Mechanism Research Of Sevoflurane Postconditioning On Myocardial Ischemia/reperfusion Injury In Vitro Rats

Part 1 Sevoflurane postconditioning alleviates myocardialischemia-reperfusion injury in rats in vitroObjective To investigate the protection of Sevoflurane postconditioning on myocardial ischemia reperfusion injury by observing the myocardial apoptosis and infarction in rats heart in vitroMethods Langendorff isolated perfused rat hearts were randomly divided into 4 groups(n=8): SHAM group, sevoflurane group(s group); myocardial I/R injury group(I/R group), sevoflurane postconditioning group(SEVOP group).(1) the SHAM group: 180 min k-h liquid continuous perfusion;(2) the group S: k-h liquid continuous perfusion 60 min, and in the 60 min give containing 2.5% sevoflurane(batch number: 2217, pill stone pharmaceutical plant’s societies, Japan) of k-h fluid infusion for 15 min, 105 min continue k-h fluid infusion;(3) the I/R group: balance 30 min, 30 min of ischemia and reperfusion 120 min;(4) SEV0 P group: in early reperfusion for k-h solution containing 2.5% sevoflurane perfusion 15 min;Record each hemodynamic parameters;TUNEL method to detect apoptosis;TTC method to calculate the myocardial infarction area;Western blot the Bcl- 2, Caspase 3 protein expression.Results compared with T0, I/R and SEVOP group in T1, T2, T3, T4 point,LVSP HR, + dp/dtmax, are lower(P < 0.05), LVEDP is increased(P < 0.05); on T1, T2, T3, T4, compared with the SHAM group, in the I/R group and SEVOP, LVSP, HR, plus or minus dp/dtma are reduced, LVEDP increased(P < 0.05);Compared with I/R group, LVSP in SEVOP group, HR, plus or minus dp/dtma, LVEDP are lower(P < 0.05).Compared with I/R group(51 + 5) %, the range of myocardial infarction in SEVOP group(31 + 4) % is lower(P < 0.05).TUNEL dyeing results, compared with the SHAM group, the I/R group and SEVOP myocardial cell TUNEL staining positive cells increased, TUNEL apoptosis index increased(P < 0.05);compared with the I/R group, SEVOP group of myocardial cell TUNEL staining positive cells less, low apoptotic index(P < 0.05).Compared with the SHAM group, the I/R group and SEVOP myocardial Bcl- 2 protein expression decreased, caspase 3 expression increases, the difference was statistically significant.(P < 0.05);compared with the I/R group, in SEVOP group the Bcl- 2 protein expression, caspase 3 protein expression drops, the difference was statistically significant.(P < 0.05)Conclusion after sevoflurane postconditioning, Left ventricular diastolic dysfunction in rats in vitro increased, increase myocardial contraction force, cardiac function improved significantly.Sevoflurane postconditioning can inhibit myocardial apoptosis and decrease the infarction area, and possesses protective effects on rats MIRI damage. Part 2 Molecular mechanisms of sevoflurane postconditioning alleviatesmyocardial ischemia-reperfusion injury in rats in vitro:NOS/NO- NHE1- MPTP pathwayObjective To evaluate the effects of sevoflurane postconditioning on myocardial ischemia/reperfusion injury in vitro rats and the role of NOS-NO-NHE1 pathway in it.Methods Seventy-two isolated rat hearts perfused in a Langendorff apparatus were randomly divided into 6 groups(n=12 each) using a random number table:sham operation group(group S), sevoflurane group(group Sev), myocardial I/R group(group I/R),sevoflurane postconditioning group(group SEVOP), sevoflurane postconditioning+ L-NAME group( group SEVOP+L-NAME),and L-NAME group( group L).The hearts were subjected to ischemia for 30 minutes followed by 2h reperfusion in the other groups except group S and Sev. In SEVOP and SEVOP+L-NAME groups, the hearts were perfused with K-H solution saturated with 2.5% sevoflurane for 15 minutes at the beginning of reperfusion, In SEVOP+L-NAME and L groups, the hearts were perfused with K-H solution saturated with 100μmol/L L-NAME for 60 minutes at the beginning of reperfusion. All myocardial specimens were made at the end of reperfusion. Detection of NO、NOS、NAD+ content using pectrophotometric method and calculate the activity of NOS. The expression of p-NHE、t-NHE1 were measured by Western bloting,The expression of NHE1-m RNA was measured by RT-PCR. Myocardial infarct size was determined by TTC at the end of reperfusion, The myocardial altrastrncture was observed by electron microscopy.Results Compared with I/R group, the content of NO、NAD+ and NOS activity increased,the expression of p-NHE、NHE1 and NHE1-m RNA were down-regulated,the myocardial infarct size was significantly decreased in group SEVOP(P<0.05).Compared with SEVOP group,the content of NO、NAD+ and NOS activity decreased,the expression of p-NHE、NHE1 and NHE1-m RNA were up-regulated,the myocardial infarct size was significantly enlarged in group SEVOP+L-NAME(P<0.05).Conclusion Sevoflurane postconditioning can enhance NOS activity,mitigate the expression of p-NHE 、 NHE1 and NHE1-m RNA, inhibiting MPTP opening, the NOS-NO-NHE1 pathway may be one of the molecular mechanisms of sevoflurane postconditioning to protect myocardial I/R injury in isolated rat hearts.Part 3 The effec of sevoflurane postconditioning on oncosis andautophagy in rats in vitroObjective To investigate the effec of sevoflurane postconditioning on oncosis and autophagy in rats in vitroMethods Langendorff isolated perfused rat hearts were randomly divided into 4 groups(n=8): control group(SHAM), sevoflurane group(group S), pure ischemia reperfusion group(I/R group), sevoflurane postconditioning group(SEVOP group), each processing method can be according to the first part of the study.Transmission electron microscope cell bulging die and autophagy;Autophagy related molecular LC3 I, Western blot test LC3 II, Beclin 1 and oncosis related molecules porimin protein expression.Results Sham group autophagosome number is 1.2± 0.7; oncosis cell number is 3.2 ±1.5;Group S autophagosome was 1.0 ±0.6, the number of expanding dead cell number was 3.0 ±1.3, compared the two groups, no statistical difference.In the ischemia reperfusion group, the number of I/R group autophagosome was 10.4 ± 2.4, oncosis cell number is 15.5 ±4.2;Sevoflurane postconditioning group SEVOP autophagosome was 6.8 ±1.8, the number of oncosis cell number is 11.7 ± 3.5, showed that after ischemia-reperfusion autophagosome and oncosis cells increased significantly, and sevoflurane postconditioning can significantly reduce the production number of both autophagosome and oncosis cells.Conclusion Only sevoflurane inhalation does not affect the production of autophagosome and oncosis cells. after ischemia-reperfusion autophagosome and oncosis cells increased significantly, and sevoflurane postconditioning can significantly reduce the number of autophagosome and oncosis cells.