Protective role of benfotiamine, a fat-soluble vitamin B1 analogue, in lipopolysaccharide-induced cytotoxic signals in murine macrophages

Abstract

This study was designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of vitamin B1, affects lipopolysaccharide (LPS)-induced inflammatory signals leading to cytotoxicity in the mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of the Bcl-2 family of proapoptotic proteins, caspase-3 activation, and PARP cleavage and altered mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor and phosphorylation and subsequent activation of p38-MAPK, stress-activated kinases (SAPK/JNK), protein kinase C, and cytoplasmic phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappaB and consequent activation and nuclear translocation of the redox-sensitive transcription factor NF-kappaB were significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and the inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE(2) was also blocked significantly. Thus, our results elucidate the molecular mechanism of the anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophages. Benfotiamine suppresses oxidative stress-induced NF-kappaB activation and prevents bacterial endotoxin-induced inflammation, indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases.

Fig.1

Chemical structures of A. thiamine and B. benfotiamine. Benfotiamine contains an open thiazole ring which helps benfotiamine readily enter the cell through plasma membrane increasing its bioavailability. Once in cytoplasm the ring closes and gives it a structure of thiamine.

Fig. 2. Benfotiamine prevents LPS-induce apoptosis in RAW cells

A. The RAW cells were treated with LPS (1 μg/mL) for 24h with or withour benfotiamine and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. The number of TUNEL-positive cells in a given field was counted to assess the apoptosis and presented as bar diagram in B, a total of 6 fields were counted. C. The RAW cells were treated with LPS (1 μg/mL) for 12h with or without benfotiamine. Apoptotic cell death was examined using the annexin V-FITC/PI and analyzed by flow-cytometry. Twenty thousand events were acquired for each sample. Bars represent percentage of total cells (n=3). *p<0.0001 Vs Control; **p<0.002 Vs LPS; ^#p<0.002 Vs Control; ^##p< 0.006 Vs LPS. C, control; CB, control+benfotiamine; L, LPS; LB, LPS+benfotiamine.

Fig. 3. Benfotiamine prevents LPS-induced activation of caspase-3, mitochondrial membrane potential (MMP) and Bcl-2 family proteins in RAW cells

A. Growth-arrested RAW cells without or with Benfotiamine were incubated with 1 μg/ml of LPS for 24 h and Caspase-3 activation and PARP cleavage were determined in the cell lysate by western blot analysis using specific antibodies. A representative blot is shown (n=4). B. For assay of MMP, the growth-arrested RAW cells were treated with LPS (2.5 μg/mL) for 4h with or without benfotiamine. The cells were harvested and washed with PBS and MMP was evaluated by staining with JC-1 dye and analyzed with flow cytometry. Twenty thousand events were acquired for each sample. The data are means ± SD; (n=3). *p<0.0007 Vs Control; **p< 0.006 Vs LPS. Cytochrome-C release in the cytosol was measured after 2 and 4 h of LPS treatment by western blot using specific antibodies. GAPDH was used as loading control. C. The expression of Bcl-2 family proteins and AIF in the cell lysate from (A) was determined by western blot analysis using specific antibodies. A representative blot is shown (n=4). C, control; CB, control+benfotiamine; L, LPS; LB, LPS+benfotiamine.

Fig. 4. Benfotiamine prevents LPS-induced ROS generation in RAW cells

The RAW cells were growth-arrested by incubating in 0.1% FBS medium with Benfotiamine (100 μM) or carrier for overnight followed by stimulation with LPS (1 μg/ml) or carrier (PBS) for 16 h. The cells were washed with cold PBS and stained with ROS-sensitive dye dihydroethidium (DHE; 2.5 μM) for 15 min at 37° C. The cells were washed and mounted with floursave mounting medium with DAPI. Photomicrographs were acquired using a fluorescence microscope (Nikon). A representative picture is shown (n=3); Magnification 200X. C, control; CB, control+benfotiamine; L, LPS; LB, LPS+benfotiamine.

Fig. 5. Benfotiamine prevents LPS-induce activation of NF-κB in RAW cells

The RAW cells were growth-arrested by incubating in 0.1% FBS medium with Benfotiamine (50 and 100 μM) or carrier for overnight and stimulated with LPS (1 μg/mL) for 1h with or without benfotiamine. Nuclear extract was prepared and equal amounts of nuclear proteins were subjected to either A. western blot or B. electrophoretic mobility gel-shift assays using specific NF-κB antibodies or oligonucleotide probes, respectively. A representative blot is shown (n=3). C. The RAW cells were transiently transfected with pNF-κB-SEAP reporter vector. The cells pre-treated without or with benfotiamine (100 μM) for 24 h and incubated with 1 μg/ml of LPS for additional 24 h. The cell culture supernatants were assayed for SEAP activity using chemiluminescence kit according to supplier’s instructions. Data represents mean ± SD (n = 3). ^#p<0.001 Vs Control; ^##p<0.01 Vs LPS. C, Control; CB₅₀, Control+benfotiamine (50 μM); CB₁₀₀, Control+benfotiamine (100 μM); L, LPS; LB₅₀, LPS+benfotiamine (50 μM); LB₁₀₀, LPS+benfotiamine (100 μM).

Fig. 6. Benfotiamine prevents LPS-induce activation of NF-κB upstream signaling kinases in RAW cells

The RAW cells were growth-arrested by incubating in 0.1% FBS medium with Benfotiamine (100 μM) or carrier for overnight and stimulated with LPS (1 μg/mL) for different time intervals with or without benfotiamine. Cytosolic extract was prepared and equal amounts of cytosolic proteins were subjected to western blot analysis using antibodies against A. phospho-IκB and unphosphorylated IκB antibodies. B. phospho-p38 (phospho-thr and phospho-tyr), phospho-SAPK/JNK, unphosphorylated p38, SAPK/JNK, and C. phospho-cFos and -cJun. GAPDH immunoblotting of the stripped membrane was done as loading control. A representative blot is shown (n=3). Ben, benfotiamine.

Fig. 7. Benfotiamine prevents LPS-induce activation of PKC in RAW cells

The RAW cells were growth-arrested by incubating in 0.1% FBS medium with Benfotiamine (100 μM) or carrier for overnight and stimulated with LPS (1 μg/mL) for 0-90 min. The cytosolic and membrane fractions were prepared and equal amounts of A. cytosolic proteins (30 μg) and B. membrane proteins (10 μg) were used for western blot analysis using antibodies against phospho-PKCβII (phospho-thr and phospho-ser) and unphosphorylated PKC. The stripped membrane was probed with GAPDH and β-actin antibodies to depict equal loading. A representative blot is shown (n=3).

Fig. 8. Benfotiamine prevents LPS-induce activation of cPLA2 in RAW cells

The RAW cells were growth-arrested and pretreated with or without benfotiamine (100 uM) in 0.1% serum medium for 24 h and stimulated with LPS (1 μg/mL) for 0-90 min. The equal amounts of A. cytosolic proteins (30 μg) and B. membrane proteins (10 μg) were subjected to western blot analysis using antibodies against phospho-cPLA2 (phospho-thr and phospho-ser) and unphosphorylated cPLA2. The stripped membrane was probed with GAPDH and β-actin antibodies to depict equal loading. A representative blot is shown (n=3).

Fig. 9. Befotiamine prevents LPS-induced NO and PGE₂ production in RAW cells. (AI and II)

NO and PGE₂ levels in the culture medium collected 24 h after LPS challenge were measured by ELISA kits as described in Methods. Each value represents the mean ± SD (n=4), ^#p<0.005 Vs Control; ^##p<0.01 Vs LPS. *p<0.0005 Vs Control; **p<0.008 Vs LPS. C, Control; CB, Control+benfotiamine; L, LPS; LB, LPS+benfotiamine. B. Growth-arrested RAW cells without or with Benfotiamine were incubated with 1 μg/ml of LPS for 24 h. The expression of COX-2 and iNOS proteins was determined by western blot analysis using specific antibodies. A representative blot is shown (n=4). C, Control; CB₅₀, Control+benfotiamine (50 μM); CB₁₀₀, Control+benfotiamine (100 μM)L, LPS; LB₅₀, LPS+benfotiamine (50 μM); LB₁₀₀, LPS+benfotiamine (100 μM).

Fig. 10

The mechanism of benfotiamine regulated inflammatory signals. Benfotiamine prevents ROS formation and thereby blocks the activation of subsequent adverse effects including mitochondrial dysfunction and activation of apoptotic markers that sets an early apoptosis. Benfotiamine could also prevent the activation of protein kinases leading to activation of NF-κB which transcribes inflammatory genes and causes inflammation.