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Randomized Controlled Trial
. 2014 Mar 10;20(8):1169-80.
doi: 10.1089/ars.2013.5198. Epub 2013 Sep 19.

NLRP3 Inflammasome Is Activated in Fibromyalgia: The Effect of Coenzyme Q10

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Free PMC article
Randomized Controlled Trial

NLRP3 Inflammasome Is Activated in Fibromyalgia: The Effect of Coenzyme Q10

Mario D Cordero et al. Antioxid Redox Signal. .
Free PMC article

Abstract

Aims: Fibromyalgia (FM) is a prevalent chronic pain syndrome characterized by generalized hyperalgesia associated with a wide spectrum of symptoms such as fatigue and joint stiffness. Diagnosis of FM is difficult due to the lack of reliable diagnostic biomarkers, while treatment is largely inadequate. We have investigated the role of coenzyme Q10 (CoQ10) deficiency and mitochondrial dysfunction in inflammasome activation in blood cells from FM patients, and in vitro and in vivo CoQ10 deficiency models.

Results: Mitochondrial dysfunction was accompanied by increased protein expression of interleukin (IL)-1β, NLRP3 (NOD-like receptor family, pyrin domain containing 3) and caspase-1 activation, and an increase of serum levels of proinflammatory cytokines (IL-1β and IL-18). CoQ10 deficiency induced by p-aminobenzoate treatment in blood mononuclear cells and mice showed NLRP3 inflammasome activation with marked algesia. A placebo-controlled trial of CoQ10 in FM patients has shown a reduced NLRP3 inflammasome activation and IL-1β and IL-18 serum levels.

Innovation: These results show an important role for the NLRP3 inflammasome in the pathogenesis of FM, and the capacity of CoQ10 in the control of inflammasome.

Conclusion: These findings provide new insights into the pathogenesis of FM and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease.

Figures

<b>FIG. 1.</b>
FIG. 1.
Mitochondrial dysfunction and oxidative stress in fibromyalgia blood mononuclear cells (BMCs). n=20 and 30 for control and fibromyalgia groups, respectively. (A) Relative expression of mitocondrial biogenesis genes (mean±SE) determined by quantitative PCR in BMCs from fibromyalgia (FM) patients. (B) Protein expression levels of mitochondrial complex I (39 kDa Subunit), complex III (Core I Subunit), cytochrome c, and 8-oxoguanine glycosylase (OGG-1, a DNA glycosylase enzyme responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG). (C) Protein levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots and normalized to the GADPH signal, using BMCs from four representative FM patients, compared with a pool of five healthy age- and sex-matched control subjects. (D) Coenzyme Q10 (CoQ10) levels in control and FM cells were determined by hexane extraction and high-performance liquid chromatography (HPLC) separation as described in Material and Methods section. (E) ATP levels in control and FM BMCs were analyzed by bioluminescence as described in Material and Methods section. (F, G) Mitochondrial reactive oxygen species (ROS) production and 8-oxoG were analyzed in BMCs from control and FM patients by flow cytometry and EIA kit as described in Material and Methods section. Data represent the mean±SD of three separate experiments. *p<0.001, **p<0.01 between control and FM patients.
<b>FIG. 2.</b>
FIG. 2.
Mitochondrial complexes enzymatic activities in fibromyalgia BMCs. n=20 and 30 for control and fibromyalgia groups, respectively. Mitochondrial enzymatic activities were determined as described in Material and Methods section. Results (mean±SD) are expressed in U/CS (units per citrate synthase). Data represent the mean±SD of three separate experiments. *p<0.001, **p<0.01 between control and FM patients.
<b>FIG. 3.</b>
FIG. 3.
Inflammasome activation in BMCs and proinflammatory cytokines in serum from FM patients. (A) NLRP3 protein levels, caspase 1 cleavage, and caspase 3 cleavage were analyzed by western blotting. We include a positive control of caspase 1 and 3 activation using lipopolysaccharides. Protein levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots and normalized to the GADPH signal, using BMCs from four representative FM patients, compared with a pool of five healthy age- and sex-matched control subjects. (B, C) NLRP3 and caspase 1 cleavage transcript expression levels were determined by real-time quantitative RT-PCR. n=20 for control and n=30 for FM groups, respectively. Data represent the mean±SD of three separate experiments.*p<0.05 between control and FM patients. (D, E) IL-1β and IL-18 levels in serum from control and FM patients were determined by ELISA as described in Material and Methods section. n=20 for control and n=30 for FM groups, respectively. (F) IL-1β protein levels in several patients and IL-1β and caspase 1 activation in patients before and after CoQ10 treatment in BMCs isolated from FM patients and cultured. Data represent the mean±SD of three separate experiments. *p<0.001 between control and FM patients.
<b>FIG. 4.</b>
FIG. 4.
Correlation of CoQ10 (A), and mitochondrial ROS levels (B) in BMCs from FM patients and IL-1β levels. n=30 for fibromyalgia groups. The correlation was established by calculating correlation coefficients.
<b>FIG. 5.</b>
FIG. 5.
Association of IL-1β serum levels and pain scores in FM patients (A) and the mouse model of CoQ10 deficiency (B). n=30 for fibromyalgia groups and n=5 for mice. The strength of the association was established by calculating correlation coefficients.
<b>FIG. 6.</b>
FIG. 6.
CoQ10 deficiency induction activates inflammasome complex in BMCs. (A, B) CoQ10 deficiency and decreased ATP levels induced by 1 mM p-aminobenzoate (PABA) treatment for 24 h in BMCs from five healthy volunteers. (C) Protein expression levels of OGG-1 and NLRP3 and induction of caspase 1 cleavage analyzed by western blotting in a homogenate cell pool of BMCs from five controls. (D) Protein expression levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots and normalized to the GADPH signal. (E, F) IL-1β and IL-18 levels in the culture media of BMCs incubated with PABA for 24 h and analyzed by ELISA as described in Material and Methods section. Data represent the mean±SD of three separate experiments. *p<0.001 between control and PABA, and between PABA and CoQ10.
<b>FIG. 7.</b>
FIG. 7.
Inflammasome activation induced by CoQ10 deficiency induction in mice. (A). CoQ levels were measured in BMCs isolated from mice treated with vehicle or PABA for 15 days (n=5 per group). *p<0.05; between vehicle and PABA-treated mice. (B) NLRP3 protein expression levels and caspase 1 cleavage were analyzed by western blotting. Protein levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots and normalized to GADPH signal, using BMCs isolated from mice treated with the vehicle or PABA. (C, D) IL-1β and IL-18 in serum levels from mice treated with the vehicle or PABA-treated were determined by ELISA as described in Material and Methods section. *p<0.001 between vehicle and PABA-treated mice. (E) Pain sensitivity in vehicle- and PABA-treated mice was evaluated in the hot plate test (1) at 45°C–52.5°C±0.5°C and with the tail flick test (2) at 45°C±0.5°C. *p<0.05; ap<0.001.
<b>FIG. 8.</b>
FIG. 8.
NLRP3 and IL-1β gene expression and IL-1β and IL-18 levels in FM patients pre- and post-treatment with oral CoQ10 versus placebo. (A) Relative gene expressions of NLRP3 and IL-1β (mean±SE) determined by quantitative PCR in BMCs from FM patients. (B) Serum IL-1β and IL-18 levels in FM patients were measured as described in Material and Methods section. Data represent the mean±SD of three separate experiments. *p<0.01 between FM patients after and before oral treatment.

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