Beta-glucogallin reduces the expression of lipopolysaccharide-induced inflammatory markers by inhibition of aldose reductase in murine macrophages and ocular tissues

Abstract

Aldose reductase (AR) catalyzes the reduction of toxic lipid aldehydes to their alcohol products and mediates inflammatory signals triggered by lipopolysaccharide (LPS). Beta-glucogallin (BGG), a recently described AR inhibitor, was purified from extracts of the Indian gooseberry (Emblica officinalis). In this study, we found that BGG showed low cytotoxicity in Raw264.7 murine macrophages and effectively inhibited AR activity as measured by a decrease in sorbitol accumulation. In addition, BGG-mediated inhibition of AR prevented LPS-induced activation of JNK and p38 and lowered ROS levels, which could inhibit LPS-induced apoptosis. Uveitis is a disease of the eye associated with chronic inflammation. In this study, we also demonstrated that treatment with BGG decreased the number of inflammatory cells that infiltrate the ocular media of mice with experimental uveitis. Accordingly, these results suggest BGG is a potential therapy for inflammatory diseases.

Fig. 1

The effect of BGG on the viability of Raw264.7 murine macrophages. Macrophages were treated with various concentrations of BGG for 24 h as described under Materials and Methods. As a positive control for toxicity, cells were incubated with 200 µM H₂O₂. Cell viability was determined by the Calcein Live assay. Data shown are means ± SEM (N = 3).

Fig. 2

The effect of aldose reductase inhibitors on regulation of sorbitol level in macrophages. Macrophages were incubated with LPS (100 ng/ml) for 12 h following 24 h pretreatment with either Sorbinil (10 µM) or BGG (50 µM). ARI-supplemented culture medium was added fresh every 12 h. The sorbitol level in the macrophage cell lysates was measured using a sorbitol colorimetric assay. The amount of sorbitol in cell lysates was normalized to total protein. *Represents the statistical significance compared to vehicle of the no LPS group. ^#Represents the statistical significance compared to vehicle of the LPS group. Data shown are means ± SEM (N = 3). ^#P < 0.05; **P < 0.01.

Fig. 3

The effect of aldose reductase inhibition on LPS-induced activation of p38 and JNK in Raw264.7 murine macrophages. Macrophages were pretreated with 10 µM Sorbinil or 50 µM BGG for 24 h followed by LPS (20 ng/ml) for the indicated times. The fold activation of p-JNK and p-p38 were normalized to total JNK and p38, respectively. For changes over time, the normalized values are expressed relative to samples taken at time zero.

Fig. 4

The effect of aldose reductase inhibition on LPS-induced oxidative stress in Raw264.7 murine macrophages. Macrophages were pretreated with 10 µM Sorbinil or 50 µM BGG for 24 h followed by stimulation with LPS (1 µg/ml) for 60 min. The level of ROS was measured fluorometrically and the fold change was compared to control. *Represents the statistical significance compared to vehicle of LPS group. ^#Represents the statistical significance compared to vehicle between no LPS and LPS groups. Data shown are means ± SEM (N = 3). *P < 0.05; ^#P < 0.05.

Fig. 5

The effect of BGG on LPS-induced uveitis in mice. (A) Inflammatory cells were stained in serial histological sections of the mouse eye as described in Materials and Methods. Cell counts were tabulated according to the anatomical location in the anterior (or aqueous) chamber (AC) or posterior (or vitreous) chamber (PC). The inflammatory cells are indicated by arrows. (B) The number of inflammatory cells is displayed as a bar chart. ^#Represents the statistical significance compared to vehicle of the AC group. *Represents the statistical significance compared to vehicle of the PC group. Data shown are means ± SEM (N = 3). ^##P < 0.01; **P < 0.01.