In this study, DOX-challenged rats showed cardiotoxicity as evidenced by increased serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) and decreased levels of antioxidants superoxide dismutase (SOD), catalase (CAT). ) and glutathione (GSH) simultaneously with increased lipid peroxidation. A significant increase in the levels of anti-inflammatory cytokines and inflammatory mediators, namely cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), was also observed in DOX-infected rats. In addition, we also investigated the CB2 receptor-dependent mechanism of cardioprotection elicited by BCP using AM630, a CB2 receptor antagonist, in a chronic DOX-cardiotoxicity model.
Doxorubicin clinical information, history and properties
DOX is most stable at a pH of 4, while it is quite unstable under very acidic or basic conditions. DOX is ineffective against brain and spinal cord tumors as it does not cross the blood-brain barrier (Bigotte, Arvidson, & Olsson, 1982). DOX is usually administered together with other chemotherapeutic drugs, and its use alone as a single agent has been reduced to avoid its main deleterious side effect (see below) (Singal, Iliskovic, Li, & Kumar, 1997).
Doxorubicin-induced cardiotoxicity and other challenges
- Dosing
- Risk factors
- Prognosis
- Diagnosis
- Histopathology of DCM
- Prevention and treatment
- Alternative strategies
Adult males have been reported to be more susceptible to dichloromethane than adult females, but the reverse appears to be true in prepubescent individuals (Jenkins et al., 2016). So far, there is no fully effective preventive measure or treatment for dichloromethane (Y. Wang et al., 2013). This technique has found some success in reducing the unwanted toxicity of DOX (Treat et al., 1990).
Mechanism of action of DOX-induced cardiotoxicity
- DNA intercalation and topoisomerase-II
- Oxidative stress
- Apoptosis
- Gene expression
- Inflammatory cytokines
Myocardial inflammation occurs primarily through activation of the innate immune system (Shi et al., 2011). ROS are also known to lead to inflammation through NF-kB activation (Tacar et al., 2013). To date, numerous anti-inflammatory agents have demonstrated their ability to partially alleviate DCM (Bruynzeel et al., 2007).
Agents of natural origin for combatting DCM
In mouse models of DOX-induced acute cardiotoxicity, a significant increase in pro-inflammatory cytokines, such as TNF-a and IL-6, was observed, along with a significant decrease in the anti-inflammatory cytokine IL- 10 (Pecoraro et al., 2016). Dexrazoxane was discovered in 1972 by Kurt Hellman and is used in combination with DOX to reduce cardiotoxicity (Shabalala et al., 2017). Recently, many reviews, including our own, have shown that plants and plant-derived phytochemicals are able to attenuate DOX-induced cardiotoxicity (Ojha et al., 2016).
Evidence of CB 2 receptor participation in cardiovascular
The anti-inflammatory effects of CB2 receptor activation on the endothelium and its inhibitory effect on monocytes/macrophages and/or leukocyte migration were reversed by pharmacological antagonism of CB2 receptors with AM630 (Hao Liu et al., 2015). Phytocannabinoids have been widely studied as potential protective agents in chronic diseases due to their multiple targets, time-tested efficacy and low cytotoxicity (Bento et al., 2011). These phytocannabinoid ligands from plants other than cannabis may provide a safe approach against DOX-induced chronic cardiotoxicity by regulating oxidative stress, immune inflammatory changes, autophagy and apoptosis (Choi et al., 2013; Béla Horváth et al., 2012 ).
Pharmacological mechanisms of BCP
The identification of BCP as a CB2 receptor agonist and nuclear receptor activator provides an incentive for evaluating BCP in various experimental disease models (Bento et al., 2011; Wu et al., 2014). Moreover, in vitro studies have shown that BCP significantly attenuated the formation of lipid peroxidation products, several orders of magnitude higher than established chain-breaking antioxidants such as α-tocopherol, probucol and α-humulene (Béla Horváth et al. , 2012). Furthermore, BCP showed efficacy in inhibiting the enzyme glutathione-S-transferase (GST) in an in vitro assay (Babu et al., 2012).
This occurs through down-regulation of transcription factors such as NF-kB and AP-1 leading to cardioprotective, chemopreventive and anticancer actions (Sain et al., 2014). Furthermore, recent studies also suggest that therapeutic targeting of CB2 receptors may provide a pharmacological mechanism for manipulating adverse immune responses, including those associated with a variety of neuropathies involving a hyper-inflammatory process (Leleu-Chavain et al., 2013). BCP acts by modulating cellular and molecular signaling pathways, altering gene expression and also interacting with biochemical and/or molecular targets (Sharma et al., 2016).
GABAergic, serotonergic and NMDA receptor-mediated activities are not triggered by BCP ( Sharma et al., 2016 ). All of these mechanisms and targets that BCP has an effect on make it a compound with great therapeutic potential against various diseases and conditions (see Sharma et al., 2016 and references therein). Spastic pain Exchange of Ca+2 (Câmara, Nascimento, .. Fonteles, 2003) Rheumatoid arthritis CB2 receptor activation (Gertsch et al., 2008).
Salvia jamensis Human leukemia cell lines (U937) (Fraternale et al., 2013) Aegle marmelos Lymphoma (Jurkat) and human. variabillima Human mouth, liver, lung, colon,.
Safety and toxicity of BCP
In multiple Salmonella strains, BCP showed no mutagenic effects at concentrations reaching up to 150 mg per plate. BCP, at concentrations up to 10 mg/plate, did not affect DNA synthesis in rat hepatocytes (Heck, Vollmuth, & Cifone, 1989).
Use of AM630 to demonstrate CB2 receptor
Aims and objectives of the study
Specific aim 2: To determine the effect of BCP in a chronic model of DOX-induced cardiotoxicity in rats and to elucidate the CB2 receptor-mediated mechanism of BCP.
Experimental animals
Experimental design
Animals in Group I received olive oil vehicle in an amount similar to the PKK treatment and served as a normal control. Animals in Group III received only BCP at a dose of 100 mg/kg per day for 5 days. BCP at a dose of 25 mg/kg was found to be more optimal in myocardial rescue, while having significant antioxidant and anti-inflammatory effects along with minimal weight loss and mortality.
In the chronic model, animals received DOX injections of 2.5 mg/kg weekly for a total of 5 weeks, reaching a cumulative dose of 12.5 mg/kg to induce cardiotoxicity. The treatment agent, BCP, was also injected into the animals six days a week for a total of five weeks to see if BCP was able to provide cardioprotection. Animals in group I received olive oil in a similar amount to BCP treatment and served as a normal control.
The animals in group II received DOX (2.5 mg/kg) alone once a week for five weeks. The animals in group III received BCP (25 mg/kg) alone six days a week for five weeks. The animals in group IV received DOX (2.5 mg/kg) once a week for five weeks and BCP (25 mg/kg) six days a week for five weeks.
Animals in group V received DOX (2.5 mg/kg) once a week for five weeks and BCP (25 mg/kg) six days a week for five weeks, and before BCP injections, animals in this group also received AM630 (1 mg/ kg), CB2.
Assessment of inflammatory markers in cardiac tissue
In both protocols (acute and chronic), blood samples were taken and centrifuged at room temperature for 10 minutes at 4000 revolutions per minute. The heart of each animal was collected and either immediately frozen in liquid nitrogen, followed by storage at −80°C, for biochemical analysis, or placed in 10% neutral buffered formalin and stored at 4°C for histological study. A manual homogenizer (TH tissue homogenizer, OMNI, USA) was used and the homogenate was centrifuged at 4°C for 30 minutes at 14,000 revolutions per minute.
The clear supernatant was collected and used to quantify inflammatory markers via the respective ELISA assay kits for TNF-a, IL-6 and IL1b (R&D Systems, Minneapolis, MN, USA). Assays were performed as instructed by the manuals provided in the respective kits.
Assessment of oxidative/nitrative stress the myocardium
Histopathological evaluation
Transmission electron microscopy (TEM)
Western blot analysis
The samples were then incubated with their corresponding secondary antibodies for 1 h at room temperature and the proteins were visualized using an enhanced chemiluminescence pico kit (Thermo Fisher Scientific, Rockford, IL, USA).
Serological evaluation
Statistical analysis
Effect of BCP on body weight of DOX-treated rats
Effect of BCP on cardiomyocyte injury marker
Effect of BCP on endogenous antioxidant enzymes
Effect of BCP on non-enzymatic antioxidant defense
Effect of BCP treatment on DOX-induced myocardial
Effect of BCP on myocardial structure
Effect of BCP on iNOS and COX-2 expression
Effect of BCP on NF-κB expression in heart
Effect of BCP on cardiomyocyte ultrastructure
Effect of BCP on body weight of DOX-treated rats
Effect of BCP on cardiomyocyte injury marker enzymes
Effect of BCP on endogenous antioxidant enzymes in
Effect of BCP on non-enzymatic antioxidant defense and
Effect of BCP treatment on DOX-induced myocardial
Effect of BCP on myocardial structure
The occurrence of fatal cardiotoxicity in all age groups is characterized by an irreversible cardiomyopathy that impairs the clinical utility of DOX and is responsible for the main cause of the DOX-related morbidity and mortality (Simůnek et al., 2009). CB2 receptor activation is also associated with nuclear translocation of the transcription factor NF-κB (Derocq et al., 2000). Recently, several of the phytocannabinoids derived from plants other than cannabis have gained interest for their favorable physicochemical, pharmacokinetic and pharmacological properties, including anti-inflammatory and antioxidant effects and.
Drug distribution in the central nervous system of normal adult mice after intravenous injection. Antispasmodic effect of Plectranthus barbatus essential oil and some main components on guinea pig ileum. Genomic and functional changes induced by activation of the peripheral cannabinoid receptor CB2 in promyelocytic HL-60 cells.
Composition and evaluation of the anti-inflammatory and anticancer activities of the essential oil of Annona sylvatica A. A critical evaluation of the proposed mechanisms of action for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Cloning and Characterization of the dnrU Ketoreductase and dnrV Genes and the doxA Cytochrome P-450 Hydroxylase Gene.
Mechanism of action of the anthracycline antitumor antibiotics doxorubicin, daunomycin, and rubidazone: Preferential inhibition of DNA polymerase α. Composition and in vitro anticancer activities of the essential leaf oil of Neolitsea variabilima from Taiwan. Cytotoxic activity and constituents of the volatile oil from the roots of Patrinia scabra Bunge.
Effect of BCP on iNOS and COX expression in heart tissue
Effect of BCP on NF-κB expression in heart tissue
DOX administration caused a remarkable increase in NF-κB expression while BCP treatment significantly decreased NF-κB expression.
Effect of BCP on CB 2 receptor expression in heart tissue
Among the many new therapeutic targets, the endogenous cannabinoid system, comprising cannabinoid ligands and cannabinoid receptors type 1 (CB1) and type 2 (CB2), is one of the newest and most promising drug targets. Among cannabinoid receptors, CB2 receptors are G-protein-coupled receptors that, upon activation, induce members of the MAPK family that trigger gene expression, including those involved in stress response, inflammation, cell survival, or proliferation (Howlett, 2005). The anti-inflammatory effects of endothelial CB2 receptor activation and its inhibitory effect on monocyte/macrophage and/or leukocyte migration were reduced by pharmacological CB2 antagonism.
However, there are no available studies evaluating the effect of CB2 receptor activation by cannabinoid ligands on DOX-induced cardiotoxicity. In recent years, a large number of experimental studies have shown that phytochemicals exert cardioprotective effects against DOX-induced cardiotoxicity due to their multiple pharmacological properties, including anti-inflammatory, antioxidant and antiapoptotic effects. It is found abundantly in various flowering plants and spices, making it one of the most readily available and accessible agents without psychoactive properties (Gertsch, 2008).
Calpain-mediated loss of dystrophin in the cardiomyocyte membrane and disruption of sarcomeric actin/myosin may contribute significantly to myocardial structural damage in DCM (Campos et al., 2011). Translation of the results to humans would be promising, as these agents have not only shown the ability to activate only CB2 receptors, but also activate PPAR-γ, a target of the thiazolidinedione class of drugs used clinically to treat diabetes. BCP showed a positive effect by reducing body weight loss and reducing oxidative stress, inflammation, and inflammatory mediators, along with decreasing serum levels of the cardiomyocyte injury marker enzymes CK-MB and LDH.
Structural basis for endotoxin-induced allosteric regulation of the innate immune receptor Toll-like receptor 4 (TLR4). Baicalein attenuates doxorubicin-induced cardiotoxicity by inhibiting myocardial oxidative stress and apoptosis in mice. Efficacy and safety of metoprolol in the treatment of doxorubicin-induced cardiomyopathy in pediatric patients.
