Estudo dos efeitos dos venenos de artrópodes (Phoneutria nigriventer e Tityus serrulatus) e suas frações sobre a regulação térmica em ratos

Abstract

Body temperature is controlled mainly by the hypothalamus ensuring the proper functioning of metabolic pathways. Accidents with Phoneutria nigriventer and Tityus serrulatus can cause various symptoms: hypothermia and/or hyperthermia, sweating and tremor that may be related to thermoregulation. This work aims to analyze and identify components of venoms acting on thermoregulation and to assess possible mechanisms of action. The venoms were separated into pools by gel filtration and gel electrophoresis to confirm the effectiveness of the separation. The venoms were tested in temperature bioassay in rats. A thermal sensor was implanted subcutaneously in the neck for recording temperature. The venom of Phoneutria nigriventer showed and hypothermic action in rats and Tityus serrulatus showed hyperthermic action. Pools of venoms were also tested; the pool II of venom both had hypothermic action and pool III, hyperthermic action. Subsequent steps were performed exclusively with the venom of Phoneutria nigriventer. The pools II and III were separated by HPLC on peaks. The peaks 8 and 13 of pool II showed hyperthermic action, as well as the peaks 30 and 31 of pool III. The peak 29 of pool III showed hypothermic action. The peaks 29, 30 and 31 were repurified and retested, but without significant results. To study the mechanisms of action, the peak 13 of pool II was tested in animals pretreated with Dantrolene, a drug used against malignant hyperthermia. The results showed that dantrolene did not decrease the temperature of healthy animals, as it acts on calcium channels that can be only modified in patients with the disease. The LPS, an exogenous pyrogen, would be tested with the peak of this hypothermic action, however the peak 29 showed no effect after repurification. The peak 13 was also tested in human glioblastoma U87, revealed not to be cytotoxic. The peaks 8 and 13, were performed by means of mass spectrometry revealing similar masses family of Tachykinins and Ctenitoxins, both actions on the nervous system. The peaks 29, 30 and 31 were tested in cultured hypothalamus neurobasal cells, all the peaks showed a statistically significant decrease in cell viability at lower concentrations after for 24 hours of incubation. At 6 hours of incubation, the statistics showed that the peaks 30 and 31 increased the viability of cells in higher concentrations. The repurified peak 30b was sequenced by Edman degradation and part of the peptide revealed 100% homology with Tx2-6 of Phoneutria nigriventer, which is an NAV inhibitor irreversible neurotoxin and cause sweating. Rats are resistant to change its temperature, thus variations in body temperature ranging only 1 degree could be considered quite significant. Molecules isolated from the venom may act for summation or synergistically and other peptides present in the venom can act in opposite effect. Study of animal toxins are a source of new biomolecules and drug development. Toxins that act on thermoregulation are little studied and need further characterized