Objective To investigate the effect of hydrogen sulfide (H 2 S or NaHS)on myo-cardial ischemia reperfusion injury induced in type 2 diabetic rats in vivo and the role of AMP-activated protein kinase (AMPK)signal pathway.Methods The induced type 2 diabetic rat models were anesthetized,left thoracotomy were performed.All the models were randomly divided into six groups (n = 14):group Sham;group IR:the left anterior descending artery was ligated 30 min, reperfused for 4 hours;group CC:prior to thoracotomy,compound c was intraperitoneally injected 250 μg/kg,then received the same treatment as group IR;group DMSO received the same treatment as compound c group but DMSO was injected intraperitoneally as control;group NaHS:the left ante-rior descending artery was injected NaHS 0.05 mg/kg then reperfused for 4 hours;group CC +NaHS:prior to thoracotomy,compound c was intraperitoneally injected 250 μg/kg,then NaHS 0.05 mg/kg injected intravenously and reperfused 4 hours.All the rat models euthanatized,infarcted area was detected by TTC assay.The AMPK,LC3 and p62 were analyzed by Western blot.Results Com-pared with group Sham,the infarcted area and concentration of AMPK,LC3 and p62 were increased in other groups (P <0.05).Compared with group IR,the infarcted area and concentration of LC3, p62 markablely decreased in group NaHS (P < 0.05 ).Compared with group NaHS,the infarcted area and concentration of LC3,p62 significantly increased but AMPK down-regulated in group CC+NaHS (P <0.05).Conclusion Hydrogen sulfide could alleviate myocardial infraction via AMPK sig-nal pathway in type 2 diabetic rats'IR models.

Objective To investigate the effect of microRNA-1 (miR-1) on the fibrosis of cardiac fibroblasts induced by high glucose. Methods The primary cultured fibroblasts from 1-3 days old Sprague-Dawley (SD) rats, were randomly divided into 4 groups (n = 3): normal glucose + lentivector-vehicle (CON+Lv-Vehicle group), normal glucose + lentivector-miR-1 (CON+Lv-miR1 group), high glucose + lentivector-vehicle (HG+Lv-Vehicle group), high glucose + lentivector-miR-1 (HG+Lv-miR1 group). Fibroblasts were cultured in glucose concentration 5.5 mmol/L and 25 mmol/L DMEM culture, and were injected lentiviral vector carrying miR-1 silencer sequence or the same volume of lentiviral vector. After 12 hours, the medium was replaced with fresh complete medium. After 3 days when transfection efficiency was up to 90%, the cellular miR-1 content was detected by real-time reverse transcription-polymerase chain reaction (qRT-PCR). The secretion of collagen Ⅰ and Ⅲ were measured by enzyme linked immunosorbent assay (ELISA). Expression of collagen Ⅰ and Ⅲ, matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), autophagy related protein LC3B-Ⅱ, P62/SQSTM1 and Cathepsin D were assessed by Western Blot. Results Compared with the CON+Lv-Vehicle group, the content of miRNA in the CON+Lv-miR1 group had no statistical significance. Compared with the CON+Lv-Vehicle group, high glucose increased the amount of miR-1 (2-ΔΔCt: 1.82±0.17 vs. 1.00±0.04), collagen Ⅰ and Ⅲ secretion (mg/L: 14.55±0.33 vs. 7.28±0.22, 157.50±13.22 vs. 61.25±8.54) and expression (gray value: 432.35±56.00 vs. 100.00±15.00, 320.35±47.00 vs. 100.00±15.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 249.0±21.0 vs. 100.0±15.0, 142.3±20.0 vs. 100.0±16.0, 178±19 vs. 100±14, 378.3±20.0 vs. 100.0±15.0), decreased the expression of lysosomal associated protein Cathepsin D (gray value: 60±14 vs. 100±10), and the differences were statistically significant (all P < 0.01). Compared with the HG+Lv-Vehicle group, the amount of miR-1 in the HG+Lv-miR1 group was significantly decreased (2-ΔΔCt: 1.21±0.10 vs. 1.82±0.17), collagen Ⅰ and Ⅲ secretion (mg/L: 10.68±0.54 vs. 14.55±0.33, 87.25±13.55 vs. 157.50±13.22) and expression (gray value: 179.41±45.00 vs. 432.35±56.00, 173.41±50.00 vs. 320.35±47.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 172.0±23.0 vs. 249.0±21.0, 90.0±17.0 vs. 142.3±20.0, 138±15 vs. 178±19, 265.0±17.0 vs. 378.3±20.0) in the HG+Lv-miR1 group were decreased and the expression of lysosomal associated protein Cathepsin D was higher (gray value: 110±17 vs. 60±14), and the differences were statistically significant (all P < 0.05). Conclusions The expression of miRNA-1 was up-regulated in cardiac fibroblasts cultured in high glucose, and miRNA-1 silencing inhibited cardiac fibroblast induced fibrosis in high glucose. The mechanism may be related to the recovery of autophagy flux, up-regulation of Cathepsin D expression and inhibition of collagen production.

Objective To evaluate the effects of sevoflurane postconditioning on mitophagy during myocardial ischemia-reperfusion (I/R) in rats.Methods Forty-two pathogen-free adult male SpragueDawley rats, weighing 250-300 g, were randomly divided into 3 groups (n=14 each) using a random number table: sham operation group (group S) , I/R group and sevoflurane postconditioning group (group SP).Myocardial I/R was induced by 30 min ligation of the left anterior descending branch of the coronary artery followed by 2 h of reperfusion.In group SP, 2.4％ sevoflurane was inhaled for 15 min starting from the onset of reperfusion, while 33％ oxygen was inhaled in group I/R.The rats were sacrificed at the end of reperfusion, and the hearts were removed for measurement of myocardial infarct size (by 1％ 2, 3, 5 triphenyltetrazolium chloride) , expression of LC3 Ⅱ/LC3 Ⅰ , Beclin-1, p62 and Parkin (by Western blot) ,and mitochondrial menbrane potential (by using JC-1 probe) , and for examination of the uhrastructure of cardiomyocytes (with transmission electron microscope).Results Compared with group S, the myocardial infarct size was significantly increased, mitochondrial membrane potential was decreased, the expression of LC3 Ⅱ/LC3 Ⅰ , Beclin-1 and Parkin was up-regulated, and the expression of p62 was down-regulated in group I/R.Compared with group I/R, the myocardial infarct size was significantly decreased, the mitochondrial membrane potential was increased, and the expression of LC3 Ⅱ/LC3 Ⅰ , Beclin-1, p62 and Parkin was down-regulated in group SP.Conclusion Sevoflurane postconditioning can mitigate I/R injury in rats, and inhibition of excessive activation of mitophagy may be involved in the nechanism.

Objective To evaluate nuclear factor (NF)-κB signaling pathway and autophagy in inhaled sevoflurane-produced delayed myocardial protection in rats.Methods Ninety-six adult male Sprague-Dawley rats,weighing 270-350 g,were randomly assigned into 6 groups (n =16 each):sham operation group (group S),ischemia-reperfusion (I/R) group,sevoflurane group (SEVO group),specific NF-κB inhibitor parthenolide (PTN)group,dimethyl sulfoxide (DMSO) group and PTN + sevoflurane group (PTN + SEVO group).The animals were anesthetized with intraperitoneal pentobarbital 50 mg/kg,intubated and mechanically ventilated.Myocardial I/R was induced by 30 min of occlusion of the left anterior descending branch of coronary artery followed by 2 h of reperfusion.In group I/R,33％ oxygen was inhaled for 2 h.In group SEVO,2.5％ sevoflurane was inhaled for 2 h.In groups PTN and DMSO,PTN 500 μg/kg and DMSO were administered intraperitoneally 15 min before oxygen inhalation respectively.In group PTN + SEVO,PTN 500 μg/kg was administered intraperitoneally 15 min before exposure to sevoflurane.Myocardial I/R was induced 24 h after intraperitoneal administration.Eight animals in each group were sacrificed immediately before ischemia and the hearts were removed to detect the NF-κB activity and expression of LC3-Ⅱ and cathepsin B.The left animals in each group were sacrificed at 2 h of reperfusion and the hearts were removed to determine the myocardial infarct size (by TTC staining).Results Compared with group S,the myocardial infarct size was significantly increased at 2 h of reperfusion in the other groups,and the NF-κB activity was significantly increased and the expression of LC3-Ⅱ and cathepsin B was up-regulated immediately before ischemia in group SEVO (P ＜ 0.05).Compared with group I/R,the NF-κB activity was significantly increased and the expression of LC3-Ⅱ and cathepsin B was up-regulated immediately before ischemia,and the myocardial infarct size was significantly reduced at 2 h of reperfusion in group SEVO (P ＜ 0.05).Compared with group SEVO,the NF-κB activity was significantly decreased and the expression of LC3-Ⅱ and cathepsin B was down-regulated immediately before ischemia,and the myocardial infarct size was significantly increased at 2 h of reperfusion in DMSO,PTN and PTN + SEVO groups (P ＜ 0.05).Conclusion NF-κB signaling pathway and autophagy are involved in inhaled sevoflurane-produced delayed nyocardial protection in rats.

Objective To investigate the effect of sevoflurane (Sero) preconditioning (Precon) on cardiomyocyte apoptosis following myocardial ischemia-reperfusion (I/R) in rats. Methods Sixty-four adult male SD rats weighing 270-350 g were randomly divided into 4 groups ( n = 16 each): group Ⅰ sham operation (group S); group Ⅱ myocardial I/R; group Ⅲ Sero and group Ⅳ Sevo-Precon + myocardial I/R. The animals were anesthetized with intraperitoneal pentobarbital 50 mg/kg, intubated and mechanically ventilated. PET CO2 was maintained at 35-45 mm Hg. Myocardial I/R was induced by 30 min occlusion of the left anterior descending branch of coronary artery followed by 2 h reperfusion in group Ⅱ and Ⅳ . In group Ⅲ the animals inhaled 2.5 % sevoflurane for 30 min while in group Ⅳ the animals inhaled 2.5% sevoflurane for 30 min at 15 min before myocardial I/R. Eight animals were killed at the end of 2 h reperfusion in each group. The hearts were removed for determination of myocardial infarct size (IS) as a percentage of area at risk (AAR) (IS/AAR) by triphenyl tetrazolium chloride staining. Myocardial apoptosis was detected using TUNEL and apoptosis index (AI) was calculated. Another 4 animals were killed before ischemia and at the end of 2 h reperfusion for determining the expression of Bcl-2 and caspase-3 protein in myocardium by Western blot. Results Sevoflurane preconditioning significantly decreased infarct size and AI in group Ⅳ as compared with group Ⅱ (group I/R). Bcl-2 protein expression was significantly decreased and caspase-3 protein expression was significantly increased after 2 h reperfusion as compared with the expression before ischemia in group I/R (group Ⅱ ). Sevoflurane preconditioning significantly reversed the I/R-induced changes in Bcl-2 and caspase-3 protein expression. Conclusion Sevoflurane preconditioning can attenuate myocardial I/R injury by decreasing myocardial apoptosis.

Objective To evaluate role of autophagosomes clearance in delayed cardioprotection by sevoflurane preconditioning in rats with ischemia-reperfusion injury in vivo.Methods Forty-five adult male Sprague-Dawley rats,weighing 270-350 g,were randomly (random number) divided into 3 groups:sham operation group (sham group),ischemia-reperfusion group (CON group),sevoflurane preconditioning group (SWOP group).Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch (LAD) of coronary artery followed by reperfusion for 2 h,and myocardial infarct size was stained by triphenyltetrazolium chloride.Cardiomyocyte apoptosis was evaluated by terminal deoxyribonucleotidyl transferase-mediated biotin-16dUTP nick-end labeling.Autophagosomes were detected under transmission electron microscope.Expression of LC3-Ⅱ,cathepsin B,p62 and cleaved caspase-3 were assessed by western blotting.Statistical analysis were performed using one or two way analysis of variance (SPSS 15.0,Chicago,USA) test followed by Dunnet-t or LSD-t test.Results Sevoflurane preconditioning reduced myocardial infarct size and the number of autophagosomes (P =0.027),attenuated cardiomyocyte apoptosis (P =0.042).Sevoflurane preconditioning decreased the level of LC3-Ⅱ (P =0.033),p62 (P =0.041)and cleaved caspase-3 (P =0.037),but increased the level of cathepsin B (P =0.046).Conclusions Delayed cardioprotection by sevoflurane preconditioning increased myocardial clearance of autophagosomes against the delayed ischemia reperfusion injury.

Objective To evaluate the effects of sevoflurane postconditioning on the expression of 70 kD aribosomalprotein S6 kinase (p70S6K) during ischemia-reperfusion (I/R) in isolated rat hearts.Methods Pathogen-free male Sprague-Dawley rats,aged 3 months,weighing 270-350 g,were used in the study.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95 ％ O2-5 ％ CO2.Ninety isolated rat hearts with I/R injury were randomly divided into 3 groups (n =30 each):sham operation group (group S),I/R group (group I/R),and sevoflurane postconditioning group (group SP).The hearts were subjected to ischemia for 30 min followed by 2 h reperfusion.In group SP,the hearts were perfused with K-H solution saturated with 3.0％ sevoflurane for 15 min starting from the end of ischemia until 15 min of reperfusion,and then with plain K-H solution for 105 min.At 2 h of reperfusion,myocardial infarct size was measured,the percentage of myocardial infarct size was calculated,and the phosphorylated p70S6K (p-p70S6K)/total p70S6K (tp70S6K) ratio,and cytoplasm,cytochrome C,and caspase-8 expression was measured.Results Compared with group S,the percentage of myocardial infarct size and p-p70S6K/t-p70S6K ratio were significantly increased,the expression of cytochrome C,and caspase-8 was up-regulated,and the expression of cytochrome C was downregulated in I/R group.Compared with I/R group,the percentage of myocardial infarct size was significantly decreased,the ratio of p-p70S6K/t-p70S6K was increased and cytochrome C expression was up-regulated,and the expression of cytochrome C and caspase-8 was down-regulated in SP group.Conclusion Sevoflurane postconditioning can mitigate I/R injury to isolated rat hearts,and up-regulation of p-p70S6K expression,inhibition of transfer of cytochrome C from mitochondria to cytoplasm,and reduced cell apoptosis are involved in the mechanism.

Objective To evaluate the role of nitric oxide in sevoflurane postconditioning-induced mitigation of autophagy and cell apoptosis during ischemia/reperfusion (I/R) in isolated rat hearts.Methods The hearts of male Sprague-Dawley rats,aged 2-3 months,weighing 250-300 g,were excised and retrogradely perfused in a Langendorff apparatus.One hundred and eight isolated rat hearts,which were successfully perfused in a Langendorff apparatus,were equally and randomly divided into 6 groups:control group (C group),sevoflurane group (S group),I/R group,sevoflurane postconditioning group (SSP group),sevoflurane postconditioning + L-NAME (non-selective nitric oxide synthase (NOS) inhibitor group (SSP + L group),and L-NAME group (L group).The hearts were perfused with K-H solution for 150 min in C group.The hearts were continuously perfused for 180 min and perfused with K-H solution containing 3％ sevoflurane for 15 min starting from 60 min of perfusion in S group.After being perfused with K-H solution for 30 min,the hearts were subjected to occlusion for 30 min followed by reperfusion for 120 min in the other groups except C and S groups.After onset of reperfusion,the hearts were perfused with K-H solution containing 3％ sevoflurane for 15 min in SSP group,the hearts were perfused with K-H solution containing 3％ sevoflurane and L-NAME 100 μmol/L for 15 and 60 min,respectively,in SSP + L group,and the hearts were perfused with K-H solution containing L-NAME 100μmol/L for 60 min in L group.Inn ediately before ischemia,and at 30,60,90 and 120 min of reperfusion,each parameter of cardiac function was recorded.At the end of reperfusion,myocardial specimens were obtained at the end of reperfusion for measurement of the infarct size,NOS activity,NO content,and expression of Bcl-2,Beclin 1 and caspase-3,for observation of formation of autophagosomes,and for examination of the pathological changes.Results Compared with C group,LVSP,+ dp/dtmax,-dp/dtmax,NOS activity and NO content were significantly decreased,and LVEDP was increased in I/R and SSP groups.Compared with I/R group,LVSP,+ dp/dtmax,-dp/dtmax,NOS activity and NO content were significantly increased,LVEDP was decreased,Bcl-2 expression was down-regulated,and the expression of Beclin 1 and caspase-3 was up-regulated in SSP group,and no significant changes were found in each index in SSP+ L and L groups.Compared with SSP group,LVSP,+ dp/dtmax,-dp/dtmax,NOS activity and NO content were significantly decreased,LVEDP was increased,Bcl-2 expression was down-regulated,and the expression of Beclin 1 and caspase-3 was up-regulated in SSP + L group.Conclusion The mechanism by which sevoflurane postconditioning reduces I/R injury may be related to promoted NO product and inhibited autophagy and cell apoptosis in isolated rat hearts.

Objective To investigate the effects of sevoflurane delayed preconditioning on caspase recruitment domain (ARC) expression during myocardial ischemia-reperfusion (I/R) in rats. Methods Sixty-four adult male SD rats weighing 270-350 g were randomly divided into 4 groups ( n = 16 each): sham operation (group S); myocardial I/R group; sevoflurane + sham operation group (group S-S) and sevoflurane delayed preconditioning + myocardial I/R group (group S-I/R) . Myocardial I/R was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 2 h of reperfusion in groups I/R and S-I/R. Group S-S inhaled 33% oxygen for 2 h, and sham operation was performed 24 h later. Group S-I/R inhaled 2.5% sevoflurane for 2 h, and then myocardial I/R was induced 24 h later. Eight animals were sacrificed at the end of 2 h reperfusion in each group and the hearts removed for determination of myocardial infarct size (IS) as a percentage of area at risk (AAR) by triphenyl tetrazolium chloride staining (IS/AAR) . Myocardial apoptosis was detected using TUNEL and apoptosis index was calculated. Another 4 animals were sacrificed immediately before ischemia and at the end of 2 h reperfusion to determine the expression of ARC and Caspase-8 in myocardium by Western blot. Results Compared with group S, the infarct size and apoptosis index were significantly increased in groups I/R and S-I/R, and ARC expression was up-regulated immediately before ischemia in groups S-S and S-I/R, and Caspase-8 expression was up-regulated at 2 h of reperfusion in group I/R ( P ＜ 0.05) . Compared with group I/R, the infarct size and apoptosis index were significantly decreased in group S-I/R, and ARC expression was up-regulated, while Caspase-8 expression was down-regulated at 2 h of reperfusion in groups S-S and S-I/R ( P ＜ 0.05) . Conclusion Sevoflurane delayed preconditioning can attenuate myocardial I/R injury through up-regulating the ARC expression and decreasing the myocardial apoptosis.

Objective To evaluate the role of the mitochondrial ATP-sensitive potassium (mito-KATP)channel in sevoflurane preconditioning-induced delayed cardioprotection against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Seventy-two adult male Sprague-Dawley rats were randomly divided into 6 groups (n =12 each):control group (group CON),I/R group,sevoflurane control group (group SEVO),sevoflurane preconditioning group (group SWO P),5-hydroxydeconoate (5-HD) + sevoflurane preconditioning group (group 5-HD+ SWOP) and 5-HD control group (group 5-HD).The rats were exposed to 33％ pure oxygen for 1 h in groups CON and I/R.The rats were exposed to 2.5％ sevoflurane for 1 h in groups SEVO and SWOP.5-HD (a mito-KATP channel inhibitor) 10 mg/kg was injected intraperitoneally 30 min before sevoflurane preconditioning in group 5-HD + SWOP.5-HD 10 mg/kg was injected intraperitoneally in group 5-HD.The hearts were immediately removed and perfused in a Langendorff apparatus.The hearts were made globally ischemic for 30 min followed by 120 min reperfusion in groups I/R,SWOP,5-HD + SWOP and 5-HD.The expression of phosphorylated protein kinase C-epsilon (p-PKC-ε) and caspase-8 was measured by Western blot immediately before ischemia (T0) and at 120 min of reperfusion (T1).The myocardial infarct volume was measured by TTC staining.Results Compared with group CON,the myocardial infarct volume was significantly increased at T1 in groups I/R,SWOP,5-HD +SWOP and 5-HD,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP and at T1 in groups I/R,SWOP,5-HD + SWOP and 5-HD,and caspase-8 expression was down-regulated at T1 in group SEVO (P ＜0.05).Compared with group I/R,the myocardial infarct volume was significantly decreased at T1 in groups SWOP and 5-HD + SWOP,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP,and caspase-8 expression was down-regulated at T1 in group SWOP (P ＜ 0.05).Compared with group SWOP,the myocardial infarct volume was significantly increased,p-PKC-ε expression was down-regulated at T0,and caspase-8 expression was up-regulated at T1 in group 5-HD + SWOP (P ＜ 0.05).Conclusion The mito-KATP channel is involved in sevoflurane preconditioning-induced delayed cardioprotection against I/R injury in isolated rat hearts through upregulation of p-PKC-ε expression before ischemia and inhibition of cell apoptosis during reperfusion.