Suppressive effects of astaxanthin against rat endotoxin-induced uveitis by inhibiting the NF-kappaB signaling pathway.

We investigated the effects of astaxanthin (AST), a carotenoid, on endotoxin-induced uveitis (EIU), and over the course of the disease measured the expression of inflammatory cytokines and chemokines in the presence or absence of AST. EIU was induced in male Lewis rats by footpad injection of lipopolysaccharide (LPS). The animals were randomly divided to 12 groups with eight animals in each. Immediately after the inoculation, AST (1, 10, or 100 mg kg(-1)) was injected intravenously. Aqueous humour was collected at 6, 12 and 24 hr after LPS inoculation and the number of infiltrating cells in the anterior chamber was counted. In addition, we assayed the concentration of protein, nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2). Immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed in order to evaluate the effects of AST on NF-kappaB activation. Rats injected with AST showed a significant decrease in the number of infiltrating cells in the anterior chamber and additionally there was a significantly lower concentration of protein, NO, TNF-alpha and PGE2 in the aqueous humour. Moreover, even early stages of EIU were suppressed by injection of AST. The number of activated NF-kappaB-positive cells was lower in iris-ciliary bodies treated with 10 or 100 mg kg(-1) AST at 3 hr after LPS injection. These results suggest that AST reduces ocular inflammation in eyes with EIU by downregulating proinflammatory factors and by inhibiting the NF-kappaB-dependent signaling pathway.

Effects of blue honeysuckle (Lonicera caerulea L.) extract on lipopolysaccharide-induced inflammation in vitro and in vivo.

The aim of the present study was to investigate the effects of blue honeysuckle extract (BHE), which contains high level of phenolic compounds, on endotoxin-induced uveitis (EIU). Male Lewis rats were randomly divided into 5 groups with 14 rats in each (eight rats for collection of aqueous humor, six rats for histologic examination). EIU was induced by a footpad injection of lipopolysaccharide (LPS). 1, 10, or 100 mg of BHE was injected intravenously immediately after LPS injection. The aqueous humor was collected at 24 h after LPS injection, the number of infiltrating cells, protein concentration, nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin (PG)-E2 levels in the aqueous humor were determined. Some eyes were enucleated for histologic examination and immunohistochemical analysis. Immunohistochemical staining with a monoclonal antibody against activated nuclear factor (NF)-kappaB was performed to evaluate the effect of BHE on NF-kappaB activation. To further clarify the anti-inflammatory effect, RAW264.7 cells (a mouse macrophage cell line) were stimulated with LPS in the presence or absence of BHE and its major phenolics, cyanidin 3-glucoside (C3G), cyanidin 3-rutinoside (C3R), chlorogenic acid (CA). Expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by Western blot method. BHE treatment significantly reduced the inflammatory cell infiltration, the protein concentration, the levels of NO, TNF-alpha and PGE2 in the aqueous humor and improved histologic status of the ocular tissue. The number of activated NF-kappaB-positive cells was lower in the iris-ciliary body treated with BHE at 3 h after LPS injection. BHE significantly suppressed the production of NO, PGE2 and TNF-alpha in the culture medium as well as the expression of iNOS and COX-2 by LPS-stimulated RAW264.7 cells in a dose-dependent fashion. C3G, C3R and CA showed no or weak inhibitory effects on the level of inflammatory mediators and the expression of iNOS and COX-2. These results suggest that BHE attenuates the degree of inflammation in eyes with EIU by inhibiting the NF-kappaB dependent signaling pathway and the subsequent production of proinflammatory mediators.

Anti-inflammatory effects of aronia extract on rat endotoxin-induced uveitis.

PURPOSE: Aronia crude extract (ACE) with high levels of polyphenol compounds has been reported to have antioxidative effects in vitro and in vivo. In this study, attention was focused on the antioxidant effect of ACE. The purpose of the present study was to investigate the effect of ACE on endotoxin-induced uveitis (EIU) in rats. In addition, the endotoxin-induced expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins was investigated in a mouse macrophage cell line (RAW 264.7) treated with ACE in vitro, to clarify the anti-inflammatory effect. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS inoculation, 1, 10, or 100 mg ACE or 10 mg prednisolone was injected intravenously. After 24 hours, the aqueous humor was collected from both eyes, and the number of infiltrating cells, protein concentration, nitric oxide (NO), prostaglandin (PG)-E2, and TNF-alpha levels in the aqueous humor were determined. RAW 264.7 cells treated with various concentrations of ACE were incubated with 10 mug/mL LPS for 24 hours. Levels of NO, PGE2, and TNF-alpha were determined by an enzyme-linked immunosorbent assay. The expression of iNOS and COX-2 proteins was analyzed by Western blot analysis. RESULTS: The number of inflammatory cells, the protein concentrations, and the levels of NO, PGE2, and TNF-alpha in the aqueous humor in the groups treated with ACE were significantly decreased in a dose-dependent manner. In addition, the anti-inflammatory effect of 100 mg ACE was as strong as that of 10 mg prednisolone. The anti-inflammatory action of ACE was stronger than that of either quercetin or anthocyanin administered alone. ACE also suppressed LPS-induced iNOS and COX-2 protein expressions in RAW 264.7 cells in vitro in a dose-dependent manner. CONCLUSIONS: The results suggest that ACE has a dose-dependent anti-ocular inflammatory effect that is due to the direct blocking of the expression of the iNOS and COX-2 enzymes and leads to the suppression of the production of NO, PGE2, and TNF-alpha.

The effects of Ginkgo biloba extract on lipopolysaccharide-induced inflammation in vitro and in vivo.

PURPOSE: Ginkgo biloba extract (GBE) contains many different flavone glycosides and terpenoides. Several previous studies have demonstrated that GBE exhibits a wide variety of biological activities, including an antioxidant action, on which we focused our attention. The aim of the present study was to investigate the efficacy of GBE on endotoxin induced uveitis in rats. The anti-inflammatory potency of GBE in vivo was compared with that of prednisolone. In addition, we also investigated nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and the expression of iNOS in a mouse macrophage cell line (RAW 264.7) treated with GBE in vitro to clarify the anti-inflammatory effect. METHODS: EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). Immediately after the LPS inoculation, either 1, 10 or 100 microg of GBE were injected intravenously. 24hr later, the aqueous humor was collected from both eyes, and the number of infiltrating cells, protein concentration and NO level in the aqueous humor was determined. The RAW 264.7 cells were pretreated with various concentrations of GBE for 24hr and subsequently incubated with LPS for 24hr. Levels of NO, PGE2 and TNF-alpha were determined by enzyme-linked immunosorbent assay. The expression of iNOS protein was analyzed by Western blotting method. RESULTS: GBE treatment in vivo decreased the concentrations of protein and NO in the aqueous humor of EIU rats. The anti-inflammatory effect of 1 mg GBE was as strong as that of same dose prednisolone. It also significantly reduced the concentration of PGE2, TNF-alpha and NO production in the medium of RAW 264.7 cells compared to that of the LPS group in vitro. The expression of iNOS protein in the 1000 microg ml(-1) of GBE treated cells decreased significantly. CONCLUSION: The present results indicate GBE suppresses the inflammation of EIU by blocking the iNOS protein expression and its anti-inflammatory effect on eye is comparable with the effect of prednisolone used in similar doses.