Regulation Of Autophagy On The Damage Triggered By Emulsified Isoflurane In Neural Stem Cells

Objective To explore the general anesthetic EI on the rat fetal neural stem cells(NSCs) proliferation which is associated with JNK and the role played by the regulation of autophagy in the influence.Methods Purchased cells were isolated from cortex of 14 days gestation rat, seeded in 96-well plates, and then incubated in an incubator. The rat fetal NSCs can be expanded to 3 to 5 passages without differentiation with more than 75% of cells retaining their undifferentiation phenotype. 1. Step(1) Establishment of the model of the rat fetal NSCs in vitro accepted EI treatment. The cultured rat fetal NSCs were randomly divided into 6 groups(n = 8 each):normal group(group N); Intralipid group(group F); EI group(including 8.12 m M EI1 group, 9.80 m M EI2 group, 12.04 m M EI3 group), 9.80 m M EI group+20u M SP600125group(group EI2SP). After incubated for 12 hours, the cellular effects of EI and cell viability were evaluated by 3-(4, 5-dimethyithiazol-2-yl)-2, 5-diphenyl-tetra-zolium bromide(MTT) reduction assay. The apoptotic rates of the rat NSCs were determined by flow cytometry, the expression of JNK1, JNK2 and apoptosis-related protein Caspase3 were observed by Western blot. Step(2) Group N, group F and group EI2 selected from Step(1) were detected by plasmid transfection technology and transmission electron microscopy forthe induction of autophagy and observed by Western blot for the expression of autophagy-related protein LC3 B and Beclin-1 in 6, 12, 24 hours respectively. 2. Establishment of autophagy interference model under EI treatment. The cultured rat fetal NSCs were randomly divided into 5 groups(n = 8 each) : normal group(group N); Intralipid group(group F); 9.80mmol/L EI group(group EI2); EI2+10m M 3-MA group(group EI2M)、EI2+25n M BAF group(group EI2B). After incubated for 12 hours, the cellular effects of EI and cell viability were evaluated by MTT reduction assay. The apoptotic rates of the rat NSCs were determined by flow cytometry and the expression of apoptosis-related protein Caspase3 was observed by Western blot.Results 1.(1)There was no statistics difference in cell viability, apoptotic rate and protein expression of Caspase3 in group N and group F. EI group has higher cell apoptotic rate(P<0.001), protein expression of Caspase3, but lower cell viability than group N(P<0.001). And significant differences can be found during three doses of EI group including cell viability(P<0.01), cell apoptotic rate(P<0.01) and protein expression of Caspase3. Compared to EI group, lower cell apoptotic rate(P<0.001), protein expression of Caspase3 but higher cell viability can be observed in group EISP(P<0.05).(2)The results from the selected groups for three different durations suggested that EI2 group has higher protein expression of LC3 B and Beclin-1(P<0.05) and higher expression of the fluorescence of GFP-LC3B( P<0.001) than group N and group F. 2. There was no statistics difference in cell viability, apoptotic rate andprotein expression of Caspase3 in group N and group F. Group EI2 has higher cell apoptotic rate(P<0.001), protein expression of Caspase3(P<0.05), but lower cell viability than group N(P<0.001). Compared to group EI2, higher cell apoptotic rate(P<0.001), protein expression of Caspase3 but lower cell viability can be observed in group EI2M(P<0.05). Compared to group EI2, higher cell apoptotic rate(P<0.001), protein expression of Caspase3 but lower cell viability can be observed in group EI2B(P<0.05).Conclusion JNK played an important role in the EI-induced cytotoxicity on the rat fetal NSCs, and autophagy has a protective effect on the neural apoptosis in this process.