The Protective Effects of the Proteasome Inhibitor Bortezomib (Velcade) on Ischemia-Reperfusion Injury in the Rat Retina

<div><p>Purpose</p><p>To evaluate the protective effects of bortezomib (Velcade) on ischemia-reperfusion (IR) injury in the rat retina.</p><p>Methods</p><p>The rats were randomized to receive treatment with saline, low-dose bortezomib (0.05 mg/kg), or high-dose bortezomib (0.2 mg/kg) before the induction of IR injury. Electroretinography (ERG) was used to assess functional changes in the retina. The expression of inflammatory mediators (iNOS, ICAM-1, MCP-1, TNF-α), anti-oxidant proteins (heme oxygenase, thioredoxin, peroxiredoxin), and pro-apoptotic proteins (p53, bax) were quantified by PCR and western blot analysis. An immunofluorescence study was performed to detect the expression of iNOS, oxidative markers (nitrotyrosine, 8-OHdG, acrolein), NF-κB p65, and CD 68. Apoptosis of retinal cells was labeled with <i>in situ</i> TUNEL staining. Neu-N staining was performed in the flat-mounted retina to evaluate the density of retinal ganglion cells.</p><p>Results</p><p>ERG showed a decreased b-wave after IR injury, and pretreatment with bortezomib, especially the high dosage, reduced the functional impairment. Bortezomib successfully reduced the elevation of inflammatory mediators, anti-oxidant proteins, pro-apoptotic proteins and oxidative markers after IR insult in a dose-dependent manner. In a similar fashion, NF-κB p65- and CD 68-positive cells were decreased by bortezomib treatment. Retinal cell apoptosis in each layer was attenuated by bortezomib. The retinal ganglion cell density was markedly decreased in the saline and low-dose bortezomib groups but was not significantly changed in the high-dose bortezomib group.</p><p>Conclusions</p><p>Bortezomib had a neuro-protective effect in retinal IR injury, possibly by inhibiting the activation of NF-κB related to IR insult and reducing the inflammatory signals and oxidative stress in the retina.</p></div

Chitosan oligosaccharides (COS) treatment attenuated reduction of electroretinogram (ERG) b- wave amplitudes.

<p>(A). Individual typical ERG records of the six groups 14 days after PQ injection. (B). Mean ERG b-wave amplitude was measured at day 3, 7 and 14 after PQ injection. The b-wave ratio was defined as b-wave amplitude of right eye/ b-wave amplitude of left eye at definite time point. The fold of b-wave ratio represented the b-wave ratio at day 3, 7 or 14 / b-wave ratio at day 0. The results represented six rats for each group in each time point. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by two-way ANOVA with post hoc Bonferroni test).</p

Effects of COS on the expression of MCP-1 and iNOS mRNA in RGC-5 cells.

<p>RGC-5 cells were stimulated with 0.5mM PQ and then treated with 50 μg/ml (low-dose) or 100 μg/ml (high-dose) COS for 12 hours. The mRNA expression levels of MCP-1 (A) and iNOS (B) were evaluated by semi-quantitative RT-PCR. The expression of MCP-1 and iNOS mRNA was significantly decreased in the low-dose and high-dose COS group than in the PQ only or PBS-treated group. In addition, the levels of iNOS and MCP-1 mRNA were more reduced in the high-dose than in the low-dose COS group. Data were expressed as mean ± SD. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA test with post hoc Bonferroni test; N=3 in each group).</p

Influence of COS on the activation of NF-κB in retina and RGC-5 cells.

<p>(A)Immunohistochemical study of the expression of the NF-κB p65 subunit in retinas. For quantitative immunohistochemistry for NF-κB p65, we first determined the p65 index, which could be measured and calculated from the following formula: Σ [(p65 staining density-threshold) × area (pixels)] / total cell number. The relative density of p65 was defined as p65 index of PQ only, COS only, PBS-treated, low-dose or high-dose COS treated groups / p65 index of normal group. Data were expressed as mean ± SD. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA with post hoc Bonferroni test; N=3 in each group). (B) COS decreased the amount of activated NF-κB translocated into nucleus in PQ-stimulated RGC-5 in a time-dependent and (C) dose-dependent manner. RGC-5 cell were stimulated with 0.5 mM PQ and treated with 50 or 100 μg/ml COS or PBS for indicated time. The amount of activated NF-κB translocated into nucleus in PQ-stimulated RGC-5 was measured by using EMSA. (D) Evaluation of the sequential change of IκB with Western blot analysis. The Y scale represents the ratio of IκB blot density to the β-actin blot density. (E) Evaluation of the sequential change of NF-κB p65 with Western blot analysis. The level of NF-κB p65 significantly reduced in RGC-5 cells after 3 hours of PQ stimulation. The Y scale represents the ratio of NF-κB p65 blot density to the β-actin blot density. Data were expressed as mean ± SD. (†<i>P</i> < 0.05 compared with PQ; ‡<i>P</i> < 0.05 compared with normal control by Student’s t-test; N=3 in each group).</p

Effects of COS treatment on oxidative damages to retina.

<p>Retinal oxidative damages were evaluated by immunohistochemical staining of (A) nitrotysine (B) acrolein, and (C) 8-hydroxy-2'-deoxyguanosine (8-OHdG) at day 14. For quantitation of immunostaining, we first determined the immunostaining index, which could be measured and calculated from the following formula: Σ [(immunostaining density-threshold) × area (pixels)] / total cell number. The relative density of immunostaining was defined as immunostaining index of PQ only, COS only, PBS-treated, low-dose or high-dose COS treated groups / immunostaining index of normal group. Treatment with COS decreased the staining for nitrotysine acrolein and 8-OHdG in the retinas at day 14. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA with post hoc Bonferroni test; N=3 in each group).</p

The influence of COS on PQ-induced cell death, apoptosis and antioxidative enzyme in RGC-5 cells.

<p>(A) Effects of COS on RGC-5 viability determined by MTT assays. RGC-5 cell were incubated for 24 h with vehicle or COS at indicated concentrations. (B) COS decreased PQ-stimulated RGC-5 cell death. RGC-5 cell were stimulated with 0.5 mM PQ and treated with 50 or 100 μg/ml COS for indicated time. (C) Inhibition by COS of PQ-induced apoptosis. RGC-5 cell were stimulated with 0.5 mM PQ and treated with 50 or 100 μg/ml COS or PBS for 6 hours. Cells apoptosis was analyzed by TUNEL stain. Percent apoptotic RGC-5 cells were obtained by counting at least 400 cells. Data were expressed as mean ± SD. ns, Non-significant compared with vehicle-treated control. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA with post hoc Bonferroni test; N=3 in each group) (D) COS activated catalase activity in PQ-stimulated RGC-5. RGC-5 cell were stimulated with 0.5 mM PQ and treated with 100 μg/ml COS for indicated time. (E) COS increased GSH levels in PQ-stimulated RGC-5. (†<i>P</i> < 0.05 compared with PQ only by Student’s t-test; N=3 in each group).</p

Effects of COS on retinal cell apoptosis.

<p>(A). Retinal apoptosis was evaluated by using TUNEL stain. In PQ only and PBS-treated group, TUNEL-positive cells were detected in GCL of the retina, whereas only sparse TUNEL-positive cells in GCL of retina were noted in COS-treated group at day 14. (B). Retinal sections and staining with anti-cleaved caspase-3 antibody. There are significantly few cleaved caspase-3-positive cells in GCL of retina in COS-treated group than in the PQ only and PBS-treated group at day 14. (C).Effects of COS on the expression of cleaved caspase-3 protein in retina. The protein level of cleaved caspase-3 in retina was evaluated by western blot analysis. The cleaved caspase-3 protein was significantly lower in the low-dose and high-dose COS treatment group than in the PQ only or PBS-treated group at day 14. (D). Effects of COS on Bax/Bcl-2 ratio. The amounts of Bax and Bcl-2 in retina were measured by ELISA and the ratio was calculated. Treatment with low-dose or high-dose of COS caused significantly decrease in Bax/Bcl-2 ratio at day 7 and 14. (*<i>P</i> < 0.05 compared with PQ only or PBS-treated group; # <i>P</i> < 0.05 compared with normal; & <i>P</i>< 0.05 by ANOVA test with post hoc Bonferroni test; N=3 for each group in each time point).</p

mRNA levels of inflammatory mediators in retinas.

<p>(A) RNA was subjected to RT-PCR using gene-specific primers for ICAM-1, MCP-1, FKN, and β-actin. (B) mRNA expression of ICAM-1, MCP-1, and FKN, normalized to the expression of β-actin. Data are presented as the mean ± SD. *, <i>p</i> < 0.05 versus the Control group; #, <i>p</i> < 0.05 versus the Diabetes group.</p

Evaluation of functional changes of the retina by electroretinography (ERG).

<p>The ERG were performed on untreated rats (Control) or STZ-induced diabetic rats treated for 8 weeks with normal saline (Diabetes), 3 mg/kg AST (AST High), 0.6 mg/kg AST (AST Low), or 0.5 mg/kg lutein (Lutein). The relative b-wave ratio was significantly decreased in the Diabetes, AST Low and Lutein groups compared with the Control group. The relative b-wave ratio in AST High group had no significant difference from Control group. The ratio in AST High group was significantly higher than that in the AST Low and the Lutein groups (<i>p</i> = 0.046 and <i>p</i> < 0.001). The data are expressed as the mean ± SD in 4 rats for each group (bar graph). *, <i>p</i> < 0.05 compared with the control group. #, <i>p</i> < 0.05 compared with the Diabetes group. Differences among groups were analyzed by one-way analysis of variance followed by Bonferroni’s test for multiple comparisons.</p

Western blot analysis of ICAM-1, MCP-1, and FKN protein expression in retinas.

<p>Retinal cell extracts were prepared from animals treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146438#pone.0146438.g001" target="_blank">Fig 1</a>. (A) Blots were probed with antibodies specific for ICAM-1, MCP-1, and FKN. β-actin was probed as a loading control. (B) The relative intensities of the bands in (A) were determined by ImageJ software and normalized to the expression of β-actin. Data are presented as the mean ± SD. *, <i>p</i> < 0.05 versus the Control group; #, <i>p</i> < 0.05 versus the Diabetes group.</p