Antioxidants inhibit SAA formation and pro-inflammatory cytokine release in a human cell model of alkaptonuria

Abstract

Objective: Alkaptonuria (AKU) is an ultra-rare autosomal recessive disease that currently lacks an appropriate therapy. Recently we provided experimental evidence that AKU is a secondary serum amyloid A (SAA)-based amyloidosis. The aim of the present work was to evaluate the use of antioxidants to inhibit SAA amyloid and pro-inflammatory cytokine release in AKU.

Methods: We adopted a human chondrocytic cell AKU model to evaluate the anti-amyloid capacity of a set of antioxidants that had previously been shown to counteract ochronosis in a serum AKU model. Amyloid presence was evaluated by Congo red staining. Homogentisic acid-induced SAA production and pro-inflammatory cytokine release (overexpressed in AKU patients) were evaluated by ELISA and multiplex systems, respectively. Lipid peroxidation was evaluated by means of a fluorescence-based assay.

Results: Our AKU model allowed us to prove the efficacy of ascorbic acid combined with N-acetylcysteine, taurine, phytic acid and lipoic acid in significantly inhibiting SAA production, pro-inflammatory cytokine release and membrane lipid peroxidation.

Conclusion: All the tested antioxidant compounds were able to reduce the production of amyloid and may be the basis for establishing new therapies for AKU amyloidosis.

Keywords: N-acetylcysteine; ascorbic acid; chondrocyte; homogentisic acid; inflammation; lipid peroxidation; lipoic acid; phytic acid; serum amyloid A; taurine.

Fig. 1

Antioxidant treatment inhibits HGA-induced amyloid formation in human chondrocytes. (A, B) HGA-treated human chondrocytes were pretreated with antioxidants and amyloid presence was revealed by CR staining. Magnification ×20. (B) Dose-response effect of treatment with N-acetylcysteine and ascorbic acid. Magnification ×20. (C) Semi-quantitative analysis of birefringence reduction. Data are the mean of three independent experiments each performed in triplicate. P < 0.05. All data were significant.

Fig. 2

HGA-treated human chondrocytes overexpress and release SAA, and Nac + Asc treatment restores control levels. Data are the mean of three independent experiments each performed in triplicate. P < 0.05.

Fig. 3

Pro-inflammatory cytokines are overexpressed in HGA-treated human chondrocytes, and Nac + Asc treatment restores control levels. Data are the mean of three independent experiments each performed in triplicate. P < 0.05.

Fig. 4

Pro-inflammatory cytokines IL-6 and IL-8 are overexpressed in chondrocytes from AKU patients. (A) IL-6 is overexpressed in chondrocytes from AKU patients compared with non-diseased cells. Data are reported as the average concentration from three patients expressed in pg/ml and are the mean of three independent experiments each performed in triplicate. P < 0.05. (B) IL-8 is overexpressed in chondrocytes from AKU patients compared with non-diseased cells. Data are reported as the average concentration from three patients expressed in pg/ml and are the mean of three independent experiments each performed in triplicate. P < 0.05.

Fig. 5

LPO assay. (A) Fluorescence micrographs of human chondrocytes treated with HGA, N-acetylcysteine + ascorbic acid (Nac/Asc), HGA in the presence of N-acetylcysteine + ascorbic acid (HGA + Nac/Asc); CTR: human chondrocytes without any treatment. For each sample, two micrographs were acquired (only specific areas are shown): red colour showing the intercalated probe in the membrane and green colour showing the intensity of LPO. Bar = 5 µm. (B) Mean and s.d. of the percentage of human chondrocytes (calculated against the total number of cells) that showed a high green fluorescence (C11-BODIPY^581/591 probe), an index of LPO, are reported.