Mechanism Of Emulsified Isoflurane Triggering An Autophagy Response To Regulate Apoptosis Via JNK Pathway In Neural Stem Cell

Objective Studies have shown that there was a link between apoptosis and autophagy, Signaling pathways play an important role in neuronal degeneration and steady state. Isoflurane, as a kind of commonly used inhaled anesthetics induced brain injury, nerve toxicity, inhibition of cell proliferation, and apoptosis to in vivo or in vitro models. This experiment is to clear how to adjust the level of autophagy and JNK pathway to reduce drug toxicity by the mechanism of apoptosis of studying the autophagy regulating JNK pathway in emulsified isoflurane(intravenous preparation of isoflurane) induction of neural stem cells. The results of this study will provide theory guideline for clinical use of drugs during the perinatal period and developing neural protection and damage repair.Methods. Cell model of Gibco?Rat Fetal Neural Stem Cell Kit was purchased from Invitrogen Ltd. We added NSCs culture solution(DMEM/F12+2%B27 addition agent+l%N2 addition agent +20 ng/ml EGF+20 ng/mlb FGF,p H 7.4), Cells were vaccinated to culture flask with 37°C, and cultivated in the condition of volume scores with 5% CO2 and 95% air, transfer of culture to six generation,, we vaccinated cells to 96 plate pore 1 day ago,modulated to 5×104 /ml. the toxicity trials were divided into 5 groups: control, emulsion control, 7.56, 9.52 and 11.48 mmol/l emulsified isoflurane, calculated OD value and observed cell apoptosis respectively in 6h, 12 h and 24 h. Percentage of viable apoptotic cell(AV+PI-) 、 non-viable apoptotic cell(AV+PI+)、dead cell(AV-PI+) and normal cell(AV-PI-) is analyzed by flow cytometer and Annexin V-FITC/PI. JNK, Bcl-2 and PARP, caspase 3 and IRE1 protein is expressed by western blotting; Punctum structures with tagged GFP-LC3 were observed by lipofectamine reagents tranfection and confocal microscope, and autophagy structure was observed by electron microscope scanning after being treating with emulsified isoflurane. Continuous data were expressed by mean± standard deviation.Results. Neural stem cell survival was decreased obviously by 7.56mmol/L,9.52mmol/L and 11.48mmol/L EI with 24 hrs compared with intralipid control in dosage dependency. There were significant difference for cell viability and inhibition. Cell survival and apoptosis(Annexin V positive cell ratio) were measured by flow cytometry in different dosage isoflurane with 24 hrs. Neural stem cells were treated with emulsified isoflurane with 7.56mmol/L,9.52mmol/L and 11.48mmol/L. P<0.05. ** P<0.01 vs. intralipid control;6h Caspase3 *P<0.05 vs. intralipid and control,12 h Caspase3**P<0.01 vs. intralipid and control,there were no difference for 3 group in 24 h. PARP with EI in 6 hrs and 24 hrs **P<0.01 vs.Control, PARP with EI in12 hrs *P<0.05 vs.Control;6hrs, JNK with EI in 12 hrs *P<0.05 vs.Control,JNK with EI in 24 hrs *P<0.05 vs. Control, JNK with EI in 24 hrs &P<0.05 vs.intralipid;JNK with EI in 24 hrs *P<0.05 vs. Control. JNK with EI in 24 hrs #P<0.05 vs. intralipid. bcl-2 protein analyzed with Western blot indicated apoptosis induced by stress in Endoplasmic Reticulum. Bcl-2 expression decreased after exposed to EI; LC3 B protein increased obviously *P< 0.05, **P< 0.01 vs. 6h, 12 h and 24 h respectively with every time point), and increased significantly in 24 hrs & P<0.05 vs.Control;p62 protein in 6h and 12 h *P< 0.05 vs. intralipid control, # P<0.05 vs. EI in 12 h, & P<0.05 vs. EI in 24 hrs;Atg5 increased obviously in EI with 6 hrs *P< 0.05 vs. intralipid;Punctum structures tagged with GFP-LC3 were observed with confocal microscope. Numbers of punctum structures in 6 hrs, 12 hrs and 24 hrs *P< 0.05, **P< 0.01 vs. intralipid, ## P<0.01 vs. EI in 6 hrs, && P<0.01 vs. EI in 12 hrs. The number of punctate GFP-LC3 dots is increased on neural stem cell induced by emulsified isoflurane with different time after transfected with a GFP-LC3 plasmid; Autophagosome formation on Embryonic neural stem cell induced by 9.52mmol/l emulsified isoflurane with different time by scanning electron microscope. OD value of EI in 12 hrs increased significantly compared with 3-MA ***P< 0.001, OD value of EI reduced obviously compared with Rapamycin group **P< 0.01. OD value of EI reduced obviously compared with control in ***P< 0.001, **P< 0.01 and ***P< 0.001 respectively,Conclusion JNK inhibitor down-regulate neural cell apoptosis by emulsified isoflurane. Inhibition of autophagy decrease the level of apoptosis induced by emulsified isoflurane in neural cell.