Ceftriaxone and MC-100093 mitigate fentanyl-induced cardiac injury in mice: Preclinical investigation of its underlying molecular mechanisms

Abstract

Drug addiction is considered a worldwide concern and one of the most prevailing causes of death globally. Opioids are highly addictive drugs, and one of the most common opioids that is frequently used clinically is fentanyl. The potential harmful effects of chronic exposure to opioids on the heart are still to be elucidated. Although β-lactam antibiotics are well recognized for their ability to fight bacteria, its protective effect in the brain and liver has been reported. In this study, we hypothesize that β-lactam antibiotic, ceftriaxone, and the novel synthetic non-antibiotic β-lactam, MC-100093, are cardioprotective against fentanyl induced-cardiac injury by upregulating xCT expression. Mice were exposed to repeated low dose (0.05 mg/kg, i.p.) of fentanyl for one week and then challenged on day 9 with higher dose of fentanyl (1 mg/kg, i.p.). This study investigated cardiac histopathology and target genes and proteins in serum and cardiac tissues in mice exposed to fentanyl overdose and β-lactams. We revealed that fentanyl treatment induced cardiac damage as evidenced by elevated cardiac enzymes (troponin I). Furthermore, fentanyl treatment caused large aggregations of inflammatory cells and elevation in the areas and volumes of myocardial fibers, indicating hypertrophy and severe cardiac damage. Ceftriaxone and MC-100093 treatment, However, induced cardioprotective effects as evidenced by marked reduction in cardiac enzymes (troponin I) and changes in histopathology. Furthermore, ceftriaxone and MC-100093 treatment decreased the levels of hypertrophic genes (α-MHC & β-MHC), apoptotic (caspase-3), and inflammatory markers (IL-6 & NF-κB). This study reports for the first time the cardioprotective effect of β-lactams against fentanyl-induced cardiac injury. Further studies are greatly encouraged to completely identify the cardioprotective properties of ceftriaxone and MC-100093.

Keywords: Apoptosis; Cardiotoxicity; Ceftriaxone; Fentanyl; Glutamate transporter-1; MC-100093; xCT.

Fig. 1

Experimental Study Design.

Fig. 2

Serum levels of cardiac enzymes. Serum obtained from all groups were examined to determine cTnI (A) & CK-MB (B) levels. Data are showed as mean ± SD (n = 4). *significant against control & ^# significant against Fen group, where *p < 0.05, ^#p < 0.05, ^##p < 0.01. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; cTn-I, Troponin I cardiac muscle; CK-MB, Creatine kinase-MB.

Fig. 3

Photomicrographs of cardiac tissue using H&E stain. (A) control cardiac cells showing normal view. (B) Animals treated with fentanyl revealing great inflammation (black arrows), vacuolar degeneration (green arrow). (C) Animals treated with fentanyl plus MC-100093 posting no pathological signs. (D) Animals treated with fentanyl and ceftriaxone showing no pathological features. (F) means myocardial fibers (H&E-400X). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Photomicrographs of cardiac muscles using Masson’s trichrome stain. (A) Control cardiac muscles showing normal depositions of connective tissue of intermediate spaces. (B) Cardiac cells of animals treated with fentanyl exhibiting great fibrosis (F) of bundles of collagenous fibers (green arrows). (C) Cardiac cells of animals treated with fentanyl and MC-compound revealing less depositions of collagenous fibers. (D) Cardiac cells of animals treated with fentanyl plus ceftriaxone posting more less depositions. (Masson’s trichrome-400X). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

β-Lactams attenuated fentanyl-induced cardiac hypertrophy. Body weight (A), heart weight (B), and heart weight to body weight ratio (C). (D) mRNA levels of α-MHC. (E) mRNA levels of β-MHC. (F) mRNA levels of β-MHC: α-MHC ratio. Data are displayed as mean ± SD. *significant against control & ^# significant against Fen group, where *p < 0.05, ****p < 0.0001, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; α −MHC, alpha Myosin heavy chain; β-MHC, beta Myosin heavy chain.

Fig. 6

β-Lactams inhibited inflammation induced by Fentanyl in the heart. (A) & (B) mRNA levels of NF-κB and IL-6, respectively. (C) & (D) Representative western blot analysis of protein levels of NF-κB and IL-6, respectively. Data are presented as mean ± SD (n = 4). *significant against control & ^# significant against Fen group, where *p < 0.05, **p < 0.01, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; NF-κB, Nuclear factor kappa-B; IL-6, Interleukin-6; β-actin, Beta actin.

Fig. 7

β-Lactams reduce oxidative stress and apoptosis in the heart. (A) Relative gene expression of SOD were measured by RT-PCR. (B) & (C) Representative western blot analysis of protein levels of SOD and CAS-3, respectively. Data are presented as mean ± SD (n = 4). *significant against control & ^# significant against Fen group, where *p < 0.05, **p < 0.01, ^#p < 0.05, ^###p < 0.001. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; SOD, Superoxide dismutase; CAS-3, Cleaved Caspase-3; β-actin, Beta actin.

Fig. 8

Effect of β-lactams on oxidative stress level. Biochemical measurements of MDA (A), CAT (B), and GSH (C). Data are showed as mean ± SD (n = 4). *significant against control & ^# significant against Fen group, where *p < 0.05, **p < 0.01, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; MDA, Malondialdehyde; GSH, Glutathione.

Fig. 9

Effect of β-lactams on xCT and GLT-1 expression in the heart. (A) & (B) mRNA levels of xCT and GLT-1, respectively. (C) & (D) Representative western blot analysis of protein levels of xCT and GLT-1, respectively. (E) Biochemical analysis of glutamate content. Data are presented as mean ± SD (n = 4). *significant against control & ^# significant against Fen group, where *p < 0.05, **p < 0.01, ^#p < 0.05, ^##p < 0.01. Fen, Fentanyl; MC, MC-100093; Cef, Ceftriaxone; xCT, Cystine/glutamic acid reverse transporter; GLT-1, Glutamate transporter-1; β-actin, Beta actin.

Fig. 10

Schematic representation of cardioprotective