Aldose reductase inhibition prevents endotoxin-induced uveitis in rats

Abstract

Purpose: The purpose of the present study was to elucidate the role of the polyol pathway enzyme aldose reductase (AR) in the mediation of ocular inflammation in a rat model of endotoxin-induced uveitis (EIU).

Methods: EIU was induced by a subcutaneous injection of 200 microg lipopolysaccharide (LPS) in male Lewis rats treated with the AR inhibitor, zopolrestat (25 mg/kg body weight, intraperitoneally) or its carrier. The rats were killed 24 hours after LPS injection, the eyes were enucleated immediately, and aqueous humor (AqH) was collected. The number of infiltrating cells, protein concentration, and levels of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin E(2) (PGE(2)) in the AqH were determined. Immunohistochemical analysis was performed in paraformaldehyde-fixed eye sections by staining with antibodies against iNOS, COX-2, TNF-alpha, NF-kappaB, and AR. The levels of reactive oxygen species (ROS) in rat eye sections were determined by dihydroethidium (hydroethidine) fluorescence staining.

Results: In the EIU rat eye AqH, both the number of infiltrating cells and protein concentrations of the inflammatory markers, TNF-alpha, NO, and PGE(2) were significantly higher than in the control rats, and inhibition of AR by zopolrestat suppressed the LPS-induced increases. The LPS-induced increased expression of AR, TNF-alpha, iNOS, and COX-2 proteins in the ciliary body, corneal epithelium, and retinal wall was also significantly inhibited by zopolrestat. Furthermore, AR inhibition prevented the LPS-induced increased levels of ROS and activation of NF-kappaB in the ciliary body, corneal epithelium, and retinal wall of the rat eye. AR inhibition also prevented the LPS-induced activation of NF-kappaB and expression of COX-2 and iNOS in the human monocyte cell line U-937.

Conclusions: The results indicate that AR inhibition suppresses the inflammation in EIU by blocking the expression and release of inflammatory markers in ocular tissues, along with the attenuation of NF-kappaB activation. This finding suggests that AR inhibition could be a novel therapeutic target for the treatment of uveitis and associated ocular inflammation.

Fig. 1. Inhibition of AR prevents LPS-induced inflammatory cell infiltration and protein concentration in aqueous humour

(I) The inflammatory cells and (II) total protein concentration in the AqH were measured using trypan-blue exclusion cell counting and Bradford methods, respectively as described in the Methods. Results are given as mean ± SD (n=6); #p<0.001 and ##p<0.05 Vs Control group; *p<0.01 and **p<0.001 Vs 24 h EIU- group. AqH, Aqueous humor; CB, ciliary body; I, Iris; C, cornea; R, Retina; V, Vitrious.

Fig. 2. Inhibition of AR prevents TNF-α secretion in EIU

TNF-α levels in the AqH collected 6 and 24 h after LPS injection were measured by using ELISA kit as described in the Methods. Each value represents mean ± SD (n=4); ^#p<0.001 vs control group and * p<0.001 vs 24 h EIU- group.

Fig 3. Inhibition of AR prevents NO and PGE₂ secretion in EIU

(AI and BI) NO and PGE₂ levels in the AqH collected 6 and 24 h after LPS injection were measured by using ELISA kits as described in Methods. Each value represents the mean ± SD (n=4), ^#p <0.001 vs control group and *p<0.01 vs 24 h EIU- group. (AII and BII) Serial sections of para-formaldehyde -fixed rat eyes were immuno-stained with antibodies against iNOS (AII) and Cox-2 (BII) and observed under EPI-800 microscope (A representative picture is shown (n=4); Magnification 200X). AqH, aqueous humor; C, Cornea; CB, ciliary body; I, Iris; V, Vitreous; R, Retina.

Fig 4. Inhibition of AR prevents expression of AR and activation of NF-κB in EIU

(A) Serial sections of para-formaldehyde -fixed rat eyes, enucleated 24 h after EIU- induction, were immuno-stained with antibodies against AR and (B) serial sections of para-formaldehyde- fixed rat eyes, enucleated 3 h after EIU-induction, were immuno-stained with antibodies against active NF-κB (phospho-p65) as described in the Methods. The antibody staining intensity was observed under EPI-800 microscope (A representative picture is shown (n=4); Magnification 200X (A) and 400X (B)). AqH, Aqueous humor; CB, ciliary body; C, Cornea; R, Retina.

Fig 5. Inhibition of AR prevents ROS generation in EIU

Serial sections of para-formaldehyde -fixed rat eyes were stained with ROS-sensitive dye dihydroethidium (DHE) for 30 min at 37°C followed by acquisition of images using a fluorescence microscope (A representative picture is shown (n=4); Magnification 200X). AqH, Aqueous humor; I, Iris; CB, Ciliary body; C, Cornea; R, Retina.

Fig. 6. Effect of inhibition of AR on the LPS-induced inflammatory response in human monocytic cells

(A). Growth- arrested U-937 cells without or with zopolrestat (10 μM each) were incubated with 1 μg/ml of LPS for 24 h. The expression of Cox-2 and iNOS proteins was determined by Western blot analysis using specific antibodies as described in the Methods. (B). U-937 cells were transiently transfected with pNF-κB-SEAP reporter vector. The cells treated without or with sorbinil and zopolrestat (10 μM each) were incubated with 1 μg/ml of LPS. After 24 h the culture supernatants were assayed for SEAP activity using chemiluminescence kit according to supplier’s instructions. Data represents mean ± SD (n = 6). #p<0.01 vs control group; ##p<0.01 vs LPS group.