Benfotiamine attenuates inflammatory response in LPS stimulated BV-2 microglia

Abstract

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Fig 1. Functional characterization of benfotiamine effects in LPS-stimulated BV-2 microglia.

(A) Real-time monitoring of BV-2 cell viability using xCELLigence RTCA analyzer. Representative graph showing the rate of proliferation in cells incubated in control medium (red line), medium with 1 μg/ml LPS (black line), or cells pretreated with benfotiamine, 50 μM (pink line), 100 μM (blue line) or 250 μM (green line) and then treated with LPS for 24 h. (B) Benfotiamine- induced alterations in cell morphology were analyzed using phase-contrast microscopy (left panels), whereas cell surface area was quantified by Phalloidin /Hoechst fluorescent staining (red/blue) microscopy (right panels), using AxioVisionRel 4.6 software. Insets: cell surface area was measured in five areas (138 × 104 μm²) per each cover-slip (n = 3) per experimental group in three independent experiments. (C) Bars present mean surface areas (± SEM) obtained from data presented in Fig. 1B. (D) Cell viability was assessed by crystal violet staining and results are displayed as percentage of control ± SEM (n = 3). *P < 0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells. Scale bar: 20 μm.

Fig 2. Effect of benfotiamine on LPS-induced production of NO.

(A) Benfotiamine suppressed LPS-induced release of NO. (B) Expression of iNOS-mRNA in LPS-stimulated BV-2 cells (black bar) and cells pretreated with benfotiamine (gray bars). The levels of iNOS-mRNA are expressed relative to the expression of GAPDH-mRNA as an internal control. (C) Expression of iNOS at the protein level, as determined by Western blot. Graph shows mean iNOS protein abundance (± SEM), from n = 3 separate determinations, expressed relative to the abundance of β-tubulin in each lane. Representative Western blot of iNOS expression. (D) Immunofluorescence labeling of BV-2 cells against iNOS. Significance inside the graphs: *p < 0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells. Scale bar: 20 μm.

Fig 3. The effect of benfotiamine on LPS—induced expression of proinflammatory effector molecules.

(A) Expression of prostaglandin—endoperoxidase synthase 2 (PTGS2) at mRNA level in BV-2 cells. Expression of PTGS2-mRNA was assessed by RT-PCR, in control culture (white bar), LPS-treated culture (black bar) and cultures pre-treated with benfotiamine, 6 h following addition of LPS. PTGS2-mRNA abundance was expressed relative to the abundance of GAPDH-mRNA, as an internal control. (B) Expression of COX-2 at the protein level, determined by Western blot analysis. Bars show Cox-2/β-actin expression ratio relative to control (100%) ± SEM, from n = 3 separate determinations. Significance levels shown inside the graphs: *p < 0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells.

Fig 4. Effect of benfotiamine on cytokines expression and the release by BV-2 cells.

Expression of TNF-α (A, B), IL-6 (C, D) and IL-10 (E, F) was analyzed at mRNA (A, C, E) and protein (B, D, F) level. Abundance of each mRNA transcript was expressed relative to GAPDH as internal control. Release of the cytokines was determined in the culture supernatants by ELISA. Bars represent mean ± SEM from n = 3 separate determinations. Significance levels shown inside the graphs: * - p < 0.05 control vs. LPS-induced BV-2 cells; # — LPS vs. benfotiamine pretreated LPS activated BV-2 cells.

Fig 5. Quantitative Western blot analysis showing the effects of benfotiamine on MAP kinase signaling pathway.

Expression levels of (A) pERK/ERK, (B) pJNK/JNK, (C) p38 and (D) pAKT/AKT were assessed 5–60 min following LPS stimulation. Bars represent mean expression ratio relative to β-tubulin ± SEM from n = 4 separate determinations. Significance levels shown inside the graphs: * - p < 0.05 control vs. LPS-induced BV-2 cells; # - LPS vs. benfotiamine pretreated LPS activated BV-2 cells.

Fig 6. Effect of benfotiamine on LPS—induced nuclear translocation of NF-κB/p65.

(A) Nuclear translocation of p65/NF-κB subunit was assessed by immunofluorescence labeling against p65 (red) and Hoechst nuclear fluorescence labeling (blue). (B) Nuclear fluorescence intensity of p65 was measured in > 200 hundred cells per experimental group, using ImageJ software and the results were presented in arbitrary units (lower graph). Data were binned (5 AU steps) according to fluorescence intensity and were represented as mean cumulative percentage ± SEM (upper graph). (C) Effect of benfotiamine on LPS—induced translocation of p65 from cytosolic to nuclear compartment was confirmed by Western blotting. Relative p65/β-tubulin abundance is expressed relative to the same abundance in control culture (100%) ± SEM from n = 4 separate determinations. Significance levels shown inside the graphs: * - p < 0.05 control vs. LPS-induced BV-2 cells; # - LPS vs. benfotiamine pretreated LPS activated BV-2 cells. Scale bar: 20 μm.

Fig 7. Effect of pharmacological inhibitors on iNOS, TNF and IL6 gene expression followed by NO, IL-6 and TNF-α production.

(A, C, E) Expression of iNOS, TNF and IL6 at mRNA level in BV-2 cells. Expression of iNOS, TNF and IL6-mRNA was assessed by RT-PCR, in control culture (white bar), LPS-treated culture (black bar), cultures pre-treated with U0126 (50 μM), SP600125 (20 μM) or LY294002 (20 μM) in presence or absence of benfotiamine (gray bars), 6 h following addition of LPS. iNOS, TNF and IL6-mRNA abundance was expressed relative to the abundance of GAPDH-mRNA, as an internal control. (B, D, F) The cultured supernatants were collected and analyzed for NO using Griess method, or TNF-α and IL6 production with ELISA. The data represent the mean ± SEM (n = 3), *P<0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells.