Mechanism Study Of Propofol Regulating Apoptosis Of Hepatic Cells And Proliferation Of Neural Stem Cells

Partâ… Propofol protects hepatic L02 cells from H₂O₂-induced apoptosis via activation of Extracellular Signal-Regulated Kinases (ERK) PathwayBackground:Propofol protects cells against ischemia/reperfusion(I/R) injury in several organs,but its effect on liver epithelial cells is few reported.Here we investigated the effect of propofol preconditioning on human hepatic L02 cells under hydrogen peroxide(H₂O₂)-induced oxidative stress and attempted to find out whether ERK pathway is involved in this process.Methods:Preconditioned or nonpreconditioned human hepatic L02 cells were exposed to H₂O₂ and the changes of apoptosis were evaluated by TUNEL assay, Caspase-3 and PARP cleavage.Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP Kinase/ERK Kinase 1/2(MEK1/2) was measured by Western blot analysis.The mRNA expression of Bcl-2,Bcl-x_L,Bad,and Bax was quantified by real-time quantitative RT-PCR.Results:Propofol preconditioning reduced the population of apoptotic cells and Caspase-3 and PARP cleavage induced by H₂O₂ in hepatic L02 cells.L02 cells treated with propofol(0.01-0.3 mM) alone,led to a dose-dependent activation of ERK and MEK,and such activation was detected within 0.5 h and eventually declined to less than 50%at 4 h.The addition of specific inhibitor PD98059 completely abolished the activation of ERK and aggravated the extent of apoptosis.Moreover,propofol treatment repressed the mRNA expression of pro-apoptotic genes Bad and Bax,and this repression could be partly reversed by PD98059.Conclusions:These findings demonstrate that propofol protects hepatic L02 cells from H₂O₂-induced apoptosis,partly through activating MEK-ERK pathway and further suppressing Bad and Bax expression. Partâ…¡Propofol acitvates endoplastic reticulum stress system related protein Grp78 expressionBackground:Based on our chapter one findings,propofol has the ability to protect the human hepital cells apopotosi by H₂O₂,we try to find out the way propofol regulates ERK activation.Some study has been reported that oxidative stress could induces endoplasic reticulum stress related gene Grp78 over expression.Grp78 is a key element for endoplasmic reticulum system to anti-apoptosis.So we want to analysis whether there is a link between propofol and endoplasmic reticulum stress.Methods:L02 cells were treated with different concentration of propofol for 4 h then were evaluated by Grp78,Grp94,PDI and calnexin by western blot analysis.L02 cells treated with TG were used as the positive control.Grp78 promoter region was amplified by PCR and subcloned into pGL3-basic.Positive transformants were identified by Xhoâ… and Hindâ…¢digestion followed by 1%agarose electrophoresis. cells were treated with 0.01-0.3 mM Propofol.After 4 h,cells were lysed and used for dual luciferase activity assay.Cells treated with Tunicamycin for 12 h were used as positive control.Results:Propofol upregulates the expression of endoplasmic reticulum stress proteins, specially the apparent increasing level of expression for Grp78.We successfully constructed the pGL3-basic-Grp78 plasmid contenting Grp78 promoter region. Positive transformants were identified by Xhoâ… and Hindâ…¢digestion followed by 1%agarose electrophoresis.L02 cells were transfected with pGL3-basic-Grp78 and pCMV-Rennila.Propofol activates Grp78 promoter-drived luciferase activity.Conclusions:Propofol initiates the reaction of endoplasmic reticulum stress.The key element Grp78 of ER system was transcriptional upregulated. Partâ…¢Propofol Inhibits Proliferation of Rat Neural Stem Cells In VitroBackground:Anesthetics have a profound effect on the neural system.Most studies focused on a short-term evaluation of cognitive function during surgery.In fact, anesthetics also affect the period from neural progenitor cell to neuron.More recently, it was reported that endogenous adult neural progenitor cells could be activated following the brain injury,although the self-repair ability is not clear identified.As anâ…£anesthetic,propofol is used during neurosurgeries because it could protect cells against ischemia/reperfusion injury.However,its role in neural stem/progenitor cells is still unknown.Here we investigated the effect of propofol on the proliferation of rat neural stem cells in vitro.Methods:Rat neural stem cells(NS cells) were derived from the forebrain of rat E14.5 embryos.NS cells were treated with propofol(0,0.01,0.03,and 0.05mM separately).At indicated time points,cell proliferation ability was analyzed by 5-bromo-2'-deoxy-uridine(BrdU) labeling and 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli um inner salt(MTS) assay.Cell cycle progression was measured using flow cytometer (FACScan) analysis.Cell apoptosis was determined by western blot analysis of poly ADP-ribose polymerase(PARP) cleavage and TUNEL.The mRNA expression of GABA_A receptor subunits was quantified by reverse transcription polymerase chain reaction analysis(RT-PCR).Activation of Chkl and Chk2 was measured by western blot analysis.Results:We successfully derived and cultured rNS cells in vitro.These cells expressed Nestin and had the ability to differentiate into neurons and glial cells. Propofol inhibits rNS cells proliferation initiating at 0.01mM concentration.Both BrdU staining and FACScan analysis showed that propofol treatment arrested rNS cells in S phase and this effect was through regulating GABA_A Receptor. Chk1-Ser317 was activated by propofol and such effect could be reversed by GABA_AR-specific antagonist bicuculine.Conclusion:Propofol inhibits neural stem cell proliferation in vitro and arrests the cell cycle in S phase.This effect is partially through activating Chk1 pathway via GABA_A receptor.