Expression of HIF-1α, Caspase-3, p53, HSP70, HSP90, and NRF2 in the canine spleen with siderofibrotic nodules: an immunohistochemical and immunofluorescence perspective on apoptosis, ferroptosis, and oxidative stress

Authors: ALI BABANEZHAD-GAJOUTI, AMIR AMNIATTALAB

Abstract: Splenic siderofibrotic nodules (SFNs) are benign tiny yellow-brownish foci found in dogs. Their pathogenesis is unclear whereas trauma and hemorrhage are assumed to be the possible causative factors. The spleen of 73 Iranian dogs of the mixed breed was examined for possible splenomegaly with SFNs. Ultrasonography, blood biochemistry, pathology, and immunohistochemistry revealed splenomegaly (n = 7), splenomegaly with SFNs (n = 6) and normal spleens (n = 60). There was no significant difference between the prevalence of splenomegaly with SFN among the examined dogs and their age or sex (p > 0.05). Ultrasonography represented a significant increase (p ≤ 0.01) in spleen size, as well as serum Malondialdehyde (MDA) level, which increased significantly (p < 0.05) in dogs with splenomegaly compared to normal animals. Using Masson's trichrome and Pearl Prussian blue staining methods, SFNs' hallmarks of fibrosis (collagen deposition) and iron deposition were confirmed. Immunohistochemically, a significant increase of S100$^{+}$ dendritic cells, CD3$^{+}$ T cells, and CD68$^{+}$ macrophages, as well as a reduction of CD20$^{+}$ B cells representing chronic splenitis with splenomegaly and SFNs (n = 6), were revealed. Further, overexpression of vWF, HIF1-α, p53, caspase 3, and HSP70 and HSP90 were detected in the spleens with SFNs compared to normal animals. HSP70, HSP90, and p53 expressed at increased levels in spleens with SFNs mediate apoptosis and ferroptosis through interaction with factors like HIF1-α. Moreover, decreased immunofluorescence intensity for NRF2 expression in the SFN group represented the promotion of ferroptosis compared to normal spleens. Altogether, in canine splenomegaly with SFN, concurrent upregulation of p53, caspase-3 active, HSP70 and HSP90 as well as downregulation of NRF2, contribute to both apoptosis and ferroptosis, involving cellular injury through the interactive complicated network. Also, enhancement of MDA level and ferroptosis may reflect long-term exposure to ROS of dogs with splenomegaly and SFNs.

Keywords: Apoptosis, canine spleen, ferroptosis, siderofibrotic nodules

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