Sevoflurane Attenuating Oxygen And Glucose Deprivation Neuronal Apoptosis Via Inducing HO-1-mRNA Expression

IntroductionNeuronal cell death after ischemia may occur via two pathways: those causingnecrosis or those causing apoptosis. The delayed-cell death, apoptosis, is an importantneuronal cell death pathway after cerebral ischemia and reperfusion, and its startingmechanism is concerned with the activation and regulation of the related apoptosisgenes, as well as the srartup of the signal transduction pathways of cell death. Therefore,it is an urgent and essential work for us to illustrate the mechanism of neuron apoptosisafter cerebral ischemia-reperfusion and seek some effective drugs to block apoptosisoccurrence and progression.Normally the heme oxygenase-1(HO-1) can not be detected in the brain. As themost inducible enzyme known currently, HO-1 can be stimulated by diverse induciblefactors, such as heme, hyperoxia, hypoxia, ischemia and reperfusion, heat shock,endotoxin, hydrogen peroxide, cytokines, UV light, heavy metals, nitric oxide, etc..Normally, characteristics of these inducers may cause oxidative stress, suggesting thatHO-1 is possibly an important part of body anti-oxidative stress in the brain. In genetherapy, HO-1 gene is effective in organ against ischemia and reperfusion injury.Furthermore, some recent researches revealed that the positive function of HO-1 anti-organic ischemia reperfusion injury has a relation with its influence on some cellapoptosis factors. At present, however, the cell signal transduction pathways thatactivated HO-1 and the mechanism of the activated HO-1 that affects the cell apoptosisprocess remain unclear.There is robust evidence that volatile anesthetics applied during cerebralischemia and reperfusion confer neuroprotection. It is generally acknowledged that themechanism of volatile anesthetics neuroprotection has something to do with depressingbrain metabolism, diluating blood vessels, inhibiting free radical generation and lipidperoxidation, as well as regulating neurotransmitters. Some recent papers proposed thatvolatile anesthetics can inhibit neuron apoptosis after ischemia, but volatile anestheticsattenuate apoptosis whether or not through inducing HO-1 gene expression is undefined.Therefore, our objective is to testify that volatile anesthetics through activating HO-1eventually inhibit neuron apoptosis. The result of our work will consummate the theoryof volatile anesthetics neuroprotection.In view of the previous reasons, the model of oxygen-glucose deprivation(OGD)in rat primary culture hippocampal neurons was established to mimic cerebral ischemiaand reperfusion in vitro. The primary culture hippocampal neurons was carried outOGD meanwhile was treated by sevoflurane, a volatile anesthetics was used in clinicalanesthesia soon. On the basis of this, we surveyed the change about OGD neuronsHO-1-mRNA expression and neurons apoptosis to demonstrate that sevofluraneneuroprotection relies on activating HO-1 enzyme, we observed OGD neurons changeof PI₃K, Akt, P^70s6k protein expression and HO-1-mRNA expression to illustrate thatsevoflurane inducing HO-1-mRNA expression depends on PI₃K/Akt/P^70s6k cell signaltransduction pathways, we investigated OGD neurons change of HO-1-mRNAexpression and Fas protein expression to explain the mechanism of activated-HO-1enzyme against apoptosis.Materials and Methods1. Materials 70 newborn (＜48 h) Wistar rats were provided by the Experimental Animal Centerof China Medical University. Cell culture chamber, hypoxia chamber, inverted phasecontrast microscope, Ohmeda excel 210 anesthesia machine, Scion Image software, 96and 6 well culture plates, RT-PCR kit, TUNEL kit, Western blot kit,Immunohistochemology kit, Dulbecco's modified Eagle's medium, horse serum, fetalbovine serum, trypsin, penicillin/streptomycin, Zinc-protoporphyrin, sevoflurane, Fasantibody, HO-1antibody, NSE antibody, PI₃-K antibody, Akt antibody, P^70S6Kantibody, ly294002, triciribin, rapamycin, MTT, DMSO.2. Methods2.1 Experiment 1Cultured 7 days hippocampal neurons, for oxygen glucose deprivation(OGD)experiments, the culture media were washed three times in a glucose-free balanced saltsolution (BSS) and placed in deoxygenated glucose-free medium and sealed under 95%N2-5% CO₂ in an anaerobic chamber equilibrated to 37℃and 100% humidity for 60min. OGD was terminated by replacement of stored medium and by returning thecultures to a standard incubator maintained at 37℃. Experimental group cells wererespectively carried out OGD (group D), OGD+ Sevoflurane (group S),OGD+Sevoflurane +Znpp (group Z).Control cells (group C1) were cultured normally.The other control cells (normal culture cells) were anesthesized with 2% sevoflurane(group C2).Group S was carried out OGD meanwhile anesthesized with 2%sevoflurane. Group Z cells was carried out OGD meanwhile culture medium was added10μmmol·L^-1 Znpp, a competive inhibitor of HO-1 enzyme,and anesthesized with 2%sevoflurane. Cultures were maintained in a CO₂ incubator for the next 24 hours.Neuron viability and apoptosis were measured. The protein expression of HO-1 andHO-1-mRNA expression were detected.2.2 Experiment 2Cultured 7 days hippocampal neurons, for oxygen glucose deprivation (OGD) experiments, the culturing media were washed three times in a glucose-free balancedsalt solution (BSS) and placed in deoxygenated glucose-free medium and sealed under95% N2-5% CO₂ in an anaerobic chamber equilibrated to 37℃and 100% humidity for45 min. OGD was terminated by replacement of stored medium and by returning thecultures to a standard incubator maintained at 37℃. Experimental group cells wererespectively carried out OGD (group D), OGD+ Sevoflurane (group S),OGD+Sevoflurane+LY294002 (group L), OGD+Sevoflurane+Triciribin (group T),OGD+Sevoflurane+Rapamycin (group R). Control cells (group C) were culturednormally. Group S was carried out OGD meanwhile anesthesized with 2% sevoflurane.Group L, group T, group R cells was carried out OGD meanwhile culture medium wasrespectively added 10μmmol·L^-1 LY294002 (PI₃-K inhibitor), 10μmmol·L^-1 Triciribin(Akt inhibitor), 10nmmol·L^-1 Rapamycin (P^70S6K inhibitor), and anesthesized with 2%sevoflurane. Cultures were maintained in a CO₂ incubator for the next 24 hours.Neuron viability and apoptosis were measured. The protein expression of PI₃-K, Akt,P^70S6K and HO-1-mRNA expression were detected.2.3 Experiment 3Cultured 7 days hippocampal neurons, for oxygen glucose deprivation(OGD)experiments, the culturing media were washed three times in a glucose-free balancedsalt solution (BSS) and placed in deoxygenated glucose-free medium and sealed under95% N2-5% CO₂ in an anaerobic chamber equilibrated to 37℃and 100% humidity for45 min. OGD was terminated by replacement of stored medium and by returning thecultures to a standard incubator maintained at 37℃. Experimental group cells wererespectively carried out OGD (group D), OGD+2% Sevoflurane (group S1), OGD+4% Sevoflurane (group S2), OGD+4% Sevoflurane+Znpp (group Z).Control cells(group C) were cultured normally. Group S1 was carried out OGD meanwhileanesthesized with 2% sevoflurane. Group S2 was carried out OGD meanwhileanesthesized with 4% sevoflurane. Group Z cells was carried out OGD meanwhileculture medium was added 10μmmol·L^-1 Znpp, a competive inhibitor of HO-1 enzyme,and anesthesized with 4% sevoflurane. Cultures were maintained in a CO₂incubator for the next 24 hours. Neuron viability and apoptosis were measured. Theprotein expression of Fas and HO-1-mRNA expression were detected.Results1. Experiment 1Group C2 hippocampal neurons' expression of HO-1-mRNA and HO-1 proteinincreased as compared with group C1 (P＜0.05). The expression of HO-1-mRNA andHO-1 protein increased, meanwhile neuron viability increased and apoptosis decreasedin group S as compared with group D (P＜0.05). The expression of HO-1-mRNA andHO-1 protein decreased, meanwhile neuron viability decreased and apoptosis increasedin group Z as compared with group S (P＜0.05).2. Experiment 2The expression of PI₃-K, Akt and P^70S6K protein and HO-1-mRNA increased,meanwhile neuron viability increased and apoptosis decreased in group S as comparedwith group D (P＜0.05). The expression of PI₃-K, Akt and P^70S6K protein andHO-1-mRNA decreased, meanwhile neuron viability decreased and apoptosis increasedin group L as compared with group S (P＜0.05). The expression of Akt and P^70S6Kprotein and HO-1-mRNA decreased, meanwhile neuron viability decreased andapoptosis increased (P＜0.05) , but PI₃-K expression had not statistics difference (P＞0.05) in group T as compared with group S. The expression of P^70S6K protein andHO-1-mRNA decreased, meanwhile neuron viability decreased and apoptosis increased(P＜0.05) , but PI₃-K and Akt expression had not statistics difference (P＞0.05) ingroup R as compared with group S.3. Experiment 3The expression of HO-1-mRNA increased, but Fas protein expression decreased,meanwhile neuron viability increased and apoptosis decreased in group S1 as comparedwith group D (P＜0.05). The expression of HO-1-mRNA increased, but Fas protein expression decreased, meanwhile neuron viability increased and apoptosis decreased ingroup S2 as compared with group S1 (P＜0.05) . The expression of HO-1-mRNAdecreased, but Fas protein expression increased, meanwhile neuron viability decreasedand apoptosis increased in group Z as compared with group S2 (P＜0.05).Conclusion1. Sevoflurane can induce rat primary culture hippocampal neurons HO-1-mRNAexpression.2. Sevoflurane, via inducing HO-1-mRNA expression, attenuates apoptosis of thehippocampal neurons of deprived-oxygen and glucose.3. Sevoflurane, via PI₃K/Akt/P^70s6k cell signal transduction pathways, inducingHO-1-mRNA expression, attenuates apoptosis of the hippocampal neurons ofdeprived-oxygen and glucose.4. Sevoflurane via inducing HO-1-mRNA expression inhibits Fas, eventuallyattenuates apoptosis of the hippocampal neurons of deprived-oxygen and glucose.