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Objective To evaluate the role of mitochondrial permeability transition pore (mPTP)in reduction of brain injury by sevoflurane postconditioning in a rat model of hemorrhagic shock and resuscitation (HSR).Methods Ninety pathogen-free healthy adult male Sprague-Dawley rats,weighing 300-350 g,were divided into 5 groups (n =18 each) using a random number table:sham operation group (group S),group HSR,sevoflurane postconditioning group (group SP),sevoflurane postconditioning plus atractyloside (ATR,a specific mPTP opener) group (group SP + ATR) and ATR group.Hemorrhagic shock was produced by withdrawing 40％ of the total blood volume from the right carotid artery over an interval of 30 min,and 1 h later the animals were resuscitated by infusion of the shed blood via the left jugular vein over 30 min.SP and SP+ATR groups were exposed to 2.4％ sevoflurane for 30 min starting from the onset of reinfusion.In ATR and SP+ATR groups,ATR 5 mg/kg was intravenously injected at 10 min before reinfusion.Six rats in each group were randomly sacrificed at 24 h after the end of autologous blood reinfusion,and the hippocampus was harvested for determination of the expression of Bcl-2 and Bax in hippocampal tissues (by Western blot) and degree of mPTP opening.At 72 h after the end of autologous blood reinfusion,the rest 6 rats in each group were selected and underwent Morris water maze test,and the cognitive function was evaluated.Results Compared with group S,the escape latency was significantly prolonged,the number of crossing the original platform and locomotor distance in the target quadrant were decreased,the expression of Bcl-2 was down-regulated,the expression of Bax was up-regulated,and the degree of mPTP opening was increased in group HSR (P＜0.05).Compared with group HSR,the escape latency was significantly shortened,the number of crossing the original platform and locomotor distance in the target quadrant were increased,the expression of Bcl-2 was up-regulated,the expression of Bax was down-regulated,and the degree of mPTP opening was decreased in group SP (P＜0.05),and no significant change was found in each parameter in ATR and SP+ATR groups (P＞0.05).Compared with group SP,the escape latency was significantly prolonged,the number of crossing the original platform and locomotor distance in the target quadrant were decreased,the expression of Bcl-2 was down-regulated,the expression of Bax was up-regulated,and the degree of mPTP opening was increased in group SP+ATR (P＜0.05).Conclusion The mechanism by which sevoflurane postconditioning ameliorates brain injury may be related to inhibiting mPTP opening in a rat model of HSR.
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Objective To evaluate the effect of carbon monoxide (CO) postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration (OGD/R) in rat hippocampai neurons and the relationship with mitochondrial permeability transition pore (mPTP)/reactive oxygen species (ROS) signaling pathway.Methods Primary hippocampal neurons were cultured in vitro,seed in 6-well or 96-well plates,and divided into 5 groups (n =24 each) using a random number table method:control group (C group),OGD/R group,CO postconditioning group (CO group),specific mPTP opener atractyloside plus CO postconditioning group (ACO group),and specific ROS inducer antimycin A plus CO postconditioning group (KCO group).Neurons were subjected to O2-glucose deprivation (OGD) for 16 h followed by restoration of O2-glucose supply for 24 h to establish the model of OGD/R injury.In group CO,neurons were exposed to 2％ CO-5％ CO2 for 3 h at 37 ℃ starting from the end of OGD,followed by normal culture for 21 h.In ACO and KCO groups,atractyloside 20 μmol/L and antimycin A 50 μmol/L were added at the end of OGD,respectively,and the other treatments were similar to those previously described in group CO.Neuronal pyroptosis rate was determined using double immunofluorescent staining cleaved caspase-1-AlexaFluor 568/DAPI after the end of treatments in each group.The neuronal survival rate was determined by MTT,opening of mPTP by Calcein-AM fluorescence,ROS content by DCFH-DA,and expression of interleukin1beta (IL-1β) and IL-18 by Western blot.Results Compared with C group,neuronal pyroptosis rate,ROS content and opening of mPTP were significantly increased,the neuronal survival rate was decreased,and the expression of IL-1β and IL-18 was up-regulated in the other groups (P＜0.05).Compared with OGD/R group,neuronal pyroptosis rate,ROS content and opening of mPTP were significantly decreased,the neuronal survival rate was increased,and the expression of IL-1β and IL-18 was down-regulated in CO,ACO and KCO groups (P＜0.05).Compared with CO group,neuronal pyroptosis rate and ROS content were significantly increased,the neuronal survival rate was decreased,and the expression of IL-1β and IL-18 was up-regulated in ACO and KCO groups,and opening of mPTP was significantly inctreased in ACO group (P＜0.05).Conclusion CO postconditioning can inhibit OGD/R-induced pyroptosis in rat hippocampal neurons,and the mechanism is related to inhibiting mPTP/ROS signaling pathway.
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Objective To evaluate the role of Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) signaling pathway in sevoflurane postconditioning-induced inhibition of mitochondrial permeability transition pore (mPTP) opening during myocardial ischemia-reperfusion (I/R)in rats.Methods Sixty pathogen-free healthy male Sprague-Dawley rats,weighing 250-300 g,were divided into 4 groups (n=15 each) using a random number table:I/R group,sevoflurane postconditioning group (group SP),AG-490 group (group AG) and sevoflurane postconditioning plus AG-490 group (group SP+AG).Myocardial I/R was induced by 30 min ligation of the left anterior descending branch of coronary artery followed by 120 min reperfusion.In group SP,2.8％ sevoflurane was inhaled for 15 min starting from 2 min before reperfusion.JAK2 inhibitor AG-490 3 mg/kg was intravenously injected at 10 min before reperfusion in group AG.In group SP+AG,AG-490 3 mg/kg was intravenously injected at 10 min before reperfusion,and 2.8％ sevoflurane was inhaled for 15 min starting from 2 min before reperfusion.At 15 min of reperfusion,5 rats were sacrificed and myocardial specimens were obtained for determination of the expression of JAK2,phosphorylated JAK2 (p-JAK2),STAT3 and phosphorylated STAT3 (p-STAT3)in myocardial tissues by Western blot.The ratios of p-JAK2 to JAK2 expression (p-JAK2/JAK2) and pSTAT3 to STAT3 expression (p-STAT3/STAT3) were calculated.Five rats were sacrificed at the end of reperfusion for measurement of myocardial infarct size.The left 5 rats were selected and sacrificed,myocardial specimens were obtained,and the opening of mPTP was detected by a calcein-cobalt quenching method.Results Compared with group I/R,the myocardial infarct size and mPTP opening were significantly decreased,and JAK2/p-JAK2 and STAT3/p-STAT3 were increased in group SP (P＜0.05),and no significant change was found in the parameters mentioned above in SP+AG and AG groups (P＞0.05).Compared with group SP,the myocardial infarct size was significantly enlarged,the extent of mnPTP opening was aggravated,and JAK2/p-JAK2 and STAT3/p-STAT3 were decreased in SP+AG and AG groups (P＜0.05).Conclusion The mechanism by which sevoflurane postconditioning inhibits the opening of mPTP during myocardial I/R is related to activation of JAK2-STAT3 signaling pathway in rats.
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Objective To evaluate the role of serine-threonine kinase (Akt)/glycogen synthase kinase-3 beta (GSK-3β) signaling pathway in isoflurane preconditioning-induced inhibition of mitochondrial permeability transition pore protein (mPTP) opening during myocardial ischemia-reperfusion (I/R) in rats.Methods Ninety-six male Sprague-Dawley rats,aged 3-4 months,weighing 200-250 g,were randomly divided into 4 groups (n =24 each) using a random number table:control group (group C);I/R group;isoflurane preconditioning group (group IPC);Akt inhibitor MK-2206 group (group MK).Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 2 h of reperfusion.In group IPC,1.5％ isoflurane was inhaled for 30 min followed by 45 min washout,and then the model of myocardial I/R injury was established.In group MK,MK-2206 300 μg/kg (in dimethyl sulfoxide) was injected intraperitoneally at 30 min before isoflurane inhalation.At 2 h of reperfusion,8 rats were selected and sacrificed,and the hearts were removed for determination of myocardial infarct size.At 2 h of reperfusion,8 rats were selected,and blood samples were collected from the right internal jugular vein for determination of serum cardiac troponin Ⅰ (cTnI) concentrations.The rats were then sacrificed,and myocardial specimens were obtained for determination of the expression of phosphorylated GSK-3β (p-GSK-3β) in cytoplasm and mitochondria (by Western blot) and co-expression of p-GSK-3β with adenine nucleotide translocator (ANT),voltage-dependent anion channel or cyclophilin D in myocardial tissues (using co-immunoprecipitation).At 2 h of reperfusion,8 rats were selected and sacrificed,myocardial cells were obtained,and the opening time of mPTP was determined with a laser scanning confocal microscope.Results Compared with group C,the myocardial infarct size and serum cTnI concentrations were significantly increased,and the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated in I/R and IPC groups,the co-expression of p-GSK-3β with ANT was significantly down-regulated,and the opening time of mPTP was shortened in group I/R,and the co-expression of p-GSK-3β with ANT was significantly up-regulated,and the opening time of mPTP was prolonged in group IPC (P＜0.05).Compared with group I/R,the myocardial infarct size and serum cTnI concentrations were significantly decreased,the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated,the co-expression of p-GSK-3β with ANT was significantly up-regulated,and the opening time of mPTP was prolonged in group IPC,and the opening time of mPTP was significantly prolonged (P＜0.05),and no significant change was found in the other parameters in group MK (P＞0.05).Compared with group IPC,the myocardial infarct size and serum cTnI concentrations were significantly increased,the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated,the co-expression of p-GSK-3β with ANT was significantly down-regulated,and the opening time of mPTP was shortened in group MK (P＜0.05).No co-expression of p-GSK-3β with voltage-dependent anion channel or cyclophilin D was found in myocardial tissues.Conclusion The mechanism by which isoflurane preconditioning inhibits mPTP opening during myocardial ischemia-reperfusion is partially related to activation of Akt/GSK-3β signaling pathway in rats.
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Objective To evaluate the role of extracellular signal-regulated kinase (ERK) signaling pathway in inhibition of oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat cortical neurons by sevoflurane and the relationship with mitochondrial permeability transition pore (mPTP).Methods The rat cortical neurons were cultured in vitro and seeded in 6-well or 12-well culture plates.The neurons were randomly divided into 4 groups (n =18 each) using a random number table:control group (group C);OGD/R group (group O);sevoflurane group (group OS);sevoflurane + ERK1/2 inhibitor PD98059 group (group OSP).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h in group O.The neurons were incubated with 2％ sevoflurane for 2 h after OGD/R in group OS.OGD/R was performed at 1 h after ERK1/2 inhibitor PD98059 30 μmol/L was added,and the neurons were incubated with 2％ sevoflurane for 2 h after OGD/R in group OSP.At 24 h of restoration of O2-glucose supply,the expression of phosphorylated ERK1/2 (p-ERK1/2) in neurons was measured by Western blot,the neuronal apoptosis was detected using Annexin V-FITC/PI double staining combined with flow cytometry,and the opening of mPTP was determined through measuring the optical density at 540 nm.The apoptosis rate was calculated.Results Compared with group C,the expression of p-ERK1/2 in neurons was significantly up-regulated,and the apoptosis rate and mPTP opening were significantly increased in group O (P＜0.05).Compared with group O,the expression of p-ERK1/2 in neurons was significantly up-regulated,and the apoptosis rate and mPTP opening were significantly decreased in group OS (P＜0.05).Compared with group OS,the expression of p-ERK1/2 in neurons was significantly down-regulated,and the apoptosis rate and mPTP opening were significantly increased in group OSP (P＜0.05).Conclusion The mechanism by which sevoflurane inhibits OGD/R-induced apoptosis in rat cortical neurons is related to inhibition of mPTP opening after activation of ERK signaling pathway.
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Objective To evaluate the role of etomidate post-conditioning on mitochondrial permeability transition pore (mPTP) in the rat cortical neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with Robo receptors.Methods The cortical neurons obtained from Sprague-Dawley rats (＜ 24 h after birth) were cultured in vitro and seeded in 6-well plates (2 ml/well).The neurons were divided into 4 groups (n=24 each) using a random number table:control group (group C),OGD/R group,etomidate post-conditioning group (group E),and etomidate post-conditioning + Robo receptor blocker group (group ER).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In E and ER groups,etomidate was added to the culture medium with the final concentration of 6 μmol/L immediately after onset of O2-glucose supply.In group ER,Robo blocker RoboN was added to the culture medium with the final concentration of 1 μg/ml at 6 h before O2-glucose deprivation.The neuronal apoptosis was detected using Hoechst/PI double staining,the viability of neurons was measured by MTT assay,and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.The mitochondria were extracted,and mitochondrial permeability transition pore (mPTP) opening was detected.Results Compared with group C,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in OGD/R,E and ER groups (P＜0.05).Compared with group OGD/R,the apoptosis rate,amount of LDH released,and mPTP opening were significantly decreased,and the cell survival rate was increased in group E,and the apoptosis and amount of LDH released were significantly decreased,and the cell survival rate was increased in group ER (P＜0.05).Compared with group E,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in group ER (P＜0.05).Conclusion Etomidate post-conditioning mitigates OGD/R-induced damage to the cortical neurons through activating Robo receptors and inhibiting mPTP opening in rats.
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Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in hepatocytes in reduction of hepatic ischemia-reperfusion (I/R) injury by limb ischemic postconditioning in rats.Methods Forty-eight healthy male Sprague-Dawley rats,weighing 240-280 g,were randomly divided into 4 groups (n =12 each) using a random number table:sham operation group (group S);I/R group;limb ischemic postconditioning group (group LIPC);limb ischemic postconditioning and atractyloside group (group LIPC + APC).In I/R,LIPC and LIPC + APC groups,I/R injury was induced by 30 min occlusion of the hepatic artery and portal vein entering the left and middle lobes of the liver,followed by 120 min of reperfusion.The animals underwent 10 min of bilateral limb ischemia starting from 20 min after liver ischemia in group LIPC.In group LIPC+APC,the animals underwent 10 min of bilateral limb ischemia starting from 20 min after liver ischemia,and atractyloside 5 mg/kg was infused simultaneously via the penile vein.Blood samples were collected from inferior vena cava at the end of reperfusion to detect plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities.The rats were sacrificed,and the left lobe of the liver was removed to detect the degree of mPTP opening and to examine the ultrastructure of hepatocytes under electron microscope.Results Compared with group S,the plasma ALT and AST activities and degree of mPTP opening were significantly increased,and the damage to the ultrastructure of hepatocytes was obvious in the other three groups.Compared with group I/R,the plasma ALT and AST activities and degree of mPTP opening were significantly decreased,and the damage to the ultrastructure of hepatocytes was reduced in LIPC and LIPC + APC groups.Compared with group LIPC,the plasma ALT and AST activities and degree of mPTP opening were significantly increased in group LIPC+APC.Conclusion Limb ischemic postconditioning reduces hepatic I/R injury through inhibiting mPTP opening in hepatocytes of rats.
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Objective To evaluate the effect of mitochondrial permeability transition pore (mPTP) in lipid emulsion-induced inversion of bupivacaine myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 6-well plates at a density of 105 cells/ml, and were randomly divided into 4 groups (6 wells in each group, 2 ml/well) using a random number table: control group (group C) , bupivacaine group (group B) , lipid emusion + bupivacaine group (group LB) , and lipid emusion + bupivacaine + atractyloside group (group LBA).Phosphate buffer solution 100 μl was added to the culture medium in group C.In group B, bupivacaine was added to the culture medium with the final concentration of 1 mmol/L.In group LB, lipid emusion and bupivacaine were added to the culture medium with the final concentrations of 1％ and 1 mmol/L, respectively.In group LBA, lipid emusion, bupivacaine and atractyloside (an mPTP opener) were added to the culture medium with the final concentrations of 1％, 1 mmol/L and 30 μmol/L, respectively.All the cells were incubated for 24 h.After the end of incubation, the expression of Bcl-2, Bax, phosphorylated Bad (p-Bad) , caspase-3, activated caspase-3, caspase-9,activated caspase-9 and cytochrome c (Cyt c) was detected using Western blot.The expression of Bcl-2 mRNA, Bax mRNA, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was detected using real-time reverse transcriptase polymerase chain reaction.The ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were calculated.Results Compared with group C,the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/ Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group B (P＜0.05) , and no significant change was found in the parameters mentioned above in group LB (P＞0.05).Compared with group B, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly decreased, the expression of p-Bad was up-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was down-regulated in LB and LBA groups (P＜ 0.05).Compared with group LB, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group LBA (P ＜ 0.05).Conclusion The mechanism underlying lipid emulsioninduced inversion of bupivacaine myocardiotoxicity is related to inhibited mPTP opening in rats.
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Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration (OGD/ R) in rat neurons.Methods Cortical neurons isolated from neonatal Sprague-Dawley rats born within 24 h, were cultured primarily and seeded in 6-well plates (2 ml/well) at a density of 1 × 106 cells/ml.The neurons were randomly divided into 5 groups (n =72 each) using a random number table: control group (group C), OGD/R group (group O), mPTP opener group (group A), mPTP opener + sevoflurane group (group AS) , and sevoflurane group (group Sev).The cells were cultured in normal culture medium in group C, and the cells were subjected to OGD for 90 min followed by restoration of O2-glucose supply for 24 h in the other groups.In A and AS groups, mPTP opener atractyloside 20 μmol/L was added immediately after OGD.In Sev and AS groups, the cells were post-conditioned with 2％ sevoflurane for 1 h immediately after OGD.The neurons were collected at 24 h of OGD in O and A groups, or at 23 h of OGD in Sev and AS groups.Annexin V-FITC/PI double staining was performed to count apoptotic neurons, apoptotic rate was calculated, and cell survival rate was measured using MTT assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescence probe as indicator.The opening of mPTP was determined through assessing the changes of mitochondrial optical density (△OD540 of mPTP).The expression of pro-apoptotic factors Bid, Bim, Puma protein and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction and Western blot.Results Compared with group C, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in the other groups.Compared with group O, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in Sev group.Compared with group Sev, apoptotic rate was significantly increased,cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in A and As groups.Compared with group A, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in group AS.Conclusion Sevoflurane mitigates OGD/R-induced apoptosis in rat neurons through inhibiting mPTP opening.
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Objective To investigate the effect of hydromorphone postconditioning on ischemiareperfusion (I/R) injury in isolated rat hearts and the role of mitochondial permeability transition pore (mPTP).Methods Male Sprague-Dawley rats, aged 2-3 months, weighing 250-320 g, were used in the study.The rats were heparinized and anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.The hearts were excised, and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 36.5-37.5 ℃.Forty isolated rat hearts were randomly divided into 4 groups (n=10 each)using a random number table: control group (group C), group I/R, hydromorphone postconditioning group (group H), and hydromorphone postconditioning + mPTP opener lonidamine group (group HL).Group C was continuously perfused with K-H solution for 120 min.Group I/R was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group I/R was perfused with K-H solution for another 30 min.Group H was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group H was perfused with K-H solution containing 0.3 μmol/L hydromorphone for 10 min, and then with K-H solution for 50 min.Group HL was perfused with K-H solution for 30 min, the perfusion was then suspended for 30 min, and group HL was perfused with K-H solution containing 0.3 μmol/L hydromorphone and 30 μmol/L lonidamine for 10 min, and then with K-H solution for 50 min.At 30 min of equilibration (T0), and 30 and 60 min of reperfusion (T2,3), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) , ±dp/dtmax, heart rate (HR), and coronary flow (CF) were measured.The concentrations of lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB) , and troponin-T (Tn-T) in the coronary effluent were determined at T0 and T3.The coronary effluent was collected at T0 and 15 min of reperfusion (T1),nicotinamide-adenine dinucleotide (NAD+) concentrations were measured using enzyme-linked immunosorbent assay to reflect the degree of mPTP opening.The myocardial infarct size was determined at T3 by TTC staining.Results Compared with group C, LVDP, HR, ±dp/dtmax and CF were significantly decreased, and LVEDP was increased at T2,3, and the concentrations of LDH, CK-MB and Tn-T in the coronary effluent, myocardial infarct size at T3, and NAD+ concentrations in the coronary effluent at T1 were increased in group I/R (P＜0.05).Compared with I/R and HL groups, LVDP, ±dp/dt CF and HR were significantly increased, and LVEDP was decreased at T2,3, and the concentrations of LDH, CK-MB and Tn-T in the coronary effluent, myocardial infarct size at T3, and NAD+ concentrations in the coronary effluent at T1 were decreased in group H (P＜0.05).Conclusion Hydromorphone postconditioning can reduce myocardial I/R injury in isolated rat hearts, and the mechanism is related to inhibition of mPTP opening.
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Objective To evaluate the effects of isoflurane postconditioning on mitochondrial permeability transition pore (mPTP) in brain tissues of neonatal rats with hypoxic-ischemic brain injury.Methods One hundred and twenty 7-day-old Sprague-Dawley rats,weighing 12-16 g,were randomly divided into 4 groups (n =30 each) using a random number table:sham operation group (group S),isoflurane group (group I),hypoxicischemic brain injury group (group HIBI),and hypoxic-ischemic brain injury + isoflurane postconditioning group (group HI).To establish hypoxic-ischemic brain injury model in the neonatal rats,the left common carotid artery ligation was carried out,and then the rats were exposed to 8％ O2 + 92％ N2 at 37 ℃ for 2 h in HIBI and HI groups.The rats inhaled 1.5 ％ isoflurane for 30 min after the model was established in group HI.The rats only inhaled 1.5％ isoflurane for 30 min in group I.At 24 h after the model was established,10 rats taken out randomly in each group were sacrificed and brains were removed to detect mPTP opening.At 7 days after the model was established,the survival rate was recorded in the rest rats.The rats were then sacrificed and brains were removed and the right and left cerebral hemispheres were weighed separately,and the ratio between left/right cerebral hemispheres was calculated.The density of normal neurons in ventral posterior inferior thalamic nucleus and hippocampal CA3 region in the left and right cerebral hemispheres were measured and the ratios of the density of normal neurons in the left to right cerebral hemisphere were calculated.Results There was no significant difference in the survival rate between the four groups (P ＞ 0.05).Compared with group S,the ratios of the density of normal neurons in the left to right cerebral hemisphere,weight of left cerebral hemisphere,and ratio between left/right cerebral hemispheres were significantly decreased,and mPTP opening was increased in group HIBI (P ＜ 0.05),and no significant changes were found in the parameters mentioned above in group I (P ＞ 0.05).Compared with group HIBI,the ratios of the density of normal neurons in the left to right cerebral hemisphere,weight of left cerebral hemisphere,and ratio between left/right cerebral hemispheres were significantly increased,and mPTP opening was decreased in group HI (P ＜ 0.05).Conclusion The mechanism by which isoflurane postconditioning reduces hypoxic-ischemic brain injury may be related to inhibition of mPTP opening in brain tissues of neonatal rats.
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Objective To enhance clinicians' intention to the importance of early diagnosis,early therapy and follow-up of type Ⅱ citrullinemia.Methods The clinical data of one adult-onset type Ⅱ citrullinemia pedigree were collected. The gene mutation type of SLC25A13 of proband and her daughter were determined by PCR and direct gene sequencing.Results The patient was a 27 years-old female,who complained of repeated dizziness, vomiting for more than 2 years and recurrent attacks of altered consciousness for about one and a half year.An abdominal ultrasonogram,liver magnetic resonance imaging and liver histology obtained by needle biopsy all determined the liver pathological changes of liver cirrhosis.Electroencephalogram showed sharp waves. The plasma amino acid showed a marked elevation of blood citrulline.Laboratory findings revealed a highly increased concentration of plasma ammonia during every episode. Mutation analysis of the SLC25A13 gene identified a homozygote of 851del4 in the patient,and heterozygote of 851del4 in her daughter. Conclusions For adults,unexplained dizziness,vomiting,but liver function still in the compensation,especially accompanied by neuropsychologic symptoms are highly suggestive of adult-onset type Ⅱ citrullinemia.SLC25A13 gene analysis contributes to the diagnosis of this disease,avoids invasive investigations and early confirmation of this disease means long-term dietary advice,genetic counseling,medical surveillance and early preparation for liver transplantation if is necessary.
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ObjectiveTo investigate the effect of dexmedetomidine postconditioning on mitochondria injury during myocardial ischemia-reperfusion (I/R) in isolated rat hearts.Methods Healthy female Wistar rats weighing 220-250 g were anesthetized with intraperitoneal 3％ pentobarbital 50 mg/kg and heparin 500 U/kg.Their hearts were excised and perfused in a Langenorff apparatus with modified K-H solution saturated with 95％ O2-5％ C02 at 37 ℃.Forty isolated rat hearts were randomly divided into 5 groups( n =8 each): I/R group(group A),dexmedetomidine 10 nmol/L group ( group B),dexmedetomidine 100 nmol/L group ( group C ),atractyloside ( the mitochondrial permeability transitionpore (mPTP) opener) group (group D)and dexmedetomidine 100 nmol/L + atractyloside group(group E).Myocardial I/R injury was induced by 40 min of global ischemia followed by 60 min of reperfusion.10 nmol/L dexmedetonidine( group B),100 nmol/L dexmedetonidine (group C),20 μmol/L atractyloside(group D) or 100 nmol/L dexmedetomidine + 20 μmol/L atractyloside (group E) was added into K-H solution and perfused for 10 min at the beginning of reperfusion.The myocardial tissues were obtained and mitochondria were isolated at the end of reperfusion for determination of activity of SOD,Na+ -K+ -ATPase,and Ca2+ -ATPase and content of MDA and Ca2+.ResultsThe activity of SOD,Na+ -K+ -ATPase and Ca2+ -ATPase was significantly higher and MDA and Ca2+ content lower in groups B and C than in group A( P ＜ 0.05).The activity of SOD,Na+ -K+ -ATPase and Ca2+ -ATPase was lower and MDA and Ca2+ content higher in groups E and D than in group C (P ＜ 0.05).There was no significant difference in activity of SOD,Na+ -K+ -ATPase and Ca2+ -ATPase and MDA and Ca2+ content between groups B and C,and between groups A and D( P ＞ 0.05).Conclusion Dexmedetomidine postconditioning can reduced mitochondria injury during myocardial I/R in isolated rat hearts through inhibiting of mPTP opening.
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Objective To investigate the role of mitochondrial permeability transition pore (mPTP) in attenuation of myocardial ischemia-reperfusion (I/R) injury by delayed preconditioning with sevoflurane in rats.Methods Eighty male SD rats, weighing 250-300 g, were randomly assigned into 5 groups ( n = 16 each): Ⅰsham operation group (group S), Ⅱ group I/R, Ⅲ sevoflurane delayed preconditioning group (group SP), Ⅳ the mPTP opener atractyloside + sevoflurane delayed preconditioning group (group A + SP), and Ⅴ atractyloside group (group A). Myocardial I/R was induced by ligation of anterior descending branch of left coronary artery for 30 min followed by 120 min of reperfusion in group I/R, SP, A + SP and A. In group SP and A + SP, 2.5%sevoflurane was inhaled for 1 h, while pure oxygen was inhaled for 1 h in the other groups, and then myocardial ischemia was performed 24 h later. In group A + SP and A, atractyloside 5 mg/kg was injected intravenously via caudal vein 15 min before ischemia. Blood samples were taken from carotid arteries for detection of serum cardiac troponin-Ⅰ (cTnI) concentrations at the end of reperfusion. Then the rats were sacrificed and hearts removed. The myocardial infarct size (IS) and expression of Bcl-2 and Bax in the myocardium were determined. Myocardial ultrastructure was examined with the electron microscope. Results Serum cTnI concentrations and Bax expression were significantly higher, the myocardial IS was significantly larger and Bcl-2 expression was significantly lower in the other groups than in group S ( P ＜ 0.05). Serum cTnI concentrations and Bax expression were significantly lower, the myocardial IS was significantly smaller and Bcl-2 expression was significantly higher in group SP than in group I/R ( P ＜ 0.05). Microscopic examination showed less damage in group SP than in group I/R. The protection provided by sevoflurane preconditioning was abolished by atractyloside. Conclusion Inhibition of mPTP opening can result in an up-regulation of Bcl-2 expression and down-regulation of Bax expression, which plays a role in attenuation of myocardial I/R injury by delayed preconditioning with sevoflurane in rats.
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Objective To explore the expression and significance of uncoupling protein (UCP)2in rats models of acute liver failure (ALF). Methods Thirty-six healthy male SD rats were randomly divided into normal control group and model group, and the model group was divided into 5 subgroups:6, 12, 24, 36 and 48 hours sub groups with 6 rats in each sub group. The rat model of ALF was established by intraperitoneal injections of D-galactosamine (D-Gal) and lipopolysaccharide (LPS).Sections of liver tissue were stained with hematoxylin and eosin and observed under optical microscope.UCP2 and UCP2 mRNA in rat liver were determined at different time points with immunohistochemical method and reverse transcription-polymerase chain reaction ( RT-PCR ),respectively. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and malondialdehyde (MDA) concentration in the liver tissues were analyzed at the same time points.Comparisons among all the experimental groups were done by SNK test. Results Infiltration of inflammatory cells and necrosis of hepatic cells were marked in model group,and ALT, AST and MDA in model group were significantly higher than those in control group [(24. 0 ± 2. 0) U/L, (82. 3±16. 9) U/L, (2. 55±0. 22)μmol/g] at all time points. And they reached a peak at 24 h [(8346. 7±1363. 1) U/L, (9766. 7±1274. 1) U/L, (8. 34±1. 13) μmol/g; all P<0. 05]. UCP2 and UCP2 mRNA expressed scarcely in the liver tissues of control group, while increased markedly from 6 to 48 hours after D-Gal/LPS challenge in model group (P<0. 05). They both reached a peak at 24 h. And the discrepancy between consecutive experimental group had statistical significance ( P < 0. 05).Conclusions The rat model of ALF was established successfully by intraperitoneal injections of D-gal and LPS. The expression levels of UCP2 mRNA and UCP2 are consistent with the extent of liver injury and the level of oxidative stress in the rat model of ALF.