Effect Of Propofol On Autophagy During Oxygen-glucose Deprivation/Reperfusion Injury In L02 Cells

Hepatic ischemia reperfusion injury(HIRI)is a common pathophysiological process. Ischemic injury, metabolism and structural damage to liver cells which occurs in reduction blood perfusion of the liver tissue not reduced but exacerbated while restoring blood perfusion. Studies have shown that apoptosis and autophagy cells are present in hepatic ischemia-reperfusion process. Aggravated cell metabolism and structural damage will be accompanied by increased autophagic activity after Hepatic ischemia / reperfusion injury.Autophagy is a degradation / recycling process which using lysosomes to degrade damaged proteins and organelles widely present in eukaryotic cells. It’s a double-edged sword in the survival of cells. Under physiological conditions, cells remaining low autophagy by lysosomes system degrades macromolecules and organelles and promotes cell survival; while under oxidative stress, ischemia / reperfusion injury and other patholoical conditions, excessive autophagy can lead to cell dysfunction, metabolic abnormalities, increasing structural damage and even cause autophagic cell death in the liver.Propofol is a non-barbiturate intravenous alkylphenols highly fat-soluble anesthetic. It is commonly used for anesthesia induction, maintenance and sedation because of its rapid onset of action, inducing steady, recovery quick and complete, less accumulation after continuous infusion. It is generally believed that propofol can reduce liver cell damage, improve liver function and protect the liver structure during ischemia and reperfusion. But the exact mechanism of its protective effect is not yet fully clear, which may be related to scavenging oxygen free radicals, inhibiting lipid peroxidation and regulating calcium balance.Objective This study: 1. To investigate the effect of oxygen-glucose deprivation/reperfusion(OGD/R) injury on autophagy in L02 cells; 2. To investigate the effect of propofol on autophagy during oxygen-glucose deprivation/reperfusion injury in L02 cells, in order to provide experimental evidence for Discussion the protection mechanism of propofol during OGD / R injury in liver.Methods 1. L02 cells were cultured to establish the model of OGD/R injury and simulate clinical hepatic ischemia-reperfusion injury. The L02 cells were randomly divided into 5 groups : normal control group(group C), oxygen-glucose deprivation 6 h/reperfusion 1h(OGD6/R1)group, oxygen-glucose deprivation 6 h/reperfusion 3 h(OGD6/R3)group, oxygen-glucose deprivation 6 h/reperfusion 6 h(OGD6/R6)group, oxygen-glucose deprivation 6 h/reperfusion 12 h(OGD6/R12)group. Then observed the form changes of the L02 cells by optical microscope; The appreciation of the company’s relative L02 cells was detected by MTT. The expression of autophagy related proteins such as LC3 and Beclin-1 were evaluated by western blot.2. L02 cells were cultured in vitro, The OGD/R model was established by oxygen-glucose deprivation 6 h and reperfusion 12 h. These L02 cells were divided into 6 groups with or not with OGD/R and/or propofol(PPF) and the concentration of PPF : control group(group C)、group OGD/R、group OGD/R+PPF10μmol/L(OGD/R+PPF10)、group OGD/R+PPF25μmol/L(OGD/R+PPF25)、group OGD/R+PPF50μmol/L(OGD/R+PPF50)、group OGD/R+PPF100μmol/L(OGD/R+PPF100), then observed the form changes of the L02 cells by optical microscope. Cell vitality was detected by MTT. The expression of autophagy relatedproteins such as LC3 and Beclin-1 were evaluated by western blot. LC3 immunofluorescence staining was used to detect autophagosome.3、Analysis of statistics: all of the date was presented as the mean ± standard deviation(SD). Differences among different groups were analyzed by one-way analysis of variance(ANOVA) using SPSS 16.0 software.P < 0.05 was considered as statistically significant.Results 1. The effect of OGD/R on the morphology of L02 cells Compared with the C group, the form damaged of L02 cells in the OGD/R group was gradually increased in a time-dependent manner.2. The effect of OGD/R on the proliferation activity of L02 cells Compared with the C group, the cells proliferation activity of L02 cells in the OGD/R group was gradually reduced in a time-dependent manner. Cell proliferation rate reduced(P<0.05)while reperfusion for 1 h; Cell proliferation rate reduced obviously(P<0.01)while reperfusion for 3h,6 h and 12 h.3. The effect of OGD/R on the expression of autophagy related proteins such as proteins LC3, Beclin-1 and P62 in L02 cells.Compared with the C group, autophagy related proteins LC3, Beclin-1 were increased while OGD6/R1. The expression of LC3 was gradually increased as the time went on and was increased gradually while OGD6/R6, reached a peak while OGD6/R12h(P<0.01);The expression of Beclin-1 was gradually increased as the time went on and was increased gradually while OGD6/R6 and OGD6/R12(P<0.01);.The expression of P62 has no obvious change while OGD6/R1 and OGD6/R3(P>0.05), began to increase sharply while OGD6/R6 and reached a peak whileOGD6/R12(P<0.01).4. PPF improved the morphology of L02 cells during OGD/R injuryThe morphology of L02 cells In C group and OGD / R group were normal; the cell of OGD / R group were wide vacuolization, floating cells increase obviously; the change of morphology was unconspicuous in low concentration(10μmol / L, 25μmol / L) treated with PPF and improved the morphology of cell in middle concentration(50μmol / L)and high concentration(100μmol / L) treated with PPF.5. PPF improved the proliferation activity of L02 cells during OGD/R injury.Compared with C group, the cells proliferation activity of L02 cells in the OGD/R group and OGD/R+PPF(10,25,50,100) were obviously reduced(P<0.01), Compared with OGD/R group, the overall survival of cells in low concentration(10μmol / L, 25μmol / L) treated with PPF was increased(P<0.05); the cells proliferation activity was obviously increased(P<0.01) in middle concentration(50μmol / L)and high concentration(100μmol / L) treated with PPF.6. The effect of PPF on the expression of autophagy-related protein LC3, Beclin-1 which was induced by OGD / R in L02 cellsCompared with C group, the expression of autophagy-related protein LC3, Beclin-1 was up regulation in OGD/R group and OGD/R+PPF(10,25,50,100)(P<0.05);Compared with OGD/R, despite in low concentration(10,25μmol / L)or high concentration(50,100μmol / L) treated with PPF, the expression of autophagy-related protein Beclin-1 was down-regulation(P<0.01);the expression of autophagy-related protein LC3 had no obvious change in low concentration(10μmol / L) treated with PPF, but down-regulation(P<0.01)while the concentration of PPF was inhanced.7. LC3 immunofluorescence staining was used to detect autophagosome.The fluorescent protein of LC3 was dispersed in control and PPF group; compared with the control and PPF group, the fluorescence intensity of LC3 was significantly higher and spotty clusters, autophagosome gathered increase after OGD / R processing; compared with OGD / R group, the fluorescence intensity of LC3 decreased autophagosome gathered reduction after administration of PPF.Conclusions1. Oxygen-glucose deprivation/reperfusion can lead to autophagic cell death;2. Propofol can mitigate the damage of L02 cell during OGD/R through inhibiting the activity of autophagy.