Prevention of endotoxin-induced uveitis in rats by benfotiamine, a lipophilic analogue of vitamin B1

Abstract

Purpose: To study the amelioration of ocular inflammation in endotoxin-induced uveitis (EIU) in rats by benfotiamine, a lipid-soluble analogue of thiamine.

Methods: EIU in Lewis rats was induced by subcutaneous injection of lipopolysaccharide (LPS) followed by treatment with benfotiamine. The rats were killed 3 or 24 hours after LPS injection, eyes were enucleated, aqueous humor (AqH) was collected, and the number of infiltrating cells, protein concentration, and inflammatory marker levels were determined. Immunohistochemical analysis of eye sections was performed to determine the expression of inducible-nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, protein kinase C (PKC), and transcription factor NF-kappaB.

Results: Infiltrating leukocytes, protein concentrations, and inflammatory cytokines and chemokines were significantly elevated in the AqH of EIU rats compared with control rats, and benfotiamine treatment suppressed these increases. Similarly increased expression of inflammatory markers iNOS and Cox-2 in ciliary body and retinal wall was also significantly inhibited by benfotiamine. The increased phosphorylation of PKC and the activation of NF-kappaB in the ciliary body and in the retinal wall of EIU rat eyes were suppressed by benfotiamine.

Conclusions: These results suggest that benfotiamine suppresses oxidative stress-induced NF-kappaB-dependent inflammatory signaling leading to uveitis. Therefore, benfotiamine could be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.

Figures

Figure 1

Benfotiamine prevents LPS-induced clinical symptoms of EIU in rats. (A) Chemical structure of benfotiamine. (B) The pathologic score of EIU in Lewis rat eyes injected with LPS in the absence and presence of benfotiamine was determined at 3 and 24 hours with a slit lamp microscope. Results are given as mean ± SD (n = 6). #P < 0.0008 versus control (C). **P < 0.005 versus EIU (Wilcoxon-Mann-Whitney test).

Figure 2

Benfotiamine prevents EIU-induced inflammatory cell infiltration and protein concentration in AqH. (A) Histopathologic changes in the anterior chamber of EIU rat eyes in the absence and presence of benfotiamine. Serial sections of paraformaldehyde-fixed rat eyes were stained with hematoxylin and eosin and were observed under a light microscope. Magnification, 200×. (B) The inflammatory cells and (C) total protein concentration in the AqH were measured 24 hours after LPS injection by using trypan-blue exclusion cell counting and Bradford methods, respectively. Results are given as mean ± SD (n = 6). #P < 0.001 versus control (C). **P < 0.001 versus EIU.

Figure 3

Benfotiamine prevents EIU-induced inflammatory cytokines and chemokines in AqH. The AqH from EIU rats was used to measure secreted cytokines and chemokines by an antibody array system. Presented here are the percentage control values for individual cytokines, taking control as 100% (n = 4) after densitometry analysis. #P < 0.001 versus control (C). *P < 0.001 versus EIU. BEN, benfotiamine; EIU, endotoxin-induced uveitis.

Figure 4

Benfotiamine prevents the expression of Cox-2 and iNOS and the activation of NF-κB and PKC in EIU. (AI, AII) Serial sections of paraformaldehyde-fixed EIU rat eyes (24 hours) were immunostained with antibodies against (AI) Cox-2 and (AII) iNOS and were observed under a light microscope. (BI, BII) Serial sections of EIU rat eyes (3 hours) were immunostained with antibodies against phospho-p65 (Ser536). (CI, CII) Phospho-PKCβII (Ser660) followed by FITC-labeled secondary antibodies and mounted with fluorescence mounting medium with DAPI. Antibody staining intensity was observed under a fluorescence microscope for FITC and DAPI. A representative microphotograph is shown (n = 4). Magnification, 200×. I, iris; CB, ciliary body; R, retina; C, control; EIU, endotoxin-induced uveitis; Ben, benfotiamine.