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Abstract
Purpose
To investigate the cellular protective effects of hypoxic preconditioning against oxidative stress in a staurosporine-differentiated RGC-5 cell line and the relevance of protein kinase C subtype expression.
Methods
The minimum staurosporine concentration and exposure time necessary to morphologically fully differentiate RGC-5 cells were determined. Cytotoxic injury was provided by oxidative stress with 800 µM hydrogen peroxide (H2 O2) for 15 hours to morphologically fully-differentiated cells. The cytoprotective effect of hypoxic preconditioning was found by exposing the cell line to 0.3% oxygen for different periods of time. Quantifiable changes in the expression of mRNAs and proteins of the isoenzymes α, β, γ, δ, ε, ζ of protein kinase C were determined before and after 1, 2, 15, and 24 hours of hypoxic preconditioning.
Results
Axonal growth in RGC-5 cells after the induction of differentiation with staurosporine caused these cells to resemble neurons. The minimal concentration and exposure time to staurosporine that evoked full differentiation of RGC-5 cells was exposure to 2 µM staurosporine for 1 hour. An LDH assay demonstrated that hypoxic preconditioning had neuroprotective effects against hydrogen peroxide-induced oxidative stress. Protein and mRNA levels of PKC isoforms α and ε increased after preconditioning.
Conclusions
Hypoxic preconditioning of staurosporine-differentiated RGC-5 cells had a cytoprotective effect against oxidative stress. The associated increase of mRNA and proteins of PKC isoenzymes α and ε suggest some functional relevance of these isoenzymes to the cytoprotective effects conferred by hypoxic preconditioning.
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 | Figure 1.The assessment of optimal level and duration in staurosporine treatment for differentiation of RGC-5: The shape of RGC-5 cell changes from fibroblast-like shape to neuron-like shape showing axon and multiple synapse with treatment of staurosporine. Staurosporine 1.0 µg for 2 hours seemed to be the minimum duration and the level for the appropriate morphological differentiation. Staurosporine 0 µg: 1 hr (A), 2 hr (B), 6 hr (C), 24 hr (D); Staurosporine 0.5 µg: 1 hr (E), 2 hr (F), 6 hr (G), 24 hr (H); Staurosporine 1.0 µg: 1 hr (I), 2 hr (J), 6 hr (K), 24 hr (L); Staurosporine 2.0 µg: 1 hr (M), 2 hr (N), 6 hr (O), 24 hr (P). |
 | Figure 2.Cytotoxicity of ischemic preconditioning (0.3% oxygen state) against oxidative stress (800 µM H2 O2 for 15 hr) in differentiated RGC-5 as demonstrated with LDH assay. With prolonged duration of ischemic preconditioning, RGC-5 survival increased up to 8 hrs of ischemic preconditioning after which cytotoxicity increased. Cytotoxicity after oxidative stress was statistically significantly decreased at 2 to 24 hours of ischemic preconditioning when compared to control (* p<0.05 by Mann-Whitney U test). |
 | Figure 3.Changes of PKC iso-enzyme mRNA before and after ischemic preconditioning (B-PC, before preconditioning; PC0hr, just after the preconditioning; PC1hr, 1 hour after preconditioning; PC2hr, 2 hours after preconditioning; PC15hr, 15 hours after preconditioning; PC24hr, 24 hours after preconditioning); We can see the elevation of level of PKC α, β, γ, δ, ε mRNA 1 or 2 hours after PC (preconditioning, 0.3% oxygen 8 hrs). The elevations are decreasing with time after PC. |
 | Figure 4.Changes of PKC iso-enzyme protein before and after ischemic preconditioning (B, before preconditioning; 0, just after the preconditioning; 1, 1 hour after preconditioning; 2, 2 hours after preconditioning; 15, 15 hours after preconditioning; 24, 24 hours after preconditioning). PKC ε increased until 2 hours after the preconditioning, and PKC α increased from 15 hours after the preconditioning. These changes are relatively correlated with increasing tendency of mRNA. |
Table 1.
Primer sequences for real-time PCR (F=forward; R=reverse)
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