Uroprotective effect of a protein isolated from seed of Morinda citrifolia (McLTP1) on hemorrhagic cystitis induced by ifosfamide in mice

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. Morinda citrifolia Linn., popularly known as noni, is a species of Rubiaceae, where it is used from the root to the fruit for therapeutic purposes. From the seeds, a thermostable protein called McLTP1 (9.4 kDa) was extracted, among its therapeutic effects, it showed anti-inflammatory, gastroprotective, antibacterial and antinociceptive activity. Thus, the objective of this study is to evaluate the protective effect and the possible mechanism of action of a protein isolated from the seed of Morinda citrifolia (McLTP1) in hemorrhagic cystitis induced by ifosfamide in mice. Hemorrhagic cystitis was induced by intraperitoneal (i.p) administration of ifosfamide (IFO) in a single dose of 400mg/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. Treatment with McLTP1 was divided into two protocols, the first to define the best dose through a dose-response curve, where a pre-treatment was performed three days before cystitis induction, with McLTP1 administered at doses of 10, 20 or 40mg/kg (i.p), and two treatments 2 and 4 hours after IFO administration, evaluating its effect on bladder wet weight, edema and hemorrhage scores, and neutrophilic infiltrate. In the second protocol, only the best dose was used for the analysis of its effect on the hemorrhagic cystitis model. After 12 hours of hemorrhagic cystitis induction, the animals were euthanized by a high anesthetic dose. Subsequently, the bladders were removed, weighed and kept in 10% buffered formalin for histological, immunohistochemical (COX-2 and TNF-), immunofluorescence (NF-kB and F4-80) analyses, or stored at -80{degrees}C for of MPO, vascular permeability, hemoblobin, cytokines (TNF-, IL-1{beta}, IL-6, IL-10, IL-4, IL-33), enzymes (iNOS and COX-2) and markers of oxidative stress (MDA, NO, GSH, SOD and CAT). The adopted experimental procedures were approved by the Animal Research Ethics Committee through protocol number 23170920-0. Treatment with McLTP1 reduced bladder wet weight at the three respective doses mentioned above, however, it was observed the reduction of toxicity parameters (macroscopic edema and hemorrhage scores) only at the lowest dose (10 mg/kg), as well as MPO activity at doses of 10 and 20 mg/kg (p<0.05). results, the lowest dose was chosen for subsequent results. McLTP1 (10 mg/kg) was able to promote permeability reduction and vascular and hemoglobin in the bladder through quantification by the evans blue method and cyanmethemoglobin, respectively (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-, IL-1{beta}, 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 (p<0.05). McLTP1 also showed antioxidant activity, being able to reduce MDA and NO and increase levels of GSH, SOD and CAT (p<0.05). From the presented data, we can infer that McLTP1 is a potential uroprotector in the prevention of ifosfamide-induced hemorrhagic cystitis in mice by reducing inflammatory parameters and antioxidant activity.