Oxidative Damage of Blood Platelets Correlates with the Degree of Psychophysical Disability in Secondary Progressive Multiple Sclerosis

Abstract

The results of past research studies show that platelets are one of the main sources of reactive oxygen species (ROS) and reactive nitrogen species (RNS) to be found in the course of many pathological states. The aim of this study was to determine the level of oxidative/nitrative stress biomarkers in blood platelets obtained from multiple sclerosis (MS) patients (n = 110) and to verify their correlation with the clinical parameters of the psychophysical disability of patients. The mitochondrial metabolism of platelets was assessed by measuring the intracellular production of ROS using the fluorescence method with DCFH-DA dye and by identification of changes in the mitochondrial membrane potential of platelets using the JC-1 dye. Moreover, we measured the mRNA expression for the gene encoding the cytochrome c oxidase subunit I (MTCO-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in platelets and megakaryocytes using the RT-qPCR method, as well as the concentration of NADPH oxidase (NOX-1) by the ELISA method. Our results proved an increased level of oxidative/nitrative damage of proteins (carbonyl groups, 3-nitrotyrosine) (p < 0.0001) and decreased level of -SH in MS (p < 0.0001) and also a pronounced correlation between these biomarkers and parameters assessed by the Expanded Disability Status Scale and the Beck's Depression Inventory. The application of fluorescence methods showed mitochondrial membrane potential disruption (p < 0.001) and higher production of ROS in platelets from MS compared to control (p < 0.0001). Our research has also confirmed the impairment of red-ox metabolism in MS, which was achieved by increasing the relative mRNA expression in platelets for the genes studied (2-fold increase for the MTCO-1 gene and 1.5-fold increase in GAPDH gene, p < 0.05), as well as the augmented concentration of NOX-1 compared to control (p < 0.0001). Our results indicate that the oxidative/nitrative damage of platelets is implicated in the pathophysiology of MS, which reflects the status of the disease.

Figure 1

The concentration of carbonyl groups in blood platelets, determined immediately after isolation from whole, untreated blood in SP MS patients (n = 40) and the control group (n = 40). The results are expressed as values obtained for individual subjects (nmol/mg of platelet proteins) with the mean value ± SD. Statistical analysis was performed using the unpaired Student-t, ^∗∗∗∗p < 0.0001.

Figure 2

The concentration of 3-NT in blood platelets, determined immediately after isolation from whole, untreated blood in SP MS patients (n = 40) and control group (n = 40). The results are expressed as values obtained for individual subjects (nmol/mg of platelet proteins) with the mean value ± SD. Statistical analysis was performed using the unpaired Student-t, ^∗∗∗∗p < 0.0001.

Figure 3

The concentration of –SH groups in blood platelets, determined immediately after isolation from whole, untreated blood in SP MS patients (n = 50) and the control group (n = 50). The results are expressed as values obtained for individual subjects (nmol/mg of platelet proteins) with the mean value ± SD. Statistical analysis was performed using the unpaired Student-t, ^∗∗∗∗p < 0.0001.

Figure 4

Regression plots of carbonyl group levels in platelet proteins obtained from SP MS patients and EDSS (a), BDI (b) scales.

Figure 5

Regression plots of 3-NT levels in platelet proteins obtained from SP MS patients and EDSS (a), BDI (b) scales.

Figure 6

The level of intracellular ROS generated in blood platelets. ROS production measured as intensity of DCF fluorescence in SP MS patients (n = 30) and control group (n = 30). Statistical analysis was performed using the Mann–Whitney U test. The results are expressed as the mean value of DCF-DA fluorescence ± SD, ^∗∗∗∗p < 0.0001.

Figure 7

Changes in MMP (ΔΨm) in blood platelets. MMP is expressed as a ratio of JC-1 aggregates (530 nm/590 nm) to JC-1 monomers (485 nm/538 nm), as quantified with a fluorescent plate reader after JC-1 staining in blood platelets obtained from SP MS patients (n = 30) and the control group (n = 30). Statistical analysis was performed using the Mann–Whitney U test. The results are shown as the mean value of fluorescence ratio (590 nm/538 nm) ± SD, ^∗∗∗∗p < 0.0001.

Figure 8

The concentration of NOX-1 in blood platelets, determined immediately after isolation from whole blood untreated. The comparison of NOX-1 expression level (ng/ml) measured in blood platelets in SP MS patients (n = 30) and control group (n = 30). The results are expressed as values obtained for individual subjects with the mean value ± SD. Statistical analysis was performed using the Mann–Whitney U test, ^∗∗∗∗p < 0.0001.

Figure 9

The mRNA level of MTCO-1 gene in blood platelets and megakaryocytes. Relative expression of mRNA for the MTCO-1 gene in platelets and megakaryocytes from patients with SP MS (n = 45) and control group (n = 45). Statistical analysis was performed using the Mann–Whitney U test. The results are expressed as the mean value of 2^−ΔCt ± SD (according to the reference gene–18SrRNA), ^∗p < 0.05.

Figure 10

The mRNA level of GAPDH gene in blood platelets and megakaryocytes. The relative expression of mRNA for the GAPDH gene in platelets and megakaryocytes from patients with SP MS (n = 45) and control group (n = 45). Statistical analysis was performed using the Mann–Whitney U test. The results are expressed as the mean value of 2^−ΔCt ± SD (according to the reference gene–18SrRNA), ^∗p < 0.05.