RESUMEN
Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.
Asunto(s)
Antivenenos/farmacología , Bothrops , Venenos de Crotálidos/antagonistas & inhibidores , Fitoterapia , Extractos Vegetales/farmacología , Tabernaemontana , Animales , Antivenenos/administración & dosificación , Antivenenos/uso terapéutico , Creatina Quinasa/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/toxicidad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Raíces de Plantas , Ratas , Ratas WistarRESUMEN
Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.