Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages

Abstract

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1

Benfotiamine prevents the release of arachidonic acid (AA) metabolites from LPS-stimulated RAW264.7 cells. The RAW254.7 cells were incubated with 3H-AA (0.1 μCi/ml) as described in methods and stimulated with 1 μg/ml LPS for 1 h. The cells were washed and incubated for 18 h in a fresh medium containing benfotiamine (100 μM). The release of metabolites in the medium was determined by measuring the radioactivity in the culture medium. Data represent mean ± S.E. (n=6). #p <0.0001 as compared with AA alone, **p<0.0001 as compared with AA+LPS-treated cells; AA; Arachidonic acid, L; LPS, B; Benfotiamine.

Figure 2

Benfotiamine prevents LPS-induced generation of cPLA2 in RAW264.7 macrophages. The RAW cells were growth-arrested in a medium containing 0.1% serum without or with benfotiamine (100 μM) and challenged with 1 μg/ml LPS. The cPLA2 levels and expression were determined in cell homogenate using the monoclonal enzyme immunoassay kit and western blotting, respectively. Data represent mean ± S.E. (n=6). #p<0.0001 Vs control; **p<0.0001 Vs LPS treated cells. Cont; Control, Ben; benfotiamine.

Figure 3

Effect of benfotiamine on LPS-induced production of PGE2, LTB4, TXB2 and 6k-PGF1α and in RAW264.7 macrophages. A-D) The RAW cells were growth-arrested in Dulbecco’s modified Eagle’s medium containing 0.1% serum with or without benfotiamine (100 μM) for overnight and challenged with 1 μg/ml LPS for 18 h. The levels of PGE2, LTB4, TXB2 and 6k-PGF1α were determined in the culture medium by using the monoclonal enzyme immune assay kits as per manufacturer’s instructions. Data represent mean ± S.E. (n=6). #p<0.0001 Vs control cells; ** p <0.0001 Vs LPS-treated cells. Cont; control, Ben; Benfotiamine.

Figure 4

Benfotiamine prevents LPS-induced expression of COX2, LOX-5, TXB synthase and PGI2 synthase in RAW cells. The RAW cells were growth-arrested by incubating in 0.1% FBS medium without or with benfotiamine (100 μM), and stimulated with 1 μg/ml LPS for 18 h. Cytosolic extracts were prepared and equal amounts of cytosolic proteins were subjected to Western blot analysis using antibodies against (A) COX-2, (B) LOX-5 (C) TXB synthase (D) PGI2 synthase and (E) GAPDH. A representative blot from each group is shown. Data represent mean ± S.E. (n=3). #p<0.0001 Vs control; ** p <0.0001 Vs LPS treated cells. B-50 and 100; Benfotiamine 50 and 100μM.

Figure 5

Effect of benfotiamine on LPS-induced COX2, LOX-5 and TXB synthase at transcriptional level in RAW macrophages. Macrophages were growth-arrested in Dulbecco’s modified Eagle’s medium containing 0.1% serum with or without benfotiamine (100 μM) overnight and challenged with 1 μg/ml LPS for 6 h. The total RNA was isolated and reverse transcriptase-PCR analysis was carried out using specific primers for COX-2, LOX-5 and TXB synthase. Equal amount of PCR products were electrophoresed with 1% agarose-TAE gels containing ethidium bromide. GADPH served as control in reverse transcriptase-PCR analysis. A representative blot from each group is shown. Data represent mean ± S.E. (n=3).* p<0.0001 Vs control; ** p <0.0001 Vs LPS treated cells. Cont; control, Ben; Benfotiamine.

Figure 6

Effect of benfotiamine on ERK1/2 phosphorylation and avtivation of p65 and Egr-1. Growth-arrested murine macrophages were treated with LPS without and with benfotiamine (100 μM) for indicated times at 37°C. Cell lysates were prepared and equal amounts of protein were subjected to western blot analysis using antibodies against A) ERK1/2, B) p65 and C) Egr-1. A representative blot from each group is shown. Data represent mean ± S.E. (n=3). # p<0.0001 Vs control; **p <0.0001 Vs LPS treated cells.

Figure 7

Effect of benfotiamine on LPS-induced protein-HNE adduct and lipid hydroperoxides formation. Macrophages were growth arrested in 0.1 % FBS and with or without benfotiamine (100μM) subsequently, the cells were exposed to LPS (1μg/ml) for 24 h. (A) Protein-HNE adduct formation was evaluated using rabbit anti-protein-HNE antibodies and FITC-labeled anti-rabbit IgG in goat. A representative blot is shown (200 X magnification). (B) Lipid hydroperoxides were measured using a microplate assay kit according to the manufacturer’s instructions. Data represent mean ± S.E. (n=6). #p<0.0001 Vs control cells; **p <0.0001 Vs LPS treated cells. Cont; control, Ben; Benfotiamine.

Figure 8

Effect of benfotiamine and specific inhibitors of COX-2 and LOX-5 on LPS-induced macrophage viability and monocyte adhesion to endothelial cells. A) Growth-arrested macrophages were pre-incubated in the presence or absence of benfotiamine (100μM), COX-2 inhibitor indomethacin (10μM) and LOX-5 inhibitor REV 5901 (10μM) for overnight at 37°C. (A) Cell viability was determined by trypan blue exclusion method. (B) Serum-starved HUVECs cells were pretreated with benfotiamine (100μM), COX-2 inhibitor indomethacin (10μM) and LOX-5 inhibitor REV 5901 (10μM) for 12 h in the absence and presence of LPS (1 ug/ml). The cells were washed and U-937 monocytes were added to each well and the incubation continued for another 12 h. Cell adhesion assays were performed by MTT assay. Data represent mean ± S.E. (n=4). #p<0.001 Vs control; *p<0.01 and **p <0.001 Vs LPS-treated cells.