DNA repair protein APE1 is involved in host response during pneumococcal meningitis and its expression can be modulated by vitamin B6

Abstract

Background: The production of reactive oxygen species (ROS) during pneumococcal meningitis (PM) leads to severe DNA damage in the neurons and is the major cause of cell death during infection. Hence, the use of antioxidants as adjuvant therapy has been investigated. Previous studies have demonstrated the possible participation of apurinic/apyrimidinic endonuclease (APE1) during PM. The aims of this study were to investigate the APE1 expression in the cortical and hippocampal tissues of infant Wistar rats infected with Streptococcus pneumoniae and its association with cell death and understand the role of vitamin B6 (vitB6) as a protective factor against cell death.

Methods: APE1 expression and oxidative stress markers were analyzed at two-time points, 20 and 24 h post infection (p.i.), in the cortex (CX) and hippocampus (HC) of rats supplemented with vitB6. Statistical analyses were performed by the nonparametric Kruskal-Wallis test using Dunn's post test.

Results: Our results showed high protein levels of APE1 in CX and HC of infected rats. In the CX, at 20 h p.i., vitB6 supplementation led to the reduction of expression of APE1 and apoptosis-inducing factor, while no significant changes in the transcript levels of caspase-3 were observed. Furthermore, levels of carbonyl content and glutamate in the CX were reduced by vitB6 supplementation at the same time point of 20 h p.i.. Since our data showed a significant effect of vitB6 on the CX at 20 h p.i. rather than that at 24 h p.i., we evaluated the effect of administering a second dose of vitB6 at 18 h p.i. and sacrifice at 24 h p.i.. Reduction in the oxidative stress and APE1 levels were observed, although the latter was not significant. Although the levels of APE1 was not significantly changed in the HC with vitB6 adjuvant therapy, vitB6 supplementation prevented the formation of the truncated form of APE1 (34 kDa) that is associated with apoptosis.

Conclusions: Our data suggest that PM affects APE1 expression, which can be modulated by vitB6. Additionally, vitB6 contributes to the reduction of glutamate and ROS levels. Besides the potential to reduce cell death and oxidative stress during neuroinflammation, vitB6 showed enhanced effect on the CX than on the HC during PM.

Keywords: APE1; Cortex; Hippocampus; Oxidative stress; Pneumococcal meningitis; Vitamin B6.

Fig. 1

Effects of pre supplementation of vitamin B6 on APE1, AIF, and Casp-3 mRNA expression levels. Gene expression was determined in the CX (a, b, c) and HC (d, e, f) by real-time PCR and the fold change was calculated using the ΔCq method. Values were transformed to percentage using the control group at 20 h post infection as 100%. Each column data expresses the mean percentage (± SEM) relative to control 20 h. Comparisons between columns which show p < 0.05 were significant in Dunn’s post test. Groups: uninfected control animals (CTRL) 20 h (n = 5), uninfected control animals (CTRL) 24 h (n = 5), infected animals (PM) without vitB6 treatment 20 h p.i. (n = 5), infected animals (PM) without vitB6 treatment 24 h p.i. (n = 5), infected animals plus vitB6 treatment (PM/pre sup vitB6) 20 h p.i. (n = 8), infected animals plus vitB6 treatment (PM/pre sup vitB6) 24 h p.i. (n = 10)

Fig. 2

Western blotting (WB) analysis of the cortex (a, b) and hippocampus (c, d) tissues from Winstar rats infected with PM under vitB6 pre-supplementation treatment: a Significant reduction in the expression of APE1 is observed in the CX/20 h in the PM/pre sup vitB6 animals compared to the PM animals; b In the CX/24 h, WB from infected animals showed different sizes of APE1 (37 and 70 kDa) which resulted in a higher expression as compared to the control group as observed in a; c In HC/20 h, a significant increase in APE1 protein level was observed in the PM animals compared to the CTRL groups; d WB from the HC showed a cleaved form of APE1 in some infected animals which was not detected in the lysates from animals which received vitB6. β-actin was measured as the loading control and was used for data normalization. Data are reported as the mean percentage relative to control of each time point ± SEM. Groups for the CX: uninfected control animals (CTRL) 20 h (n = 5), uninfected control animals (CTRL) 24 h (n = 3), infected animals (PM) without vitB6 treatment 20 h p.i. (n = 5), infected animals (PM) without vitB6 treatment 24 h p.i. (n = 3), infected animals plus vitB6 treatment (PM/pre sup vitB6) 20 h p.i. (n = 9), and infected animals plus vitB6 treatment (PM/pre sup vitB6) 24 h p.i. (n = 3). Groups for the HC: uninfected control animals (CTRL) 20 h (n = 3), uninfected control animals (CTRL) 24 h (n = 3), infected animals (PM) without vitB6 treatment 20 h p.i. (n = 5), infected animals (PM) without vitB6 treatment 24 h p.i. (n = 3), infected animals plus vitB6 treatment (PM/pre sup vitB6) 20 h p.i. (n = 4), and infected animals plus vitB6 treatment (PM/pre sup vitB6) 24 h p.i. (n = 3)

Fig. 3

Levels of glutamate (a, b) and carbonyl content (c, d) in the CX and HC tissue from animals with PM after vitB6 supplementation. Glutamate level is significant increased during infection in both tissues and vitB6 has an effective influence up to 20 h p.i.. Reduction of carbonyl content were significant only in the CX/20 h in PM/pre sup vitB6. In contrast, in the HC, the vitB6 is associated to the highest level of carbonyls. Values were transformed to percentage using the control group at 20 h post infection as 100%. Each column data expresses the mean percentage (± SEM) relative to control 20 h from at least three or more animals. Comparison between columns which show p < 0.05 were significant in Dunn’s post test. Groups for the CX: uninfected control animals (CTRL) 20 h (n = 3), uninfected control animals (CTRL) 24 h (n = 5), infected animals (PM) without vitB6 treatment 20 h p.i. (n = 5), infected animals (PM) without vitB6 treatment 24 h p.i. (n = 5), infected animals plus vitB6 treatment (PM/pre sup vitB6) 20 h p.i. (n = 9), and infected animals plus vitB6 treatment (PM/pre sup vitB6) 24 h p.i. (n = 9). Groups for the HC: uninfected control animals (CTRL) 20 h (n = 3), uninfected control animals (CTRL) 24 h (n = 3), infected animals (PM) without vitB6 treatment 20 h p.i. (n = 3), infected animals (PM) without vitB6 treatment 24 h p.i. (n = 3), infected animals plus vitB6 treatment (PM/pre sup vitB6) 20 h p.i. (n = 9), and infected animals plus vitB6 treatment (PM/pre sup vitB6) 24 h p.i. (n = 4)

Fig. 4

Effect of an additional dose of vitB6 on APE1 protein expression (a) and carbonyl content (b) in the CX/24 h. Western blot images reveal a reduction in the expression of different APE1 forms after vitB6 pre/post supplementation as indicated in the graph (a). Carbonyl content was significantly reduced after an extra dose of vitB6 (b). Each column from carbonyl data expresses the mean percentage (± SEM) relative to control CX/20 h (showed in Fig. 3c). Comparison between columns which show p < 0.05 were significant in Dunn’s post test. Groups: uninfected control animals (CTRL) (n = 3), infected animals (PM) without vitB6 treatment (n = 6), infected animals plus vitB6 treatment (PM/pre sup vitB6) (n = 9), and infected animals plus second dose of vitB6 treatment (PM/pre pos sup vitB6) (n = 5)