Quercetin modulates keratoconus metabolism in vitro

Abstract

Corneal scarring is the result of a disease, infection or injury. The resulting scars cause significant loss of vision or even blindness. To-date, the most successful treatment is corneal transplantation, but it does not come without side effects. One of the corneal dystrophies that are correlated with corneal scarring is keratoconus (KC). The onset of the disease is still unknown; however, altered cellular metabolism has been linked to promoting the fibrotic phenotype and therefore scarring. We have previously shown that human keratoconus cells (HKCs) have altered metabolic activity when compared to normal human corneal fibroblasts (HCFs). In our current study, we present evidence that quercetin, a natural flavonoid, is a strong candidate for regulating metabolic activity of both HCFs and HKCs in vitro and therefore a potential therapeutic to target the altered cellular metabolism characteristic of HKCs. Targeted mass spectrometry-based metabolomics was performed on HCFs and HKCs with and without quercetin treatment in order to identify variations in metabolite flux. Overall, our study reveals a novel therapeutic target OF Quercetin on corneal stromal cell metabolism in both healthy and diseased states. Clearly, further studies are necessary in order to dissect the mechanism of action of quercetin.

Keywords: cell metabolism; cornea; fibrosis; keratoconus; metabolomics; quercetin; urea cycle.

Figure 1

Metabolite enrichment analysis showing pathways upregulated in (A) HCFs and (B) HKCs following 10-μM quercetin treatment. Data was analysed using MetaboAnalyst using only metabolites upregulated >twofold. Data is representative of three independent experiments, n = 3

Figure 2

Metabolite enrichment analysis showing pathways downregulated in (A) HCFs and (B) HKCs following 10-μM quercetin treatment. Data was analysed using MetaboAnalyst using only metabolites downregulated >twofold. Data is representative of three independent experiments, n = 3

Figure 3

(A) Schematic depicting the role of quercetin in modulating metabolic flux in glycolysis in HCFs and HKCs. (B) Quantification of fold changes in glucose-6-phosphate (G-6-P), glyceraldehyde-3-phosphate (Glyc-3-P), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate (3-P-Gly), glucose-1-phosphate (G-1-P) and glycolate in HCFs and HKCs following quercetin treatment. Statistical significance denoted by * = p <0.05 determined by an unpaired Student T-test. Q = quercetin. Data is representative of three independent experiments, n = 3

Figure 4

(A) Schematic depicting the role of quercetin in modulating metabolic flux in the pentose phosphate pathway in HCFs and HKCs. (B) Quantification of fold changes in glucose-6-phosphate (G-6-P), glyceraldehyde-3-phosphate, erythrose-4-phosphate (Ery-4-P) and glyceraldehyde-3-phosphate (Gly-3-P) following quercetin treatment. Statistical significance denoted by * = p <0.05 determined by an unpaired Student T-test. Q = quercetin. Data is representative of three independent experiments, n = 3

Figure 5

(A) Schematic of the TCA cycle showing the effect of quercetin in modulating concentrations of key metabolites in HCFs and HKCs. (B) Quantification of fold changes in citrate, isocitrate, malate and oxaloacetate in HCFs and HKCs following quercetin treatment. Statistical significance denoted by * = p <0.05 determined by an unpaired Student T-test. Q = quercetin. Data is representative of three independent experiments, n = 3

Figure 6

(A) Schematic of the urea cycle showing the effect of quercetin in modulating concentrations of key metabolites in HCFs and HKCs. (B) Quantification of fold changes in arginine (Arg), carbamoyl phosphate (Carbamoyl-P) and fumarate. Statistical significance denoted by * = p <0.05 determined by an unpaired Student T-test. Q = quercetin. Data is representative of three independent experiments, n = 3

Figure 7

Relative flux of (A) AMP, (B) ADP and (C) ATP in HCFs and HKCs measured by LC-MS/MS. (D) Ratios of ATP/ADP and (E) ATP/AMP in HCFs and HKCs following quercetin or vehicle (control) treatment, as measured by LC-MS/MS. Values normalized to HCF control. n = 3, error bars represent standard error of the mean. Statistical significance denoted by * = p <0.05 determined by an unpaired Student T-test. Q = quercetin. Data is representative of three independent experiments, n = 3