Caracterização bioquímica e mecanismo de ação do efeito protetor in vivo de proteínas do látex de Calotropis procera (Ait.) R. Br. sobre infecção letal induzida por Salmonella enterica subespécie enterica sorotipo Typhimurium

Abstract

Latex of the medicinal plant Calotropis procera (Apocynaceae) possesses molecules displaying different pharmacological activities, including pro- and anti-inflammatory. In this study immune inflammatory modulation of the soluble protein fraction (LP) recovered from the latex was investigated in experimental models of sepsis induced by CLP and inoculation of the S. Typhimurium. Biochemical aspects of laticifers proteins were investigated and biochemical, pharmacological and histopathological parameters were evaluated in experimental animals. LP protected animals and a unique dose (30 mg/Kg; i.p.) lead to 100% survival while non-treated infected animals (Salmonella group) reached 100% mortality within 24 h. Protection was only observed when LP was given 24 earlier of infection. LP did not exhibit in vitro bactericide activity suggesting indirect mechanism of action, probably by immune modulation. After 4 and 24 h of infection bacteria was similarly disseminated in liver, spleen and peritoneal fluid, local of bacteria inoculum. However, in blood viable bacteria was reduced only in animals given laticifers proteins. The protective effect of LP was also observed in its three sub-fraction, denominated of PI, PII and PIII, obtained after protein fractionation by ion exchange chromatography on a CM-Sepharose performed at pH 5.0. Neither, heat-treatment (100 °C, 30 min) nor inhibition of its endogenous proteolytic enzymes, by iodoacetamide, eliminated the protective effect of LP. Nitric oxide (NO) an important signaling molecule was augmented in serum of non-treated animals whereas it was unaltered in control and LP/PI-treated animals 24 h after infection. Increasing of NO in serum is known to directly contribute to inhibit neutrophil migration in septic animals. Accordingly, failure of neutrophil migration to the infectious focus in septic animals was confirmed. Conversely, in animals given LP and PI, influx of neutrophils was evident. In addition, NO was also elevated in the infectious focus of LP/PI treated animals, probably due neutrophil activity as part of their microbicidal activity. Activity of adenosine deaminase (ADA) was measured locally after 4 and 24 h of infection was initiated. ADA was augmented in LP/PI treated animals and unaltered in non treated animals. Thrombocytopenia was observed in non treated animals but not in LP/PI protected animals. Neutrophilia and lymphopenia were documented in non-treated animals. Neutrophilia would result of high NO content in serum, which inhibits neutrophil migration and lymphopenia is part of immune suppression caused by sepsis. Neutrophilia was observed 7 days after infection in animals given LP/PI, suggesting a hematopoiesis stimulus. Lymphopenia first observed (24 h) in septic animals was only detected after 7 days in PL/PI treated animals. Activity of oxalacetic-glutamic transaminase was altered in all groups while glutamic-pyruvic transaminase not. Lactate dehydrogenase was augmented in all groups. Histopathological examination of liver and spleen revealed tissue damage caused by sepsis, but these alterations appeared later (7 days) in PL/PI-treated animals. Results reported in this study suggest that PL modulates immune inflammatory activity in septic animals by an indirect mechanism that remains to be investigated. Activity of LP leads to animal survival, even under infection. It is proposed that LP modulates NO synthesis, reducing NO levels in serum of infected animals and abolishing the failure of neutrophil migration. The pharmacological properties of LP previously described in classical models of inflammation, is now confirmed in a model of systemic inflammatory response elicited by microbial infection.