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Curcumin attenuates MSU crystal-induced inflammation by inhibiting the degradation of IκBα and blocking mitochondrial damage - PubMed

  • ️Tue Jan 01 2019

Curcumin attenuates MSU crystal-induced inflammation by inhibiting the degradation of IκBα and blocking mitochondrial damage

Baofeng Chen et al. Arthritis Res Ther. 2019.

Abstract

Background: Gouty arthritis is characterized by the deposition of monosodium urate (MSU) within synovial joints and tissues due to increased urate concentrations. In this study, we explored the effect of the natural compound curcumin on the MSU crystal-stimulated inflammatory response.

Methods: THP-1-derived macrophages and murine RAW264.7 macrophages were pretreated with curcumin for 1 h and then stimulated with MSU suspensions for 24 h. The protein level of TLR4, MyD88, and IκBα, the activation of the NF-κB signaling pathway, the expression of the NF-κB downstream inflammatory cytokines, and the activity of NLRP3 inflammasome were measured by western blotting and ELISA. THP-1 and RAW264.7 cells were loaded with MitoTracker Green to measure mitochondrial content, and MitoTracker Red to detect mitochondrial membrane potential. To measure mitochondrial reactive oxygen species (ROS) levels, cells were loaded with MitoSOX Red, which is a mitochondrial superoxide indicator. The effects of curcumin on mouse models of acute gout induced by the injection of MSU crystals into the footpad and synovial space of the ankle, paw and ankle joint swelling, lymphocyte infiltration, and MPO activity were evaluated.

Results: Curcumin treatment markedly inhibited the degradation of IκBα, the activation of NF-κB signaling pathway, and the expression levels of the NF-κB downstream inflammatory genes such as IL-1β, IL-6, TNF-α, COX-2, and PGE2 in the MSU-stimulated THP-1-derived macrophages. Curcumin administration protected THP-1 and RAW264.7 cells from MSU induced mitochondrial damage through preventing mitochondrial membrane potential reduction, decreasing mitochondria ROS, and then inhibited the activity of NLRP3 inflammasome. Intraperitoneal administration of curcumin alleviated MSU crystal-induced paw and ankle joint swelling, inflammatory cell infiltration, and MPO activity in mouse models of acute gout. These results correlated with the inhibition of the degradation of IκBα, the phosphorylation levels of NF-κB subunits (p65 and p50), and the activity of NLRP3 inflammasome.

Conclusion: Curcumin administration effectively alleviated MSU-induced inflammation by suppressing the degradation of IκBα, the activation NF-κB signaling pathway, the damage of mitochondria, and the activity of NLRP3 inflammasome. Our results provide a new strategy in which curcumin therapy may be helpful in the prevention of acute episodes of gout.

Keywords: Curcumin; Gout; IκBα; MSU; NF-κB.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1

Effects of curcumin on MSU-induced degradation of IκBα and NF-κB activation in THP-1 cells. a THP-1 cells were pretreated with curcumin for 1 h and then stimulated with MSU suspensions. Total protein extracted from THP-1 cells was reacted with anti-IκBα by western blotting. b Densitometry analysis of IκBα. c THP-1 cells were pretreated with curcumin for 1 h and then stimulated with MSU suspensions. Cytosolic and nuclear protein extracted from THP-1 cells was respectively reacted with anti-phosphorylated P50 (p-P50), anti-phosphorylated P65 (p-P65), anti-P50, and anti-P65 by western blotting. c–i Densitometry analysis of p-P50, p-P65, P50, and P65 protein. The data represent the mean ± SEM for three experiments

Fig. 2
Fig. 2

The effects of curcumin on the P50 and P65 nuclear translocation in MSU-stimulated RAW264.7 cells. a The percentages of p50 in the RAW264.7 nucleus were quantified in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin). b The percentages of p65 in the RAW264.7 nucleus were quantified in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin). Blue shows nuclei staining with DAPI. Scale bar: 10 μm. # Significantly different from absence both MSU and curcumin. * Significantly different from absence of curcumin, P < 0.05

Fig. 3
Fig. 3

Effects of curcumin on the expression of MSU-induced NF-κB downstream inflammatory cytokines. a mRNA expression of the NF-κB downstream inflammatory cytokines IL-1β, IL-6, TNF-α, PGE2, and COX-2 in THP-1-derived macrophages treated with curcumin and MSU suspensions (n = 3, mean ± SEM). b Measurement of the secretion of the NF-κB downstream inflammatory cytokines IL-1β, IL-6, TNF-α, and PGE2 in macrophages treated with curcumin and MSU suspensions (n = 3, mean ± SEM). c Representative western blot analysis of COX-2 in MSU-stimulated THP-1-derived macrophages with pretreatment of curcumin. d Densitometry measurements of protein analysis. The data represent mean ± SEM for three experiments. #Significantly different from absence both MSU and curcumin. *Significantly different from absence of curcumin, P < 0.05

Fig. 4
Fig. 4

Curcumin inhibits the mRNA expression of MSU crystals induced inflammatory cytokines in the PBMCs. a The relative mRNA of IL-1β (n = 3, mean ± SEM). b The relative mRNA of IL-6 (n = 3, mean ± SEM). c The relative mRNA of TNF-α (n = 3, mean ± SEM). d The relative mRNA of COX-2 (n = 3, mean ± SEM). The data represent the mean ± SEM for three experiments. #Significantly different from absence both MSU and curcumin. *Significantly different from absence of curcumin, P < 0.05

Fig. 5
Fig. 5

Curcumin blocks the expression levels of TLR4, MyD88, and NLRP3 in the MSU-induced THP-1 cells. a The relative mRNA expression of TLR4, MyD88, and NLRP3 in the MSU-stimulated THP-1 cells pretreated with curcumin (n = 3, mean ± SEM). b Effects of curcumin on the MSU-stimulated protein levels of TLR4, MyD88, and NLRP3 in the MSU-stimulated THP-1 cells; the protein levels were determined by western blot analysis. c Densitometry analysis of TLR4, MyD88, and NLRP3 protein. The data are expressed as mean ± SEM for three experiments. #Significantly different from absence both MSU and curcumin. *Significantly different from absence of curcumin, P < 0.05

Fig. 6
Fig. 6

Curcumin suppresses the expression of SOD2, Caspase-1 activation, and IL-1β maturation in the THP-1 cells. a Effects of curcumin on the MSU-stimulated expression levels of SOD2, p20 subunit of Caspase-1, and active p17 form of IL-1β; the expression levels were analyzed by western blot. b–d Densitometric analysis was used to quantify the level of SOD2, cleavage of caspase-1, and active p17 form of IL-1β. The data are expressed as mean ± SEM for three experiments. #Significantly different from absence both MSU and curcumin. *Significantly different from absence of curcumin, P < 0.05

Fig. 7
Fig. 7

Curcumin inhibits the activity of T-SOD, Caspase-1 activation, and secretion of IL-1β in the MSU-stimulated RAW264.7 cells. a Effects of curcumin on the MSU-stimulated protein levels of p20 subunit of Caspase-1. b the activity of T-SOD (n = 3, mean ± SEM). c Densitometry analysis of p20 subunit of Caspase-1, the results represent the mean ± SEM for three experiments. P < 0.05. d Supernatants were analyzed by ELISA for IL-1β (n = 3, mean ± SEM). #Significantly different from absence both MSU and curcumin. *Significantly different from absence of curcumin, P < 0.05

Fig. 8
Fig. 8

Curcumin blocks the decrease of mitochondrial membrane potential and reduces the production of mitochondrial ROS in the MSU-stimulated THP-1 cells. a Representative micrographs of MitoTracker Red- and MitoTracker Green-labeled THP-1 cells in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin); blue shows nuclei staining with Hoechst 33342. Scale bar: 10 μm. b The relative fluorescence intensities of MitoTracker Green were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoTracker Green in the MSU-untreated THP-1 cells. c The relative fluorescence intensities of MitoTracker Red were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoTracker Green in the MSU-untreated THP-1 cells. d The ratio of relative fluorescence intensities of MitoTracker Red to the relative fluorescence intensities of MitoTracker Green. e Representative micrographs of MitoSOX Red- and MitoTracker Green-labeled THP-1 cells in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin). f The relative fluorescence intensities of MitoSOX Red were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoSOX Red in the MSU-untreated THP-1 cells; blue shows nuclei staining with Hoechst 33342. For all measurements, the values were from three independent experiments with four fields of view in each experiment

Fig. 9
Fig. 9

Curcumin prevents the mitochondrial MMP collapse and lowers the production of mitochondrial ROS in the MSU-stimulated RAW264.7 cells. a Representative micrographs of MitoTracker Red- and MitoTracker Green-labeled THP-1 cells in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin); blue shows nuclei staining with Hoechst 33342. Scale bar: 10 μm. b The relative fluorescence intensities of MitoTracker Green were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoTracker Green in the MSU-untreated THP-1 cells. c The relative fluorescence intensities of MitoTracker Red were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoTracker Green in the MSU-untreated THP-1 cells. d The ratio of relative fluorescence intensities of MitoTracker Red to the relative fluorescence intensities of MitoTracker Green. e Representative micrographs of MitoSOX Red- and MitoTracker Green-labeled THP-1 cells in the absence (Control) or presence of MSU (0.2 mg/ml) or combinations (0.2 mg/ml MSU + 5 μM curcumin). f The relative fluorescence intensities of MitoSOX Red were quantified in MSU-treated THP-1 cells which were divided by the fluorescence intensities of MitoSOX Red in the MSU-untreated THP-1 cells; blue shows nuclei staining with Hoechst 33342. All the values were from three independent experiments with four fields of view in each experiment

Fig. 10
Fig. 10

Curcumin alleviates the swelling of ankle and footpad in MSU-induced arthritis. a MSU suspensions were injected into the right rear ankle joints of C57BL/6 mice, while the same volume of PBS was injected into the left ankles. The swelling is expressed as the right-left/left ratio and a ratio > 0.15 indicates inflammation. Data are expressed as the mean ± SEM of six mice per group. b A total of 1 mg/40 μl MSU suspensions were injected into the right footpad of mice, and foot thickness was detected 24 h after MSU administration (n = 6 for each group). c The ankle joint swelling index is expressed as the MSU-injected joint/PBS-injected joint ratio. d The paw swelling index is expressed as the MSU-injected paw/PBS-injected paw ratio. e Myeloperoxidase (MPO) activity of the homogenates of footpad tissue (n = 4 per group, mean ± SEM)

Fig. 11
Fig. 11

Curcumin inhibits the inflammatory cells infiltration in the MSU injection of mice footpad. a Representative photographs of HE staining of footpads from the MSU + vehicle group (× 100 original magnification). b MSU + vehicle group (200 × original magnification), arrow indicates abundant inflammatory cells in the footpad tissue section. c Representative photographs of HE staining of footpads from the MSU + curcumin group (× 100 original magnification). d MSU + curcumin group (× 200 original magnification), arrow indicates fewer inflammatory cells in the footpad tissue section

Fig. 12
Fig. 12

Curcumin blocks the MSU-induced degradation of IκBα and NF-κB activation in the footpad tissue of mice with MSU-induced arthritis. a The total protein extracted from the footpad tissue was analyzed by immunoblotting for degradation of IκBα and NF-κB activation. b–h Densitometry measurements of protein analysis. The data represent the mean ± SEM of four mice per group. #Significantly different from vehicle (Veh) alone, P < 0.05. *Significantly different from MSU alone, P < 0.05

Fig. 13
Fig. 13

Curcumin suppresses the protein levels of MSU-induced COX-2, TLR4, MyD88, and NLRP3 in the footpad tissue of mice with MSU-induced arthritis. a Total protein was examined by immunoblotting for the protein level of COX-2. b Total protein was examined by immunoblotting for the protein levels of TLR4, MyD88, and NLRP3. c, d, e, f Densitometry analysis of TLR4, MyD88, and NLRP3. The data are expressed as the mean ± SEM of four mice per group. #Significantly different from vehicle (Veh) alone, P < 0.05. *Significantly different from MSU alone, P < 0.05

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