Efeito uroprotetor de uma proteína isolada das sementes de noni (Morinda citrifolia L.) na cistite hemorrágica induzida por ifosfamida em camundongos

Abstract

Hemorrhagic cystitis is a side effect of chemotherapy induced by an antineoplastic agent from the oxazaphosphorine group (ifosfamide and cyclophosphamide), resulting from the formation of the urotoxic metabolite acrolein. Thus, the search for vegetable proteins can be a therapeutic alternative. Morinda citrifolia Linn., popularly known as noni, is a species of the Rubiaceae family, this species is used from the root to the fruit for therapeutic purposes. From the seeds, a thermostable protein called McLTP1 (9.4 kDa) was purified, among its therapeutic effects, it showed anti-inflammatory, gastroprotective, antibacterial and antinociceptive activity. Thus, the aim of this study was to evaluate the uroprotective effect of McLTP1 on hemorrhagic cystitis induced by ifosfamide in mice, investigating the mechanisms of action involved. Hemorrhagic cystitis was induced by intraperitoneal (i.p) administration of ifosfamide (IFO) in a single dose of 400 mg/kg, according to a standardized protocol, in male Balb/c mice. The experimental group treated with the uroprotective drug, Mesna (80 mg/kg; i.p), received a pretreatment 30 minutes before, 4 and 8 hours after IFO. A pre-treatment was carried out three days before the induction of cystitis with McLTP1 administered at doses of 10, 20 and 40 mg/kg (i.p), and two treatments 4 and 8 hours after IFO administration. After 12 hours of hemorrhagic cystitis induction, the animals were euthanized by a high anesthetic dose. Subsequently, the bladders were removed, weighed, analyzed macroscopically (edema and hemorrhage), then kept in 10% buffered formalin for histological, immunohistochemical (COX-2 and TNF-α), immunofluorescence (NF-kB and F4-80) or stored at -80ºC for measurement of MPO, hemoglobin, cytokines (TNF-α, IL-1β, IL-6, IL-10, IL-4, IL-33), enzymes (iNOS and COX-2) and oxidative stress markers (MDA, NO, GSH, SOD and CAT). The adopted experimental procedures were approved by the Committee on Ethics in the Use of Animals (CEUA/NPDM) through protocol number 23170920-0. Treatment with McLTP1 significantly reduced bladder wet weight at the three respective doses mentioned above, however, a reduction in toxicity parameters (macroscopic scores of edema and hemorrhage) was observed only at the lowest dose (10 mg/kg), as well as the activity of MPO at doses of 10 and 20 mg/kg (p<0.05). Given these results, the lowest dose was chosen for subsequent experiments. McLTP1 (10 mg/kg) was able to reduce vascular permeability and hemoglobin in the bladder (p<0.05). In addition, it had a protective effect by attenuating inflammatory scores and preserving the structure of the urothelium. The anti-inflammatory activity was demonstrated through the significant decrease of the cytokines TNF-α, IL-1β, IL-6 and increase of IL-10; reduced expression of COX-2, NF-kB and F4/80, and gene expression of IL-33, IL-4 and iNOS. McLTP1 also showed antioxidant activity, being able to reduce MDA and NO and increase levels of GSH, SOD and CAT. From the presented data, we can infer that McLTP1 is a uroprotector in the prevention of ifosfamide-induced hemorrhagic cystitis in mice by reducing inflammatory parameters and antioxidant activity.