Biochemical Insights into the Effects of a Small Molecule Drug Candidate on Imatinib-Induced Cardiac Inflammation

Abstract

BGP-15, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor exerts cardioprotective effects; however, the underlying mechanisms remain unclear. Therefore, our study aimed to investigate the effects of BGP-15 on the imatinib (Imtb)-induced cardiac inflammation at the biochemical level. Male rats were divided to control, Imtb-treated (60 mg/kg/day for 14 days), and Imtb + BGP-15-treated animals. In this group Imtb was co-administered with BGP-15 at the dose of 10 mg/kg/day. At the end of the experiment, nuclear factor-kappa B/p65 (NF-κB/p65), nuclear transcription factor erythroid-2 related factor (Nrf2), heme oxygenase-1 (HO-1), high mobility group box 1 (HMGB1), and myeloperoxidase (MPO) were measured by Western blot. Chemokine and interleukins (ILs) were determined by Legendplex. Additionally, cardiac specific changes were visualized by immunohistochemistry. We demonstrated that Imtb increased NF-κB/p65, IL-6, IL-1β, IL-18, MCP-1, HMGB1, as well as the expression and activity of MPO. Conversely, the expressions of antioxidant Nrf2 and HO-1 were decreased. Administration of BGP-15 effectively mitigated these inflammatory alterations by significantly reducing pro-inflammatory cytokines and MPO activity, while simultaneously restoring and enhancing the levels of Nrf2 and HO-1, thereby promoting antioxidant defenses. The immunohistochemical staining further supported these biochemical changes. Our study provides new and comprehensive biochemical insight for managing Imtb-induced inflammatory responses via BGP-15-induced PARP1 inhibition.

Keywords: BGP-15; PARP-1; imatinib; inflammation.

Figure 1

Effect of imatinib and BGP-15-induced changes on the concentration of PARP1 in the heart. Data are represented as the means ± SEMs. n = 7–8. * p < 0.05: statistical significance between CTRL and Imtb groups; # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. PARP1: Poly(ADP-ribose) polymerase-1, CTRL: control, Imtb: imatinib.

Figure 2

Effect of imatinib and BGP-15-induced changes on the expressions of (a): NF-κB/p65 and (b): Nrf2 in the heart. (c): Representative fluorescent micrographs illustrate NFκB/p65 (red) and Nrf2 (green) staining in rat ventricular myocardium. Nuclei were counterstained with DAPI (blue). Scale bars indicate 100 µm. Data are represented as the means ± SEMs. n = 6–7 for NF-κB/p65 and n = 6–7 for Nrf2. * p < 0.05: statistical significance between CTRL and Imtb groups; # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. NF-κB/p65: nuclear factor κB/p65 domain, Nrf2: nuclear transcription factor erythroid-2 related factor, CTRL: control, Imtb: imatinib.

Figure 3

Effect of imatinib and BGP-15-induced changes on the expression of HO-1 in the heart (a). (b): Representative fluorescent micrographs illustrate HO-1 (red) staining in rat ventricular myocardium. Nuclei were counterstained with DAPI (blue). Scale bars indicate 100 µm. Data are represented as the means ± SEMs. n = 6–7. # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. HO-1: heme oxygenase-1, CTRL: control, Imtb: imatinib.

Figure 4

Effect of imatinib and BGP-15-induced changes on the concentrations of pro-inflammatory cytokines in the heart. (a): IL-6 concentration, (b): representative fluorescent micrographs illustrate IL-6 (red) staining in rat ventricular myocardium. Nuclei were counterstained with DAPI (blue). Scale bars indicate 50 µm. (c): IL-1β concentration, (d): representative fluorescent micrographs illustrate IL-1β (red) staining in rat ventricular myocardium. Nuclei were counterstained with DAPI (blue). Scale bars indicate 100 µm. (e): IL-18 concentration, (f): MCP-1 concentration. Data are represented as the means ± SEMs. n = 5–6. # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. IL-6: interleukin-6, IL-1β: interleukin-1beta, IL-18: interleukin-18, MCP-1: monocyte chemoattractant protein-1, CTRL: control, Imtb: imatinib.

Figure 5

Effect of imatinib and BGP-15-induced changes on the expression of HMGB1 in the heart. Data are represented as the means ± SEMs. n = 5–8. * p < 0.05: statistical significance between CTRL and Imtb groups; * p < 0.05: statistical significance between CTRL and Imtb groups; # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. HMGB1: high mobility group box 1, CTRL: control, Imtb: imatinib.

Figure 6

Effect of imatinib and BGP-15-induced changes on (a): the expression and (b): activity of MPO enzyme in the heart. Data are represented as the means ± SEMs. n = 5–8 for MPO expression and n = 6–8 for MPO activity. * p < 0.05: statistical significance between CTRL and Imtb groups; * p < 0.05: statistical significance between CTRL and Imtb groups; # p < 0.05: statistical significance between Imtb and Imtb + BGP-15 groups. MPO: myeloperoxidase, CTRL: control, Imtb: imatinib.

Figure 7

PARP1 plays a major role in imatinib-mediated inflammatory processes and also serves as a key target of BGP-15. Imatinib-induced mitochondrial damage results in ROS accumulation, which induces PARP1 activation. PARP1 augments NF-κB-mediated gene expression by its nuclear coactivator function; thus, pro-inflammatory genes, such as IL-6, IL-18, MCP-1, and IL-1β are upregulated. IL-1β and IL-18 and play a role in immune response by recruiting and activating neutrophils. Thus, neutrophil infiltration and inflammation can be associated with increased MPO level. There is a strongly connected interplay between NF-κB and Nrf2. As a consequence of NF-κB activation, Nrf2-dependent HO-1 gene transcription is down-regulated, which decreases the antioxidant defense mechanisms. Furthermore, PARP1 catalyzes PARylation of HMGB1, which facilitates its dissociation from chromatin and translocation to cytosol. Cytoplasmic HMGB1 may bind to receptors, such as TLR, and further augment the downstream NF-κB signaling. In contrast to the imatinib-induced effects, BGP-15, a small molecule drug candidate, is able to reduce inflammatory responses through the inhibition of PARP1. NF-κB: nuclear factor-κappa B, Nrf2: nuclear transcription factor erythroid-2 related factor, HMGB1: high mobility group box 1, IL-6: interleukin-6, IL-1β: interleukin-1beta, IL-18: interleukin-18, MCP-1: monocyte chemoattractant protein-1, HO: heme oxygenase, MPO: myeloperoxidase, ARE: antioxidant response element.