effects of the selective mitochondrial ATP-sensitive K+ channels opener diazoxide on mitochondrial respiratory function and enzyme activity in isolated rat myocardium under ischemia/reperfusion.METHODS: Observation was made on rat hearts perfused with Langendorff apparatus.72 Sprague-Dawley(SD) rats were randomly divided into 4 groups: normal group(NOR),ischemia reperfusion(IR),diazoxide group(DIA) and 5-hydroxydecanoate(5-HD) antagonized diazoxide group(5HD-DIA).Hearts isolated from SD rats were mounted on a Langendorff apparatus and started with a 20 min perfusion for equilibration.NOR went on perfusion for another 100 min after equilibration.IR underwent 40 min global ischemia and followed by 30 min reperfusion after 30 min stabilization.DIA was administered with K-H solution containing diazoxide at concentration of 50 ?mol/L for 10 min before ischemia and reperfusion.5HD-DIA was infused with 100 ?mol/L 5-HD(a specific mitochondrial ATP sensitive K+ channel blocker) and the same procedure was carried out as DIA group.Hearts were taken down to extract mitochondrial at the end-equation,before ischemia and at the end-reperfusion for determination of mitochondrial respiratory function and the enzyme activity of mitochondria.RESULTS: At the end of reperfusion,mitochondrial respiratory function(mitochondrial respiratory control rate,P/O ratio and state 3 respiration) and mitochondrial enzyme activity(NADH oxidase,succinate oxidase and cytochrome C oxidase) in DIA group were better than those in IR group and 5HD-DIA group(P0.05).CONCLUSION: Preconditioning with mitochondrial ATP sensitive potassium channel opener,diazoxide,protects rat heart mitochondria against ischemia-reperfusion injury.The mechanisms are involved in the safeguarding of respiratory function and activity of enzymes of respiratory chain.
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Objective To evaluate the myocardial protective effect of hyperpolarized heart arrest at different temperature during cardiopulmonary bypass CPB.Methods Eighteen dogs were randomly allocated to be infused with St.Thomas solution containing 50?mol/L pinacidil and 5mmol/L K + at 37℃ (group A) or 4℃ (group B), or the standard St.Thomas solution containing K + 16mmol/L at 4℃ (group C) respectively, through aortic root after aortic clamping . The global surgical ischemia lasted 60min with the declamping of 20min .The activities of serum myocardial enzymes, and levels of lipid peroxide and adenine nucleotide of myocardium were measured.Results All paramaters showed to occur serious ischemic and reperfusion damages during the whole procedures in group C, while there were the mild damages in two hyperpolarized groups, especially in group B.Conclusions Myocardial protective effect of hyperpolarized arrest induced with pinacidil, an ATP sensitive potassium channel opener, is superior to that of traditional hyperkalemic cardioplegia , which is much more prominent in hypothermic state.
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Objective To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on plasma concentrations of proinflammatory cytokines and myocardial energy metabolism induced by cardiopulmonary bypass (CPB) .Methods Twelve healthy mongrel dogs of both sexes weighing 13.5-17.5 kg were randomly divided into 2 groups (n = 6 in each group) : PDTC group and control group. The animals were anesthetized with intraperitoneal 2.5% pentobarbital sodium 25 mg?kg-1, intubated and mechanically ventilated. In PDTC group PDTC 30 mg?kg-1 was given i.v. after tracheal intubation while in control group normal saline was given instead of PDTC. Aorta was clamped for 60 min and then undamped for 60 min reperfusion during CPB. Blood samples were taken before (baseline), 30 and 60 min after aortic clamping and 30 and 60 min after aortic unclamping for determination of plasma concentrations of TNF-? and IL-1?. Myocardial tissue was obtained before and 60 min after aortic clamping and 60 min after aortic unclamping for determination of myocardial content of adenine nucleotide (ATP, ADP, AMP, TAN, EC) and expression of ICAM-1 protein.Results Plasma TNF-? concentration was increased after aortic cross-clamping as compared to the baseline value before clamping in both groups but the TNF-? concentration was significantly lower in PDTC group than in control group (P
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AIM: To study the protective effect of hyperpolarized cardioplegic arrest on reperfused rat heart performance and to investigate the role of mitochondrial ATP-sensitive K+ channels(mitoKATP) opening in the protection of hyperpolarized cardioplegia against ischemia/reperfusion damage.METHODS: Forty Sprague-Dawley rats were randomized into five groups(n=8 in each group): control group(Con);depolarized arrest group(D);hyperpolarized arrest group(H);depolarized cardioplegia with 5-hydroxydecanoate(5-HD) group(5HD+D);hyperpolarized cardioplegia with 5-HD group(5HD+H).The rat hearts were quickly removed to Langendorff apparatus.The heart perfusion was performed for 20 min with 37 ℃ Krebs-Henseleit buffer balanced with gas mixture(O2∶CO2=95%∶5%) at 5.8 kPa perfusion pressure,then cardial arrest was induced by different cardioplegic solution.Hearts were subjected to ischemia at 37 ℃ for 40 min followed by 30 min reperfusion.(1) The hemodynamics was detected at recovery after 30 min reperfusion.(2) Before ischemia and at the end-reperfusion,tissue was harvested for mitochondrial isolation and ultrastructure was observed by transmission electron microscopy(TEM).(3) Production of reactive oxygen species(ROS) was also determined at different time points.RESULTS:(1) Compared with end-equilibration,30 min reperfusion caused significant differences in left ventricular developed pressure(LADP),left ventricular end-diastolic pressure(LVEDP),double product(DP),heart rate (HR),coronary flow(CF)(P