Epigenetic regulation of high glucose-induced proinflammatory cytokine production in monocytes by curcumin

Abstract

Diabetes is a proinflammatory state. We have previously shown increased monocyte proinflammatory cytokines in patients with Type 1 and Type 2 diabetes. High glucose induces proinflammatory cytokines via epigenetic changes. Curcumin, a polyphenol responsible for the yellow color of the spice turmeric, is known to exert potent anti-inflammatory activity in vitro. Recent studies indicate that it may regulate chromatin remodeling by inhibiting histone acetylation. In this study, we aimed to test the effect of curcumin on histone acetylation and proinflammatory cytokine secretion under high-glucose conditions in human monocytes. Human monocytic (THP-1) cells were cultured in presence of mannitol (osmolar control, mannitol) or normoglycemic (NG, 5.5 mmol/L glucose) or hyperglycemic (HG, 25 mmol/L glucose) conditions in absence or presence of curcumin (1.5-12.5 μM) for 72 h. Cytokine level, nuclear factor κB (NF-κB) transactivation, histone deacetylases (HDACs) activity, histone acetylases (HATs) activity were measured by western blots, quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining. HG significantly induced histone acetylation, NF-κB activity and proinflammatory cytokine (interleukin 6, tumor necrosis factor α and MCP-1) release from THP-1 cells. Curcumin suppressed NF-κB binding and cytokine release in THP-1 cells. Also, since p300 histone acetyltransferase is a coactivator of NF-κB, we examined its acetylation. Curcumin treatment also significantly reduced HAT activity, level of p300 and acetylated CBP/p300 gene expression, and induced HDAC2 expression by curcumin. These results indicate that curcumin decreases HG-induced cytokine production in monocytes via epigenetic changes involving NF-κB. In conclusion, curcumin supplementation by reducing vascular inflammation may prevent diabetic complications.

Figure 1. Curcumin treatment suppresses cytokine release in HG-induced THP-1cells

Human monocytic (THP-1) cells(1 × 10⁵ cells/ml) were cultured in presence of osmolar control (19.5 mmol/L mannitol) or normal glycemic (NG, 5.5 mmol/L glucose ) or hyperglycemic ( HG, 25 mmol/L) conditions in absence or presence of curcumin for 72 h as described in Methods. After that the media was saved. Cytokines were measured with a Milliplex ™ MAP Assay Kit (Millipore,USA)) according to the manufacturer's instructions. Values were calculated based on a standard curve constructed for the assay. Results were shown as mean ± SD of 5 different experiments. *a: p<0.05 compared to NG; *p<0.05; **p<0.01 compared to HG

Figure 2. Modulation of HDACs and HATs activity by curcumin treatment in HG-induced THP-1 cells

Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and nuclear lysates were prepared. 10 μg and 50 μg of nuclear lysate protein from each group were taken for determination of HDACs and HATs activity, respectively. The experiment was done according to the manufacturer's instructions. Absorbance was taken at 405 nm and 440 nm by using ELISA reader. Results were shown as mean ± SD of 5 different experiments. *a: p<0.05 compared to NG; *p<0.05; **p<0.01 compared to HG

Figure 3. Effect of curcumin on HDAC2 gene expression level in HG –induced THP-1 cells

(A) Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and nuclear lysates were prepared. protein was subjected to SDS-PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP). The immunoblot shown here are representative of three independent experiments with similar results. (B) Cells were fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4 °C with p300 as described in Methods. Data from a typical experiment of 3 are shown; Magnification × 400. *a: p<0.05 compared to NG; *p<0.05 compared to HG.. (C) In order to measure Immunostaining intensity of HDAC2, p300 and acetylated p65, images were captured with a Nikon eclipse TE200 camera (Japan). The signal intensity was measured using ImageJ software. *a: p<0.05 compared to NG; *p<0.05 compared to HG..

Figure 4. Effect of curcumin on p300 and acetylated CBP/p300 gene expression level in HG –induced THP-1 cells

(A) Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and nuclear lysates were prepared. Protein was subjected to SDS-PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP). The immunoblot shown here is representative of three independent experiments with similar results. (B) Cells were fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4 °C with p300 as described in Methods. Data from a typical experiment of 3 are shown; Magnification × 400. *a: p<0.05 compared to NG; *p<0.05 compared to HG. (C) In order to measure Immunostaining intensity of p300, images were captured with a Nikon eclipse TE200 camera (Japan). The signal intensity was measured using ImageJ software *a: p<0.05 compared to NG; *p<0.05 compared to HG.

Figure 4. Effect of curcumin on p300 and acetylated CBP/p300 gene expression level in HG –induced THP-1 cells

(A) Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and nuclear lysates were prepared. Protein was subjected to SDS-PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP). The immunoblot shown here is representative of three independent experiments with similar results. (B) Cells were fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4 °C with p300 as described in Methods. Data from a typical experiment of 3 are shown; Magnification × 400. *a: p<0.05 compared to NG; *p<0.05 compared to HG. (C) In order to measure Immunostaining intensity of p300, images were captured with a Nikon eclipse TE200 camera (Japan). The signal intensity was measured using ImageJ software *a: p<0.05 compared to NG; *p<0.05 compared to HG.

Figure 5. Effect of curcumin on HDAC2 and p300 mRNA level in HG –induced THP-1 cells

Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and total RNA were prepared. Template cDNAs are obtained by reverse transcription (RT) using QuantiTect reverse trasnscription kit (Qiagen). Detection of cDNAs was done by PCR reactions using primers designed from for candidate transcript detection. HDAC2 and p300. Real-time RT-PCR experiments was done in triplicate using SYBRR Green chemistry and a Mastercycler ep gradient S (eppendorf). The quantity of each sample was normalized to the levels of GAPDH transcripts. qPCR data were calculated by Delta-Delta CT method. Values are expressed as mean ± standard deviation (n = 3; technical replicates). Data represent mean ± SD. *a: p<0.05 compared to NG; *p<0.05 compared to HG..

Figure 6. Suppression of NF-κBp65 activation pathway by curcumin in HG-induced THP-1 cells

(A)Following treatment of cells with various concentrations of curcumin for 72 h as described in Methods, cells were harvested and nuclear lysates were prepared. Protein was subjected to SDS-PAGE as detailed in Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP). The immunoblot shown here are representative of three independent experiments with similar results. (B) The commercially available kit for NF-κB/p65 (Active Motif, Carlsbad, CA) contains the specific oligos with the specific consensus sequence for NF-κB/p65 binding. Five micrograms of nuclear lysate protein from each group was taken for quantification of NF-κB activity. The experiment was done according to the manufacturer's instructions. Absorbance was taken at 450 nm by using ELISA reader (Multiscan MCC/340, Fisher Scientific). (C) Cells were fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4 °C with p300 as described in Methods. Data from a typical experiment of 3 are shown; Magnification × 400. *a: p<0.05 compared to NG; *p<0.05 compared to HG. (D) In order to measure Immunostaining intensity of acetylated p65, images were captured with a Nikon eclipse TE200 camera (Japan). The signal intensity was measured using ImageJ software. *a: p<0.05 compared to NG; *p<0.05 compared to HG..

Figure 7. Effect of curcumin on chromatin events at the promoters of inflammatory genes

ChIP assays were performed using Chip-IT™ Express (Active Motif) according to the manufacturer's instructions. Immunoprecipitations were performed overnight at 4°C with 5 μL of p300 antibody. DNA was subjected to PCR. ChIP assays showed the recruitment of p300 to the TNF-α (A) and IL-6 (B) promoters. Results of 1 typical experiment of 3 are shown. Values from ChIP with anti-p300 antibody represents the fold difference relative to those from IgG control antibody. Data are the average of three independent experiments each, and error bars represent standard deviations. *a: p<0.05 compared to NG; *p<0.05 compared to HG..

Figure 8. Proposed epigenetic mechanisms via which curcumin regulates NF-κB signaling pathway leading to decreased expression of pro-inflammatory genes

High glucose activates the NF-κB signaling pathway leading to pro-inflammatory gene expression. Curcumin treatment of HG-induced cells activates the HDACs activity, especially HDAC2, and suppresses HAT activity, especially p300, leading to deacetylation of p65 NF-κB, subsequently suppressing proinflammatory cytokine release.