A crude methanolic extract from the parotoid gland secretion of Rhaebo guttatus stimulates the production of reactive species and pro‑inflammatory cytokines by peritoneal macrophages

The parotoid gland secretion of toads of the family Bufonidae contain numerous pharmacologically active biocompounds. Rhaebo guttatus (R. guttatus) is a common species of this family in Brazil; however, this species has been poorly studied. Thus, the present study aimed to assess the immunomodulatory activity of a methanol extract from the parotoid gland secretion of R. guttatus in Swiss mice. Non‑isogenic Swiss mice were administered (100 µl/mouse/day, orally) of the R. guttatus extract (8, 16, or 32 µg/ml), the vehicle (0.5% Tween‑20, vehicle group), or distilled water (control group) for 7 or 30 days. After the treatment period, the mice were sacrificed, and the hearts, liver, lungs and kidneys were removed for histopathological analysis. Cells from peritoneal lavage and spleen were used for immunological analyses. The results revealed that the treatment did not affect water or feed consumption or body weight of the mice. The lungs of mice treated with 8 µg/ml for 30 days exhibited a reduction in relative weight compared to those of the control group. Histopathological analysis revealed that the extract induced edema, blood clots and leukocyte infiltrates, mainly after 30 days at doses of 8 and 32 µg/ml. The administration of the extract promoted the production of the pro‑inflammatory cytokines, IL‑12 and TNF‑α, and reduced the production of IL‑10 and IL‑4. The spontaneous release of hydrogen peroxide by peritoneal macrophages was also induced in the mice treated with 16 µg/ml extract for 30 days. On the whole, the present study demonstrates that a crude methanolic extract from the parotoid gland secretion of R. guttatus has immunomodulatory activity and low toxicity, and may have potential as a stimulator of the immune system for immunosuppression conditions.