The Expression Of Focal Adhesion Kinase In Human Lens Epithelial Cells Treated With Hydrogen Peroxide

BackgroundLens epithelial cells (lens epithelial cells, LECs) are the monolayer cuboidal cells located under the lens capsule, whose metabolism are activity, and have the ability of proliferation, differentiation, and healing after the trauma. Lens epithelial cells play roles in providing nutrition, metabolizing, repairing damage and keeping transparency of lens. Destroying the normal structure and function of the LECs may lead to different types of lens opacity. In previous studies, comparing normal LECs with cataract LECs, people found that there were three characteristics in cataract patients: change in cell morphology, decrease in cell density and multilayer arrangement of cells caused by proliferation. These abnormal changes may lead to lens opacification. Therefore, to study the biological characteristics such as morphology, structure and apoptosis of LECs is very important for studying cataract.The formation of cataract is strongly related to oxidative stress. It is believed that excessive oxidative stress is the common pathway leading to cataract, which activate the intracellular signaling pathways cascade and finally leads to LECs injury. Hydrogen peroxide (H2O2) is the most important substance causing oxidative stress. It is reported that H2O2concentration is approximately20-30μmol/L in normal aqueous humor compare to660μmol/L in cataract aqueous humor, which is30times higher. Owning to oxidative stress, permeability of LECs membrane increases, the intracellular proteins leak out, intracellular environment changes, and the stability of cells decrease; At the same time, proteins structure changes which affect cell physiological function, DNA damage leads to cell’s apoptosis and thus nutrients supply of the whole lens is affected. All of these changes finally lead to lens opacification. Oxidative stress provokes a vicious cycle of cataract formation process, so that it is essential to study it’s mechanism in LECs.Focal adhesion kinase——FAK is a cytoplastic non-receptor tyrosine kinases. It has been nearly20years since FAK discovery, which regulates embryonic cell differentiation, cell adhesion, migration, fibrosis, proliferation and apoptosis. Several studies have proved that:(1) FAK is important for cell migration and adhesion. Cell migration is dependent on the interaction of ECM-integrin and FAK.(2) In cell proliferation, a large number of reports indicate that activation of FAK stimulate MAPK or PI-3K signaling pathway activation,which is necessary for cells proliferation.(3) Inhibition of FAK can induce apoptosis, which may be associated with PI3-K/Akt-1and MEK/Erk signaling pathway. In all, FAK works in the upstream of several signaling pathways, plays a very important role in regulating cells’biological behavior.ObjectiveOxidative stress model is made by treating cells with hydrogen peroxide. During this procedure, cells’ proliferation, migration, apoptosis, and morphological changes are studied, at the same time, the dynamic expression and activation of FAK are observed in order to figure out whether it regulate LECs’biological behavior in oxidative stress.Method:1Cells cultivation:human lens epithelial cells (HLECs) cell lines were purchased from ATCC. The cells were cultured in low-sugar DMEM medium containing10%FBS at37℃under an atmosphere containing5%CO2.When the cells reached80%-90%confluence, cells were treated with serum-free DMEM containing H2O20,30,50,70,100,300,500,700,1000μmol/L for0,30min,3h,6h,12h,24h.2, CCK-8assay:cells were seeded in96-well culture plates at a density of1×108/L and incubated for24h. Use DMEM as negative control group and other groups were treated with DMEM containing30,50,70,100,300,500,700,1000μmol/L H2O2for0,30min,3h,6h,12h,24h. After treatment, blank group (without cells, but only DMEM with CCK-8) was added and cell viability was detected by CCK-8assay. A value was detect by spectrophotometer at the wavelength of450nm. Cell viability could be calculated base on the formula:. Viability (%)=(A value of the experimental group-A value of blank group)/(A value of negative control group-A value of blank group)×100%.3, Cell migration assay:100μL sterile pipette tips was used to scratch on cells in6well plate. Floating cells and cytokines were washed away by PBS. Treat cells with DMEM as control group and DMEM containing100,300,500,700,1000μmol/L H2O2as treated group. Took pictures of the cells at time0,12h,24h; the distance of cell migration was measured by imaging system.4, Cell apoptosis rate detection:Cells were seeded in6-well plates, and then treated with DMEM containing0,100,1000μmol/L H2O2for24h. Cells were collected、 washed、and treated with Annexin V and PI under the guidance of the protocol. According to the, Annexin V/PI assay, cells’ apoptosis rate was detected by flow cytometry.5, Focal adhesion kinase (FAK) distribution was observed under laser confocal microscope:cells were seeded on cover glasses in6-well plates. And then cells were incubated with DMEM containing0,100,300,500,700,1000μmol/L H2O2for24h. Used PBS to wash cells for three times and fixed cells with4%paraformaldehyde for5min. Then cells were washed by PBS again. Fixed cells with methanol (-80℃) in-20℃for15min, washed by PBS again. Incubated with goat serum for1h, after washed by PBS, incubated with FAK antibody (1:200) overnight at4℃. After washing out the antibody, Hoechst33258was used to stain the nuclear for1hour, and then incubated with FITC-fluorescence antibody for30min. Cells were observed under laser confocal microscope.6, The dynamic expression of FAK and phosphorylated FAK was detected by western blot assay:cells were treated with DMEM containing0,100,300,500,700,1000μmol/L H2O2for30min,3h,6h,12h,24h. The cells were collected and proteins were extracted respectively.15μL protein samples were used for western blot, and then proteins were transferred to PVDF membrane; After transgerred, the membrane was incubated with3%BSA at room temperature for1h and then1:1000FAK antibody at-4℃overnight; Used TBST to wash membrane three times, and incubated with second antibodies (1:5000) at room temperature for2h; After washing the membrane for three times, enhancing chemiluminescence was used to bright the bands. And then expose them on X-ray film to get the image of the bands. Density of the bands were analysed by Fluor Chem program.7, All statistical analyses were performed using the SPSS13.0statistical software package. Differences in cell viability, migration, apoptosis, FAK protein levels were assessed by the one-way ANOVA and differences among concentration groups were assessed using the LSD test (homogeneity of variance) or or Dunnelt’s T3test (unequal variances). Two-way ANOVA was used to assess whether there was an interaction effect between concentraions and time pionts in migration assay. p<0.05was considered to be indicative of significance.Results1, After24hours of high concentration hydrogen peroxide treatment, cells’ morphology changed:cells become sparse, contractive, and was elongated, edges became stiff. Meanwhile, the boundary between the nucleu and the cytoplasm was not so obvious. FAK was redistributed to the elongated parts.2. Within12hours, there was a toxin effect induced by hydrogen peroxide whose concentration is higher than500μmol/L; Afeter24hs treatment, there were statistical differences of survival rate among different concentration gourps (F=17.96, p<0.01):compared with control group, the survival rates in30,70,100,300μmol/L group increased,which were1.24±0.03%(p<0.01),1.35±0.08%(p<0.01),1.75±0.19%(p<0.01) and1.37±0.17%(p=0.04), however100μmol/L group did not differ from50,70,300μmol/L group (p50=0.20, p70=0.051, p300=0.10). In500,700,1000μmol/L group, cell viability decreased compared with the control group (p500<0.01, p700<0.01,p1000<0.01). 3. After24hours treatment, the migration speed of100μmol/L group reached to62.23±1.99units/hours (p<0.01) which was higer than other groups. We found that both time (F=27.76, p<0.01) and H2O2concentration (F=23.34, p<0.01) would influence What’s more, there was an interaction effect (F=9.61, p<0.01) between the time and concentration.4, The overall apoptosis rate was different among groups (F=7.49, p=0.02). The apoptosis rate in100μmol/L group was2.40±0.01%, which was lower compared with5.04±0.00%in control group and4.61±0.01%in1000μmol/L group (p=0.01, p=0.02).The diffierence between1000μmol/L group and control group was not statistically significant.5The expression of FAK changed after24hours H2O2treatment. The FAK expression increased in100,300μmol/L group, while in700,1000μmol/L group it decreased which was statistically significant (p<0.05). FAK was phosphorylated in this process, and its degree of phosphorylation was in a time and dose-dependent manner.ConclusionThere are dual effects on lens epithelial cells with hydrogen peroxide treatment:100μmol/L H2O2can increase the expression of FAK in cells, promotes cell proliferation, migration, inhibit apoptosis.1000μmol/L hydrogen peroxide inhibit intracellular FAK expression, inhibit cell physiological function, and can induce death of cells.