Ceftriaxone, a beta-lactam antibiotic, modulates apoptosis pathways and oxidative stress in a rat model of neuropathic pain

Abstract

Purpose: In our previous study, ceftriaxone, a beta-lactam antibiotic, elicited antinociceptive effects in the chronic constriction injury (CCI) of neuropathic pain. In this study, we assessed apoptosis and oxidative stress in the spinal cord of neuropathic rats treated with ceftriaxone.

Methods: 45 male Wistar rats were divided as naïve, sham, normal saline-treated CCI rats, and CCI animals treated with the effective dose of ceftriaxone. Involvement of Bax, Bcl2, and caspases 3 and 9, important contributors of programmed cell death (apoptosis), was determined using western blotting at days 3 and 7. The markers of oxidative stress including malondialdehyde (MDA) and reduced glutathione (GSH) were measured on days 3 and 7.

Results: Increased Bax/Bcl2 ratio and cleaved active forms of caspases 3 and 9 were observed in the spinal cord of CCI rats on day 3. Ceftriaxone attenuated the increased levels of Bax and cleaved forms of caspases 3 and 9, while it increased Bcl2 levels. Bax and active forms of caspases declined by day 7. Consequently, comparison among groups showed no difference at this time. CCI enhanced MDA and decreased GSH on days 3 and 7, while ceftriaxone protected against the CCI-induced oxidative stress.

Conclusion: Our results suggest that ceftriaxone, an upregulator/activator of GLT1, could concomitantly reduce oxidative stress and apoptosis and producing its new analogs lacking antimicrobial activity may represent a novel approach for neuropathic pain treatment.

Figure 1

Effect of ceftriaxone on the spinal cord protein levels of Bax (21 kDa) and Bcl2 (26 kDa) and relative density of Bax/Bcl2, following western immunoblotting on days 3 (a and b, resp.) and 7 (c and d, resp.) after CCI. Administration of ceftriaxone (Cef 200 mg/kg, i.p.) started when CCI was induced and continued for 7 successive days. The semiquantitative analysis of protein levels was carried out by the “Gel Doc 2000 UV System” (Alliance 4.7). Each lane was loaded with 100 μg of proteins. β-actin is the loading protein control. Data are mean ± SEM (n = 3/group). One-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. ^# P < 0.05 difference between CCI-NS and sham/naive groups. *P < 0.05 CCI-Cef versus NS-CCI group (control).

Figure 2

Effect of ceftriaxone on the spinal cord protein levels of procaspase-9 (47 kDa) and cleavage products (37 and 35 kDa) and relative density following western immunoblotting on days 3 (a and b, resp.) and 7 (c and d, resp.) after CCI. Administration of ceftriaxone (Cef 200 mg/kg, i.p.) started when CCI was induced and continued for 7 successive days. The semiquantitative analysis of protein levels was carried out by the “Gel Doc 2000 UV System” (Alliance 4.7). Each lane was loaded with 100 μg of proteins. β-Actin is the loading protein control. Data were mean ± SEM (n = 3/group). One-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. ^## P < 0.01 difference between CCI-NS and sham/naive animals. *P < 0.05, **P < 0.05 CCI-Cef versus NS-CCI group (control).

Figure 3

Effect of ceftriaxone on the spinal cord protein levels of procaspase-3 (37 kDa) and cleavage product (17 kDa) and relative density following western immunoblotting on days 3 (a and b, resp.) and 7 (c and d, resp.) after CCI. Administration of ceftriaxone (Cef 200 mg/kg, i.p.) started when CCI was induced and continued for 7 successive days. The semiquantitative analysis of protein levels was carried out by the “Gel Doc 2000 UV System” (Alliance 4.7). Each lane was loaded with 100 μg of proteins. β-Actin is the loading protein control. Data were mean ± SEM (n = 3/group). One-way ANOVA followed by Tukey's post hoc test was used for multiple comparisons. ^# P < 0.05 difference between CCI-NS and sham/naive animals. *P < 0.05 CCI-Cef versus NS-CCI group (control).