Luteolin attenuates endotoxin-induced uveitis in Lewis rats

Abstract

The aim of the present study was to investigate the efficacy of luteolin on endotoxin-induced uveitis (EIU) in rats. EIU was induced in Lewis rats by subcutaneous injections of lipopolysaccharide (LPS). One hr before the LPS injection, 0.1, 1 or 10 mg/kg luteolin or 1 mg/kg prednisolone was intraperitoneally injected. We investigated its effect upon clinical scores, cellular infiltration and protein leakage, as well as on the level of tumor necrosis factor (TNF)-α, nitric oxide (NO) and prostaglandin (PG) E2 in the aqueous humor (AqH). Histologic examination and immunohistochemical analysis in the iris-ciliary body (ICB) were performed to determine the expressions of cyclooxygenase (COX)-2 and inducible NO synthase (iNOS), and then the activated nuclear factor (NF)-κB p65, I kappa B (IκB)-α degradation, phosphorylated (p)-IκB kinase (IKK) α/β and activator protein (AP)-1 c-Jun. Luteolin suppressed, in a dose-dependent manner, the clinical scores, number of inflammatory cells, the protein concentration, and the TNF-α, NO and PGE2 levels in the AqH and improved the histiologic status of the ocular tissue. Luteolin suppressed the expression of iNOS and COX-2 and the activated NF-κB p65, IκB-α degradation, p-IKKα/β and AP-1 p-c-Jun in the ICB. The anti-inflammatory potency of 10 mg/kg luteolin was as strong as that observed with 1 mg/kg prednisolone. These results demonstrate that luteolin attenuates ocular inflammation by inhibiting expression and release of inflammatory markers, along with the inhibition of the activated NF-κB pathway and at least partly AP-1 activity in the ICB.

Fig. 1.

Effect of luteolin on clinical scores (A), infiltrating cells (B) and protein concentration (C) in aqueous humor 24 hr after LPS injection. Each value represents the mean ± SD (n=6). No clinical signs and infiltrating cells in aqueous humor were investigated from LPS non-treated group (LPS (−) group). * P<0.05, ** P<0.01 and ***P<0.001, compared with the LPS group.

Fig. 2.

Histologic evaluation of EIU treated with luteolin. A: histologic changes in the anterior segment of the eye in an animal 24 hr after LPS injection. Rats without LPS injection (a) showed no inflammation (LPS (−) group). Severe inflammatory cell infiltration was observed in the LPS injected rats (b). A reduction in inflammation was observed in the anterior segment of rats treated with 10 mg/kg luteolin (c) and 1 mg/kg prednisolone (d). AC: anterior chamber; CB: ciliary body; HE staining; Bars: 100 µm; Arrows: inflammatory cells. B: effect of various luteolin dosages on histologic grading of EIU. Each value represents the mean ± SD (n=8). **P<0.01 and ***P<0.001, compared with the LPS group.

Fig. 3.

Effect of luteolin on TNF-α (A), NO (B) and PGE2 (C) levels in the aqueous humor. The aqueous humor was collected 24 hr after LPS injection. Each value represents the mean ± SD (n=5). * P<0.05, ** P<0.01 and ***P<0.001, compared with the LPS group.

Fig. 4.

Effects of luteolin on expression of iNOS and COX-2 in the ICB. Paraffin-embedded serial sections 24 hr after LPS injection were immunostained with antibodies against iNOS and COX-2. Luteolin prevented the expression of iNOS and COX-2 in the ICB. Bars: 25 µm.

Fig. 5.

Effects of luteolin on NF-κB pathway and AP-1activity in the ICB. Serial sections of paraformaldehyde in PBS fixed rat eye enucleated 3 hr after LPS injection were immunostained with antibodies against active NF-κB p65, IκB-α, phosphorylation of IKKα/β and AP-1 c-Jun, respectively. Luteolin inhibited the expression of activated NF-κB p65- and Ap-1 p-c-Jun-positive cells in the ICB. Luteolin inhibited IκB-α degradation and phosphorylation of IKKα/β. Bars: 25 µm. Arrows: activated NF-κB p65 positive cells. Arrowheads: phosphorylation of AP-1 c-Jun positive cells.