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Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium
Journal of Ethnopharmacology 129 (2010) 327–334
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Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium José V. Lima-Filho a,∗ , Joyce M. Patriota a , Ayrles F.B. Silva a , Nicodemos T. Filho b , Raquel S.B. Oliveira c , Nylane M.N. Alencar d , Márcio V. Ramos c,∗∗ a Laboratório de Microbiologia e Imunologia, Departamento de Biologia, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, Recife CEP 52171-900, PE, Brazil b Laboratório de Imunoparasitologia Keizo Azami (Lab. Anatomopatologia), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235 - Cidade Universitária, Recife CEP 50670-901, PE, Brazil c Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza CEP 60451-970, CE, Brazil d Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Brazil
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Article history: Received 30 November 2009 Received in revised form 11 February 2010 Accepted 27 March 2010 Available online 3 April 2010 Keywords: Bacteria Cytokines Infection Inflammation Latex SEPSIS
a b s t r a c t Aim of the study: The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. Materials and methods: Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 106 CFU/ml of Salmonella Typhimurium using the same route of administration. Results: All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 104 cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-␣ in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1. Conclusions: LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-␣). LP inhibited IL-1 release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The shrub Calotropis procera (R. Br.) is a plant of the Asclepiadaceae family, and has been used extensively in folk medicine
∗ Corresponding author. Tel.: +55 31 81 33206312. ∗∗ Corresponding author. Tel.: +55 85 33669789. E-mail addresses: [email protected] (J.V. Lima-Filho), [email protected] (M.V. Ramos). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.03.038
of East countries. Hepato-protection, treatment of rheumatism and aches, local anesthesia and treatment of skin disease infections are among the scientifically reported activities (Mossa et al., 1991; Pathak and Argal, 2007; Qureshi et al., 2007). The plant is widely known because it releases important amounts of latex from its green parts when it is injured. This latex, as well as organic and aqueous latex extracts, has been investigated through in vitro and in vivo experimental models to establish pharmacological and toxicological aspects. Thus, different authors have claimed a set of properties for the latex of Calotropis procera
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including healing, anti-cancer, antipyretic, analgesic together with inflammatory and anti-inflammatory properties, among many others (Rasik et al., 1999; Larhsini et al., 2002; Alencar et al., 2006; Choedon et al., 2006). On the other hand, disadvantageous toxicity, hyperalgesia and lethal effects have also been observed in animals treated with the whole latex (Biedner et al., 1977; El Badwi et al., 1998; Kumar and Sehgal, 2007). The effects of the latex of Calotropis procera on immune responses have been particularly investigated. The latex-methanol extract reduced the inflammatory stimulus induced by Freud’s adjuvant on the experimental arthritis model (Kumar and Roy, 2007). Furthermore, the antiinflammatory capacity appears to be similar to Fenilbutazone (PBZ) in acute and chronic models of inflammation (Sangraula et al., 2002; Arya and Kumar, 2005). However, dialysis and centrifugation of the latex provide a set of proteins that shows different effects on cell-mediated immunity. Administrating the dialyzable fraction (DL) to rats induces an inflammatory response whereas the non-dialyzable fraction (LP) suppresses this effect when assayed at the same doses (Alencar et al., 2006). The inflammatory stimuli carried by DL fraction also appear to be dependent on histamine and prostraglandin, which trigger neutrophil recruitment and stimulate cellular vascularization (Shivkar and Kumar, 2003; Kumar and Shivkar, 2004). Moreover, histamine has been detected in the latex (Shivkar and Kumar, 2003). It has been reported that LP fraction reduces neutrophil migration induced by carrageenin on the paw edema model (Alencar et al., 2004, 2006). Gram-negative bacterial infections can induce sepsis as a seriously pathological consequence of the spread of bacteria in the bloodstream. This condition is accompanied by a systemic host inflammatory response characterized by the release of inflammatory plus pro-inflammatory cytokines due to stimulus of the lipopolyssacharide (LPS) present in the bacterial cell wall. Since a balanced inflammation is necessary to control infection and to keep the host from succumbing to septic shock, the management of sepsis has been done through the use of anti-inflammatory agents or substances that enhance immune cells for bacterial clearance (Freeman and Natanson, 2000). As an alternative, natural products reported to have influence on the immune system have also been investigated, such as sugarcane extracts (Motobu et al., 2006), plant flavonoids (Van Dien et al., 2001), synthetic peptides derived from the beetle Allomyrina dichotoma defensin (Koyama et al., 2006) and glutamine (Wang et al., 2008). Despite the amount of information available concerning the immune-modulatory properties of the latex of Calotropis procera, there have not been any reports of such effects on a systemic infection. The murine model of infection by Salmonella enterica serovar Typhimurium produces the symptoms of typhoid fever and represents a reliable model to evaluate the immune response because it induces a rapid systemic disease after small inoculums. It is easily detected and quantified in several organs such as the spleen and liver, where the bacteria survive inside nonactivated macrophages. Damage to organs can also be assessed through histopathological examination and it can be studied without causing septic shock (Jones, 1996; Groisman and Ochman, 1997; Portillo, 2001). In view of the relevant anti-inflammatory activity previously found in LP and the continuing search for new products capable of fighting the lethal effects produced on the course of systemic infections, the aim of the present study was to evaluate the possible benefits of LP in controlling of the inflammatory immune response induced by Salmonella enterica serovar Typhimurium by using the murine model that represents a more realistic model of inflammation instead of using carrageen of others inflammogens.
2. Materials and methods 2.1. Latex and laticifer proteins Samples of the latex of Calotropis procera were collected in Fortaleza in the state of Ceará, Brazil. The voucher (sample specimen No. 32663) was deposited at Prisco Bezerra Herbarium of the Universidade Federal do Ceará, Brazil and the plant was authenticated by a taxonomist from the Department of Biology. Fresh latex of Calotropis procera was collected from healthy plants by small incisions near the youngest leaves and left to flow off into distilled water in order to obtain a mixture 1:1 (v/v). Samples were centrifuged (5000 × g) at 10 ◦ C in a refrigerated bench top centrifuge for 10 min. The precipitated material, showing rubber aspect, was discarded while the suspended phase was submitted to exhaustive dialysis (cut off 8000 Da) against distilled water at 8 ◦ C for 60 h, and then centrifuged as previously mentioned. The new precipitate material was eliminated and the soluble phase devoid of rubber was freeze-dried. This fraction comprising almost all of the soluble proteins of the latex was named laticifer proteins (LP). The biochemical characterization of this material was described previously in different studies (Alencar et al., 2004, 2006; Oliveira et al., 2007). All further experiments were performed using the lyophilized material in appropriated solutions.
2.2. Microorganism Salmonella enterica Subsp. enterica serovar Typhimurium was isolated from a human clinical case at Fundac¸ão Ezequiel Dias (FUNED, Belo Horizonte, MG, Brazil) and was a kind gift from Dr. Jacques Robert Nicoli (Universidade Federal de Minas Gerais). The bacteria were maintained at −18 ◦ C in Brain Heart Infusion (BHI) medium containing 50% glycerol. During the experiments, bacteria were activated after culture in BHI broth for 24 h at 37 ◦ C.
2.3. Animals Adult male Swiss mice weighing approximately 30 g were used in this study. A minimum number of animals (n = 10 per group) was used in all experiments in order to adhere to the modern rules for the use of animals in experimentation. The animals were kept in an animal house with controlled lighting (12-h light–dark cycles), temperature (25 ◦ C) and humidity (60–70%) with free access to water and commercial sterile diet (Purina, Paulínia, SP, Brazil). The mice were handled according to established experimental procedures, which were performed according to the standards rules in the “Guide for the Care and Use of Laboratory Animals” of the National Research Council, following approve of Institutional Animal Ethics Committee.
2.4. In vitro antibacterial activity of LP Possible antibacterial activity of LP against Salmonella Typhimurium was evaluated by using the broth dilution method (Koneman et al., 2001). LP fraction was suspended in PBS and added to tubes with Mueller Hinton broth containing Salmonella Typhimurium at 105 Colony Forming Units (CFU)/ml. The tested LP concentrations ranged from 3.9 g/ml to 2 mg/ml. All assays were performed in duplicate and control tubes were free of LP. The minimum inhibitory concentration was estimated as the lowest concentration that caused visible growth inhibition after an incubation period of 24 h at 37 ◦ C.
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2.5. Experimental design Animals were given LP by intraperitoneal (i.p.), subcutaneous (s.c.) or orally (p.o.). For the complementary determinations, intraperitoneal injection was used. A single dose of LP fraction at 30 or 60 mg/kg body weight, in 0.2 ml Phosphate Buffered Saline – sterile, pH 7.2 (PBS) was given to each mouse from the two experimental groups (n = 10), 24 h before or after challenge with Salmonella Typhimurium. The control group was inoculated with 0.2 ml PBS according to the same schedule for the experimental one. Salmonella Typhimurium was grown in BHI broth at 37 ◦ C and serially diluted to attain a bacterial suspension containing 107 CFU/ml. Animals from both the experimental and control groups were challenged with 0.2 ml of this bacterial suspension (106 CFU/ml) by the intraperitoneal route to provoke a systemic infection. To investigate the protective effect of LP given by different routes of administration, bacterial inoculum was 107 CFU/ml in 0.2 ml instead of 106 CFU/ml. Experiments were conducted for 7 or 28 days. 2.6. Evaluation of clinical signs and survival
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was maintained at 37 ◦ C (5% CO2 ). After 24 h, the cells were incubated in fresh medium or in medium containing LP (500 g/ml) for 1 h. The supernatants were then discarded and after three further washes, the cells were incubated with medium (1.5 ml) or LPS (1 g/ml) for 5 h. The supernatants were used for the cytokine assay. Cell viability of each macrophage culture was analyzed by the Trypan blue exclusion method. 2.11. Cytokine determination The amount of Interleukin-12 (IL-12) and Tumor Necrosis Factor-␣ (TNF-␣) was estimated in serum of animals sacrificed on days 1 and 5 after bacteria inoculums. TNF-␣ and Interleukin-1-betha (IL-1) were quantified on supernatant of the macrophages. Assays were performed by using commercial kits of ELISA immunoassay (R&D Systems). Color was developed with ophenylenediamine (1,2-benzenediamine; Sigma) and absorbance at 450 nm was determined by an ELISA plate reader (ELISA READER, BIORAD).
Animals that were submitted to a long-term assay (28 days) from initial inoculation with Salmonella Typhimurium were evaluated for survival rate. Clinical signs such as prostration, weight loss and diarrhea were also observed daily throughout the experiment. Histopathological examination, counting of bacteria in spleen and liver and cytokine titration in serum were performed on day 5. The survival animals were sacrificed on days 15 and 28 to evaluate clearance of bacteria in the spleen and liver. 2.7. Analysis of early infection In additional experiments, mice were treated with LP fraction (i.p.) and infected by Salmonella 24 h later as described above. The animals from the control and LP groups were sacrificed 4 and 24 after infection and bacteria enumeration was performed in blood, peritoneal fluid, spleen and liver. An aliquot of blood was taken for cytokine titration in serum of animals (24 h). 2.8. Histopathological examination Samples of tissues from the liver and spleen from mice sacrificed on day 5 post-infection were fixed in 10% formaldehyde in PBS and processed to embed them in paraffin. The histopathological sections (5 m) were stained with hematoxylin-eosin. The slides were coded and examined by a single pathologist, who was unaware of the experimental conditions of all the groups. 2.9. Salmonella enumeration The liver and spleen were dissected, weighed and macerated in PBS (1:10 or 1:100, w/v). Samples of blood and peritoneal fluid were used as collected. Serial decimal dilutions were made and 0.1 ml aliquots were plated onto MacConkey agar (Oxoide). The colonies were counted after incubation at 37 ◦ C for 24 h and the results expressed as CFU/g of organs (spleen and liver) and CFU/ml (blood and peritoneal fluid). 2.10. Analysis in macrophage-culture cells activated by LP The method described by Cunha and Ferreira (1986) was followed. Peritoneal macrophages of mice were harvested with RPMI (pH 7.4) 4 days after i.p. injection of 3% thioglycolate (3 ml/cavity) and cultured in plastic tissue culture dishes (24 wells) at 37 ◦ C (5% CO2 ). Non-adherent cells were removed by three washes with 1 ml of RPMI medium and the adherent population (95% macrophages)
Fig. 1. (A) Protective effect of LP on mice after lethal challenge with Salmonella enterica Subsp. enterica serovar Typhimurium. Animals were treated intraperitoneally with LP (30 or 60 mg/kg) 24 h before bacteria inoculums (0.2 ml; 106 CFU/ml). Control animals were given PBS. (B) Protective effect of LP (60 mg/kg) given by different routes 24 h before bacteria inoculums (0.2 ml; 107 CFU/ml). Results were statistically assessed by Mantel–Cox log rank test (*p < 0.05) and are representative of three independent experiments (n = 10). All experimental statistically differed from controls.
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2.12. Statistical analyses The data, excepting survival curves, are reported as mean ± S.E.M. Statistical significance was assessed by analyzing the variance (ANOVA) followed by Bonferroni’s test or the Student’s t-test. The level of significance was determined as p < 0.05. Survival curves of mice after bacterial inoculums were expressed as percentages (%) of survival and statistically assessed by Mantel–Cox log rank test (p < 0.05) and are representative of three independent experiments.
3. Results Laticifer proteins obtained from Calotropis procera were used in all experiments. This material has been previously shown to be a potent anti-inflammatory agent. In this study LP was examined as a protective agent against bacterial lethal infection. LP did not inhibit in vitro growth of Salmonella Typhimurium at the highest concentration of 2 mg/ml. Thus, LP fraction does not act directly on the microorganism. The animals that were experimentally infected with a bacterial inoculums (0.2 ml) corresponding to 106 CFU/ml died within 6 days (Fig. 1A). When pre-treated (i.p.) with LP (30 mg/kg), mortality was significantly (p < 0.05) delayed when compared to control but the mortality rate reached 100% on day 12. Very surprisingly, the laticifer proteins given at 60 mg/kg completely prevented the death of mice (p < 0.05), which exhibited only discrete signs of infection commonly seen in infected animals which disappeared latter. These animals remained apparently healthy throughout the 28 days after challenge when the experiment was finished. It is worth noting that the protective effect of LP (60 mg/kg) was achieved by a single dose administered 24 h before bacterial infection. On the other hand, LP given at 30 mg/kg did not exhibit the same protective effect and the animals presented typical signs of infection such as weight loss and prostration. However, the time of death of these animals was delayed by LP treatment (30 mg/kg) as shown in Fig. 1A. Thus pro-
tective effect mediated by LP seems to be dose-dependent. When assayed orally or by subcutaneous route LP remained protective but at a lesser extent compared to protection observed in animals given LP by intraperitoneal route (Fig. 1B). Survival rate after 7 days of animals infected with 107 CFU/ml was only discretely lower (10%) than that of animals infected with 106 CFU/ml, both receiving i.p. LP injection (Fig. 1A versus Fig. 1B). This was certainly correlated with the inoculums that was higher (107 CFU/ml) in the second experiment. Thus, the protective effect of LP is probably dependent of the initial bacterial population. Animals receiving LP 24 h after bacteria inoculums did not resist and mortality rate was documented to be similar to the control (data not shown). This result and the lack of LP activity on in vitro bacteria growth are consistent with the idea that LP fraction acts by immunomodulatory route. Histopathological analysis of the spleen and liver of mice that were given 60 mg/kg of LP or PBS revealed tissue necrosis and inflammatory infiltrates (Fig. 2C and D). These harmful effects were very similar in both the experimental and control groups (Fig. 2A and B versus Fig. 2C and D) but this pattern was compared only on day 5 after initiating infection, since on day 6 the control reached 100% mortality. The number of necrosis foci together with inflammatory infiltrates on day 5 after infection was comparatively lower in the LP-treated mice. The spleen and liver were also examined to determine population numbers of Salmonella in organs. Samples of tissues corresponding to 4 and 24 h and days 5, 15 and 28 were examined. At the early stages (4 and 24 h) of infection, bacteria were estimated to be at similar levels among the groups. This profile was also observed on day 5 (data not shown). To determine the persistence of bacteria in both organs of survival mice, samples corresponding to days 15 and 28 after initial infection were also examined. As previously shown (Fig. 1A), 60 mg/kg of LP completely prevented the death of the animals. However, bacteria were present in both the spleen and liver on day 15 (Fig. 3). Furthermore, the pathogen was still present in high numbers in the spleen of the remaining experimental group, 28 days after challenge, with approximately 104 CFU/g of organ. On day 28 after infection, the bacteria were
Fig. 2. Histopathological damage of organs hematoxylin-eosin stained. Slides of liver (A and C) and spleen (B and D) of control treated with PBS (A and B) and experimental mice (n = 10) treated with LP (60 mg/kg) (C and D) respectively, after 5 days of the initial infection by Salmonella enterica Subsp. enterica serovar Typhimurium (0.2 ml; 106 CFU/ml). The arrows show inflammatory infiltrates and stars indicate necrosis foci. Original magnification, 50×. Splenic macrophages are in detail (1000×). Bar = 50 m.
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Fig. 3. Population numbers of Salmonella enterica Subsp. enterica serovar Typhimurium on days 15 and 28 after initial infection, measured in the spleen and liver of survival experimental mice (n = 10) previously treated with LP (60 mg/kg). Data were statistically accessed by Student’s t-test (*p < 0.05).
reduced in liver tissues (Fig. 3). As expected, the amount of bacteria in the peritoneal fluid (local of inoculums) was elevated after 4 h. This population was maintained after 24 h (Fig. 4A). This kinetic was not observed on blood. Four hours after intraperitoneal inoculums, bacteria levels in the blood were already diminished and after 24 h drastically reduced and statistically different to the control (Fig. 4B). In liver and spleen, bacterial population was determined to be elevated and similar to the controls (data not shown). In an attempt to investigate immunological aspects of the protective effects of LP, the levels of TNF-␣ and IL-12, two known pro-inflammatory cytokines involved in response triggered after Salmonella infection, were determined in serum. The levels of TNF␣ were similar at 24 h and on day 5 in both groups, when infection reached maximum in the control group (Fig. 5A). IL-12 was unaltered at 24 h and thus LP did not modulate this cytokine in the early stage of infection. However, IL-12 was discretely diminished on day 5 in the serum of animals that had received 60 mg/kg of LP (Fig. 5B). Cultured macrophages were stimulated with LPS and treated with LP (500 g/ml) and used to investigate the possible modulating effect of LP on activated macrophages. The levels of TNF-␣ and IL-1 were measured in the supernatant of culture medium. As expected, macrophages incubated with LPS released TNF-␣. Pretreatment with LP did not modify the level of soluble TNF-␣. This result is in accordance with that seen in the serum of LP-treated animals (Fig. 6A). However, cultured macrophages stimulated by LPS and pre-treated with LP released significantly less IL-1 (Fig. 6B). Inhibition of IL-1 production by in vivo pre-treatment of animals with LP would be therefore determined. 4. Discussion LP fraction comprises almost all soluble proteins found in the latex of Calotropis procera. This protein fraction has been shown to exhibit expressive anti-inflammatory and antinociceptive activities in addition to in vitro anti-cancer properties (Alencar et al., 2004; Soares et al., 2005; Oliveira et al., 2007). LP fraction therefore seems to be a promising source of bioactive proteins and thus, a valuable tool to be used in different inflammatory models in order to better characterize the still unknown action mechanisms. This study was undertaken in order to determine if the anti-inflammatory activity of LP could positively contribute to the protection of experimen-
Fig. 4. Viable bacterial counts in the peritoneal fluid (A) and blood (B) of LP or PBStreated mice following infection with Salmonella enterica serovar Typhimurium. Male Swiss mice (n = 10) were treated with LP (60 mg/kg), or PBS 24 h before the challenge with Salmonella Typhimurium (0.2 ml; 106 CFU/ml). Enumeration of colony forming units was carried out onto MacConkey Agar plates after 4 or 24 h postinfection. The number of the viable bacterial was statistically accessed by Student’s t-test (*p < 0.05).
tal animals in the course of a bacterial infection, here induced by Salmonella. The control of salmonelosis in the murine model is mainly dependent on cell-mediated immunity (Jones, 1996). The clearance of bacteria in the liver and spleen is dependent on CD4+ T cells, especially in the late phase of infection (Nauciel, 1990; Hess and Kaufmann, 1996; Withanage et al., 2005). Thus, Interferon-␥ (IFN-␥), produced by either T cells or natural killer cells is important in early infection by restricting growth of bacteria inside macrophages. However, IFN-␥ has a limited role in clearing bacteria disseminated in organs (Mastroeni et al., 1996; Portillo, 2001). Moreover, IFN-␥ is stimulated by IL-12 derived from macrophages that also promote activation of the NK cell effector functions, T cell citotoxicity via interaction to APCs and macrophage activation (Pie et al., 1997; John et al., 2002). Macrophage activation is also dependent on Tumor Necrosis Factor ␣ (TNF-␣) triggering NADPH-oxidase pathway, important in the late phase of infection and its inhibition augments the susceptibility of mice to salmonelosis (Vasquez-Torres et al., 2001; Sebastiani et al., 2002; Mastroeni and Ménager, 2003; Fidan et al., 2008). On the other hand, after 5–7 days of infection, the bacterial population usually increases in the spleen and liver of mice, which are susceptible to infection by
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Fig. 5. Levels of TNF-␣ (A) and IL-12 (B) measured in the serum of mice (n = 10) previously treated with LP (60 mg/kg) or PBS 24 h before the lethal challenge with Salmonella enterica serovar Typhimurium (0.2 ml; 106 CFU/ml). Data was statistically accessed by Student’s t-test (*p < 0.05).
Salmonella Typhimurium (Pie et al., 1997). Bacterial proliferation in the bloodstream induces a high inflammatory stimulus despite bacterial lipid-A, the main consequence being toxic shock usually due to accumulation of pro-inflammatory cytokines such TNF-␣ and IL-1 (Jones, 1996). We observed that LP attenuated signs of infection and at the higher dose tested protected animals of septic shock. LP did not exhibit direct effect on the bacteria and lost its protective effects when given 24 h after bacteria inoculums. Both observations suggested that LP fraction signalizes immunomodulatory responses instead of directly eliminate bacteria. It was observed that neither TNF-␣ nor IL-12 played a key role in the activation of the protective immune response in LP-treated mice against Salmonella Typhimurium (Fig. 5). This was reinforced by the high population of bacteria in the liver and spleen of the animals on days 1 and 5 after initiating infection, confirming that protection was not due to the increase in the bactericidal capacity of macrophages induced by these cytokines. However, IL-12 was slightly decreased after 5 days of infection in the experimental animals (p < 0.05). Since it is not reasonable to expect LP to remain present in the bloodstream for 28 days, it is likely that LP fraction acts by signaling cellular events involved in the control of inflammation induced by bacteria during infection, preventing septic shock and death of the animals. This suggestion is reinforced by our previously findings that
Fig. 6. Levels of TNF-␣ (A) and IL-1 (B) measured in supernatants of macrophage cultures previously treated with LP (500 g/ml) or RPMI 1 h before incubation with LPS (1 g/ml). Data were statistically accessed by ANOVA followed by Bonferrroni’s test (*p < 0.05).
intravenous injection of LP fraction of Calotropis procera inhibits neutrophil migration induced by dialyzable fraction of Calotropis procera (DL) or carrageenin to rat’s peritoneal cavities by inhibiting TNF-␣ release (Alencar et al., 2006). This effect should be similar to thalidomide, which down-regulates TNF-␣ production. However, in this study the measured serum levels of TNF-␣ were similar for both the control and experimental animals, showing this cytokine was less important on control of systemic inflammatory response syndrome (SIRS) on LP-treated mice (Fig. 5A). The drastic reduction of bacteria in bloodstream observed 24 h after the initial infection in animals receiving LP suggested that the protective pathway of LP was initiated in the initial stages of infection. As cited before, the belief that TNF-␣ was not directly involved in the protective effect of LP was also supported by the lost of the protective effect of LP when it was given 24 h after bacteria inoculums. Recently, another study reported that rats given LP intravenously exhibited increase of nitric oxide (NO) in serum, associated with strong anti-inflammatory activity (Ramos et al., 2009). NO is a multifunctional molecule which concentration and local of production interfere in its effects. As a result it is not certain that intraperitoneal injection of LP modulates NO in serum. More experimental evidence will be necessary to investigate this
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hypothesis. Despite the interesting protective effect of LP on infected mice as reported here, poor information was given on its protective mechanism. The full effectiveness of LP (60 mg/kg) in protecting mice by a single administration before experimental infection with Salmonella produced a number of issues to be considered in order to investigate the protective mechanism of the action. Since prevention of septic shock due to down-regulation of immune responses in LP-treated mice could involve others pro-inflammatory cytokines, the release of IL-1 and TNF-␣ was evaluated in macrophage culture supernatants pre-treated with LP and stimulated by LPS. As observed in the in vivo experiments, titration of TNF-␣ in culture of macrophages showed similar content in both the control and experimental cells after LPS stimulus. On the other hand, the level of soluble IL-1 was decreased in the supernatant of cells treated with LP (Fig. 6B). Mononuclear phagocytes stimulated by bacterial LPS and by other cytokines such as TNF-␣ are the major source of IL-1. This cytokine mediates host inflammatory responses to infections together with TNF-␣ in innate immunity (Jones, 1996; Cavaillon and Annane, 2006). In high amounts it can promote fever, synthesis of acute phase plasma proteins by the liver, and cachexia. However, IL-1 does not cause septic shock or apoptotic death of cells by itself. Despite this observation, in vivo modulation of this pro-inflammatory cytokine in LP-treated animals deserves additional approaches. It is also relevant to cite that the protective effect of LP was preserved when animals were treated orally. In this respect, many studies have already demonstrated anti-inflammatory activity of latex extracts of Calotropis procera (Kumar and Roy, 2007). The latex of Calotropis procera is introduced in folk medicine and used by different ways. It is topically applied to treat skin infections or teeth pain. We have found local usages to control diabetes by oral ingestion after diluting in water. However, there is a prevalence of use on inflammatory-related problems. In this case, the ethnopharmacological use of Calotropis procera does not deserve confirmation, since Kumar and co-workers have largely confirmed this folk usage by different experimental approaches and inflammatory models investigated (Kumar and Roy, 2009; Bharti et al., 2010). It is therefore important to advance in understanding mechanisms underlying anti-inflammation; identifying molecules involved and establishing safe use of latex. Here the potential of latex to treat infections was evaluated. Biochemical and functional studies of LP from Calotropis procera are currently under investigation. This material is a rich source of proteins involved in different metabolic pathways typically related to the plant metabolism, including defensive strategies against infections and predators. However, this protein fraction has become a relevant pharmacological tool due to the cumulative benefic properties recently described. At the present it is not possible to nominate or discuss putative proteins involved in the protective effects observed in this study. Despite such a limitation, the results described in this study appear as a relevant contribution to the search for new sources of biological compounds that could be useful in fighting bacterial infections and modulation of the immune responses. This work opens a new window of research since latex is found in many plants and offers now a new biological material to be investigated as a source of molecules capable of modulating immune responses. Furthermore, new approaches are currently being developed to investigate the involvement of NO in the protective effect of LP reported here. In this study, we report for the first time the protective role of the laticifer proteins of Calotropis procera in the control of a systemic murine infection caused by Salmonella enterica serovar Typhimurium. The protective activity of LP was not associated to the increase in the bactericidal capacity of macrophages and preliminary evidence suggested that the protective activity may be due
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to down-regulation of pro-inflammatory cytokines such IL-12 and IL-1 which prevented animals succumbing to toxic shock. Nevertheless, it is not clear yet the involvement of other inflammatory mediators and the effects of LP on NO in this model. The characterization of the biological properties, especially those cross-related to modulation of the immune system performed by latex proteins of Calotropis procera in the control of infections, is currently being investigated by this research group. Acknowledgements Biochemical, functional and applied studies of the latex from Calotropis procera have been supported by grants from FUNCAP, CNPq, CAPES, PADCT, RENORBIO and IFS (M.V.R.). The authors are in debt with Mr. Brian Stephen Curry who critically reviewed the language of the manuscript. References Alencar, N.M., Figueiredo, I.S., Vale, M.R., Bitencurt, F.S., Oliveira, J.S., Ribeiro, R.A., Ramos, M.V., 2004. Anti-inflammatory effect of the latex from Calotropis procera in three different experimental models: peritonitis, paw edema and hemorrhagic cystitis. Planta Medica 70, 1144–1149. Alencar, N.M., Oliveira, J.S., Mesquita, R.O., Lima, M.W., Vale, M.R., Etchells, J.P., Freitas, C.D.T., Ramos, M.V., 2006. Pro- and anti-inflammatory activities of the latex from Calotropis procera (Ait.) R.Br. are triggered by compounds fractionated by dialysis. Inflammation Research 55, 559–564. Arya, S., Kumar, V.L., 2005. Anti-inflammatory efficacy of extracts of latex of Calotropis procera against different mediators of inflammation. Mediators of Inflammation 2005, 228–232. Biedner, B., Rothkoff, L., Witztum, A., 1977. Calotropis procera (Sodom apple) latex keratoconjunctivitis. Israel Journal of Medical Science 13, 914–916. Bharti, S., Wahane, V.D., Kumar, V.L., 2010. Protective effect of Calotropis procera latex extracts on experimentally induced gastric ulcers in rats. Journal of Ethnopharmacology 127, 440–444. Cavaillon, J.M., Annane, D., 2006. Compartmentalization of the inflammatory response in sepsis and SIRS. Journal of Endotoxin Research 12, 151–170. Choedon, T., Mathan, G., Arya, S., Kumar, V.L., Kumar, V., 2006. Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma. World Journal Gastroenterology 12, 2517–2522. Cunha, F.Q., Ferreira, S.H., 1986. The release of a neutrophil chemotactic factor from peritoneal macrophage by endotoxin: inhibition by glucocorticoids. European Journal of Pharmacology 129, 65–76. El Badwi, Samia, M.A., Adam, S.E., Shigidi, M.T., Hapke, H.J., 1998. Studies on laticiferous plants: toxic effects in goats of Calotropis procera latex given by different routes of administration. Dtsch Tierarztl Wochenschr 105, 425–427. Fidan, I., Yesilyurt, E., Gurelik, F.C., Erdal, B., Imir, T., 2008. Effects of recombinant interferon-␥ on cytokine secretion from monocyte-derived macrophages infected with Salmonella typhi. Comparative Immunology and Microbiology Infectious Diseases 31, 467–475. Freeman, B.D., Natanson, C., 2000. Anti-inflammatory therapy in sepsis and septic shock. Expert Opinion on Investigational Drugs 9, 1651–1663. Groisman, E.A., Ochman, H., 1997. How Salmonella became a pathogen. Trends Microbiology 5, 343–349. Hess, J., Kaufmann, S.H., 1996. Salmonella enterica infection. Research Immunology 147, 581–586. John, B., Rajagopal, D., Pashine, A., Rath, S., George, A., Bal, V., 2002. Role of IL-12independent and IL-12-dependent pathways in regulating generation of the IFNgamma component of T cell responses to Salmonella typhimurium. Journal of Immunology 169, 2545–2552. Jones, B.D., 1996. Salmonellosis: host immune responses and bacterial virulence determinants. Annual Review of Immunology 14, 533–561. Koneman, E.W., Allen, S.D., Janda, W.M., 2001. Diagnóstico microbiológico-texto e atlas colorido, 5th ed. Médica e Científica, Rio de Janeiro, 1465 pp. Koyama, Y., Motobu, M., Hikosaka, K., Yamada, M., Nakamura, K., Saido-Sakanaka, H., Asaoka, A., Yamakawa, M., Sekikawa, K., Kitani, H., Shimura, K., Nakai, Y., Hirota, Y., 2006. Protective effects of antimicrobial peptides derived from the beetle Allomyrina dichotoma defensin on endotoxic shock in mice. International Immunopharmacology 6, 234–240. Kumar, V.L., Shivkar, Y.M., 2004. Involvement of prostaglandins in inflammation induced by latex of Calotropis procera. Mediators of Inflammation 13, 151–155. Kumar, V.L., Sehgal, R., 2007. Calotropis procera latex-induced inflammatory hyperalgesia – effect of bradyzide and morphine. Autonomic and Autacoid Pharmacology 27, 143–149. Kumar, V.L., Roy, S., 2007. Calotropis procera latex extract affords protection against inflammation and oxidative stress in Freund’s complete adjuvant-induced monoarthritis in rats. Mediators of Inflammation 2007, 47523. Kumar, V.L., Roy, S., 2009. Protective effect of latex of Calotropis procera in Freund’s Complete Adjuvant induced monoarthritis. Phytotherapy Research 23, 1–5.
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Larhsini, M., Markouk, M., Jaouhari, J.T., Bekkouche, K., Lazrek, H.B., Jana, M., 2002. The antipyretic activity of some Moroccan medicinal plants. Phytotherapy Research 1, S97–S98. Mastroeni, P., Harrison, J.A., Chabalgoity, J.A., Hormaeche, C.E., 1996. Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid. Infection and Immunology 64, 189–196. Mastroeni, P., Ménager, N., 2003. Development of acquired immunity to Salmonella. Journal of Medical Microbiology 52, 453–459. Mossa, J.S., Tariq, M., Mohsin, A., Ageel, A.M., al-Yahya, M.A., al-Said, M.S., Rafatullah, S., 1991. Pharmacological studies on aerial parts of Calotropis procera. The American Journal of Chinese Medicine 19, 223–231. Motobu, M., Amer, S., Koyama, Y., Hikosaka, K., Sameshima, T., Yamada, M., Nakamura, K., Koge, K., Kang, C.B., Hayasidani, H., Hirota, Y., 2006. Protective effects of sugar cane extract on endotoxic shock in mice. Phytotherapy Research 20, 359–363. Nauciel, C., 1990. Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection. Journal of Immunology 145, 1265–1269. Oliveira, J.S., Bezerra, D.P., Freitas, C.D.T., Marinho-Filho, J.D.B., Moraes, O.M., Pessoa, C., Costa-Lotufo, L.V., Ramos, M.V., 2007. In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br. Toxicology In Vitro 21, 1563–1573. Pathak, A.K., Argal, A., 2007. Analgesic activity of Calotropis gigantea flower. Fitoterapia 78, 40–402. Pie, S., Truffa-Bachi, P., Pla, M., Nauciel, C., 1997. Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance. Infection and Immunity 65, 4509–4514. Portillo, F.G., 2001. Salmonella intracellular proliferation: where, when and how? Microbes and Infection 3, 1305–1311. Qureshi, A.A., Prakash, T., Patil, T., Viswanath-Swamy, A.H., Gouda, A.V., Prabhu, K., Setty, S.R., 2007. Hepatoprotective and antioxidant activities of flowers of Calotropis procera (Ait) R. Br. in CCl4 induced hepatic damage. Indian Journal of Experimental Biology 45, 304–310. Ramos, M.V., Oliveira, J.S., Figueiredo, J.G., Figueiredo, I.S., Kumar, V.L., Bitencourt, F.S., Cunha, F.Q., Oliveira, R.S.B., Bomfim, L.R., Lima-Filho, J.V., Alencar, N.M.N.,
2009. Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model. Journal of Ethnopharmacology 125, 387–392. Rasik, A.M., Raghubir, R., Gupta, A., Shukla, A., Dubey, M.P., Srivastava, S., Jain, H.K., Kulshrestha, D.K., 1999. Healing potential of Calotropis procera on dermal wounds in Guinea pigs. Journal of Ethnopharmacology 68, 261–266. Sangraula, H., Dewan, S., Kumar, V.L., 2002. Evaluation of anti-inflammatory activity of latex of Calotropis procera in different models of inflammation. Inflammopharmacology 9, 257–264. Sebastiani, G., Blais, V., Sancho, V., Vogel, S.N., Stevenson, M.M., Gros, P., Lapointe, J.M., Rivest, S., Malo, D., 2002. Host immune response to Salmonella enterica serovar Typhimurium infection in mice derived from wild strains. Infection and Immunity 70, 1997–2009. Shivkar, Y.M., Kumar, V.L., 2003. Histamine mediates the pro-inflammatory effect of latex of Calotropis procera in rats. Mediators of Inflammation 12, 299–302. Soares, P.M., Lima, S.R., Matos, S.G., Andrade, M.M., Patrocínio, M.C., Freitas, C.D.T., Ramos, M.V., Criddle, D.N., Cardi, B.A., Carvalho, K.M., Assreuy, A.M., Vasconcelos, S.M., 2005. Antinociceptive activity of Calotropis procera latex in mice. Journal of Ethnopharmacology 99, 125–129. Van Dien, M., Takahashi, K., Mu, M.M., Koide, N., Sugiyama, T., Mori, I., Yoshida, T., Yokochi, T., 2001. Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine sensitized mice. Microbiology and Immunology 45, 751–756. Vasquez-Torres, A., Fantuzzi, G., Edwards, C.K., Dinarello, C.A., Fang, F.C., 2001. Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagossomes in tumor necrosis factor p55 receptor-deficient macrophages. Proceedings of National Academy of Sciences of USA 98, 2561–2565. Wang, X.M., Liang, M.F., Yuan, Y., Jiang, W., 2008. The different effects of glutamine on macrophage cytokines release in vivo and in vitro. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 20, 456–460. Withanage, G.S.K., Wigley, P., Kaiser, P., Mastroeni, P., Brooks, H., Powers, C., Beal, R., Barrow, P., Maskell, D., McConnell, I., 2005. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infection and Immunity 73, 5173–5182.
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Proteins from latex of Calotropis procera prevent septic shock due to lethal infection by Salmonella enterica serovar Typhimurium José V. Lima-Filho a,∗ , Joyce M. Patriota a , Ayrles F.B. Silva a , Nicodemos T. Filho b , Raquel S.B. Oliveira c , Nylane M.N. Alencar d , Márcio V. Ramos c,∗∗ a Laboratório de Microbiologia e Imunologia, Departamento de Biologia, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, Recife CEP 52171-900, PE, Brazil b Laboratório de Imunoparasitologia Keizo Azami (Lab. Anatomopatologia), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235 - Cidade Universitária, Recife CEP 50670-901, PE, Brazil c Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Cx. Postal 6033, Fortaleza CEP 60451-970, CE, Brazil d Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Brazil
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Article history: Received 30 November 2009 Received in revised form 11 February 2010 Accepted 27 March 2010 Available online 3 April 2010 Keywords: Bacteria Cytokines Infection Inflammation Latex SEPSIS
a b s t r a c t Aim of the study: The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. Materials and methods: Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 106 CFU/ml of Salmonella Typhimurium using the same route of administration. Results: All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 104 cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-␣ in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1. Conclusions: LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-␣). LP inhibited IL-1 release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The shrub Calotropis procera (R. Br.) is a plant of the Asclepiadaceae family, and has been used extensively in folk medicine
∗ Corresponding author. Tel.: +55 31 81 33206312. ∗∗ Corresponding author. Tel.: +55 85 33669789. E-mail addresses: [email protected] (J.V. Lima-Filho), [email protected] (M.V. Ramos). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.03.038
of East countries. Hepato-protection, treatment of rheumatism and aches, local anesthesia and treatment of skin disease infections are among the scientifically reported activities (Mossa et al., 1991; Pathak and Argal, 2007; Qureshi et al., 2007). The plant is widely known because it releases important amounts of latex from its green parts when it is injured. This latex, as well as organic and aqueous latex extracts, has been investigated through in vitro and in vivo experimental models to establish pharmacological and toxicological aspects. Thus, different authors have claimed a set of properties for the latex of Calotropis procera
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including healing, anti-cancer, antipyretic, analgesic together with inflammatory and anti-inflammatory properties, among many others (Rasik et al., 1999; Larhsini et al., 2002; Alencar et al., 2006; Choedon et al., 2006). On the other hand, disadvantageous toxicity, hyperalgesia and lethal effects have also been observed in animals treated with the whole latex (Biedner et al., 1977; El Badwi et al., 1998; Kumar and Sehgal, 2007). The effects of the latex of Calotropis procera on immune responses have been particularly investigated. The latex-methanol extract reduced the inflammatory stimulus induced by Freud’s adjuvant on the experimental arthritis model (Kumar and Roy, 2007). Furthermore, the antiinflammatory capacity appears to be similar to Fenilbutazone (PBZ) in acute and chronic models of inflammation (Sangraula et al., 2002; Arya and Kumar, 2005). However, dialysis and centrifugation of the latex provide a set of proteins that shows different effects on cell-mediated immunity. Administrating the dialyzable fraction (DL) to rats induces an inflammatory response whereas the non-dialyzable fraction (LP) suppresses this effect when assayed at the same doses (Alencar et al., 2006). The inflammatory stimuli carried by DL fraction also appear to be dependent on histamine and prostraglandin, which trigger neutrophil recruitment and stimulate cellular vascularization (Shivkar and Kumar, 2003; Kumar and Shivkar, 2004). Moreover, histamine has been detected in the latex (Shivkar and Kumar, 2003). It has been reported that LP fraction reduces neutrophil migration induced by carrageenin on the paw edema model (Alencar et al., 2004, 2006). Gram-negative bacterial infections can induce sepsis as a seriously pathological consequence of the spread of bacteria in the bloodstream. This condition is accompanied by a systemic host inflammatory response characterized by the release of inflammatory plus pro-inflammatory cytokines due to stimulus of the lipopolyssacharide (LPS) present in the bacterial cell wall. Since a balanced inflammation is necessary to control infection and to keep the host from succumbing to septic shock, the management of sepsis has been done through the use of anti-inflammatory agents or substances that enhance immune cells for bacterial clearance (Freeman and Natanson, 2000). As an alternative, natural products reported to have influence on the immune system have also been investigated, such as sugarcane extracts (Motobu et al., 2006), plant flavonoids (Van Dien et al., 2001), synthetic peptides derived from the beetle Allomyrina dichotoma defensin (Koyama et al., 2006) and glutamine (Wang et al., 2008). Despite the amount of information available concerning the immune-modulatory properties of the latex of Calotropis procera, there have not been any reports of such effects on a systemic infection. The murine model of infection by Salmonella enterica serovar Typhimurium produces the symptoms of typhoid fever and represents a reliable model to evaluate the immune response because it induces a rapid systemic disease after small inoculums. It is easily detected and quantified in several organs such as the spleen and liver, where the bacteria survive inside nonactivated macrophages. Damage to organs can also be assessed through histopathological examination and it can be studied without causing septic shock (Jones, 1996; Groisman and Ochman, 1997; Portillo, 2001). In view of the relevant anti-inflammatory activity previously found in LP and the continuing search for new products capable of fighting the lethal effects produced on the course of systemic infections, the aim of the present study was to evaluate the possible benefits of LP in controlling of the inflammatory immune response induced by Salmonella enterica serovar Typhimurium by using the murine model that represents a more realistic model of inflammation instead of using carrageen of others inflammogens.
2. Materials and methods 2.1. Latex and laticifer proteins Samples of the latex of Calotropis procera were collected in Fortaleza in the state of Ceará, Brazil. The voucher (sample specimen No. 32663) was deposited at Prisco Bezerra Herbarium of the Universidade Federal do Ceará, Brazil and the plant was authenticated by a taxonomist from the Department of Biology. Fresh latex of Calotropis procera was collected from healthy plants by small incisions near the youngest leaves and left to flow off into distilled water in order to obtain a mixture 1:1 (v/v). Samples were centrifuged (5000 × g) at 10 ◦ C in a refrigerated bench top centrifuge for 10 min. The precipitated material, showing rubber aspect, was discarded while the suspended phase was submitted to exhaustive dialysis (cut off 8000 Da) against distilled water at 8 ◦ C for 60 h, and then centrifuged as previously mentioned. The new precipitate material was eliminated and the soluble phase devoid of rubber was freeze-dried. This fraction comprising almost all of the soluble proteins of the latex was named laticifer proteins (LP). The biochemical characterization of this material was described previously in different studies (Alencar et al., 2004, 2006; Oliveira et al., 2007). All further experiments were performed using the lyophilized material in appropriated solutions.
2.2. Microorganism Salmonella enterica Subsp. enterica serovar Typhimurium was isolated from a human clinical case at Fundac¸ão Ezequiel Dias (FUNED, Belo Horizonte, MG, Brazil) and was a kind gift from Dr. Jacques Robert Nicoli (Universidade Federal de Minas Gerais). The bacteria were maintained at −18 ◦ C in Brain Heart Infusion (BHI) medium containing 50% glycerol. During the experiments, bacteria were activated after culture in BHI broth for 24 h at 37 ◦ C.
2.3. Animals Adult male Swiss mice weighing approximately 30 g were used in this study. A minimum number of animals (n = 10 per group) was used in all experiments in order to adhere to the modern rules for the use of animals in experimentation. The animals were kept in an animal house with controlled lighting (12-h light–dark cycles), temperature (25 ◦ C) and humidity (60–70%) with free access to water and commercial sterile diet (Purina, Paulínia, SP, Brazil). The mice were handled according to established experimental procedures, which were performed according to the standards rules in the “Guide for the Care and Use of Laboratory Animals” of the National Research Council, following approve of Institutional Animal Ethics Committee.
2.4. In vitro antibacterial activity of LP Possible antibacterial activity of LP against Salmonella Typhimurium was evaluated by using the broth dilution method (Koneman et al., 2001). LP fraction was suspended in PBS and added to tubes with Mueller Hinton broth containing Salmonella Typhimurium at 105 Colony Forming Units (CFU)/ml. The tested LP concentrations ranged from 3.9 g/ml to 2 mg/ml. All assays were performed in duplicate and control tubes were free of LP. The minimum inhibitory concentration was estimated as the lowest concentration that caused visible growth inhibition after an incubation period of 24 h at 37 ◦ C.
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2.5. Experimental design Animals were given LP by intraperitoneal (i.p.), subcutaneous (s.c.) or orally (p.o.). For the complementary determinations, intraperitoneal injection was used. A single dose of LP fraction at 30 or 60 mg/kg body weight, in 0.2 ml Phosphate Buffered Saline – sterile, pH 7.2 (PBS) was given to each mouse from the two experimental groups (n = 10), 24 h before or after challenge with Salmonella Typhimurium. The control group was inoculated with 0.2 ml PBS according to the same schedule for the experimental one. Salmonella Typhimurium was grown in BHI broth at 37 ◦ C and serially diluted to attain a bacterial suspension containing 107 CFU/ml. Animals from both the experimental and control groups were challenged with 0.2 ml of this bacterial suspension (106 CFU/ml) by the intraperitoneal route to provoke a systemic infection. To investigate the protective effect of LP given by different routes of administration, bacterial inoculum was 107 CFU/ml in 0.2 ml instead of 106 CFU/ml. Experiments were conducted for 7 or 28 days. 2.6. Evaluation of clinical signs and survival
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was maintained at 37 ◦ C (5% CO2 ). After 24 h, the cells were incubated in fresh medium or in medium containing LP (500 g/ml) for 1 h. The supernatants were then discarded and after three further washes, the cells were incubated with medium (1.5 ml) or LPS (1 g/ml) for 5 h. The supernatants were used for the cytokine assay. Cell viability of each macrophage culture was analyzed by the Trypan blue exclusion method. 2.11. Cytokine determination The amount of Interleukin-12 (IL-12) and Tumor Necrosis Factor-␣ (TNF-␣) was estimated in serum of animals sacrificed on days 1 and 5 after bacteria inoculums. TNF-␣ and Interleukin-1-betha (IL-1) were quantified on supernatant of the macrophages. Assays were performed by using commercial kits of ELISA immunoassay (R&D Systems). Color was developed with ophenylenediamine (1,2-benzenediamine; Sigma) and absorbance at 450 nm was determined by an ELISA plate reader (ELISA READER, BIORAD).
Animals that were submitted to a long-term assay (28 days) from initial inoculation with Salmonella Typhimurium were evaluated for survival rate. Clinical signs such as prostration, weight loss and diarrhea were also observed daily throughout the experiment. Histopathological examination, counting of bacteria in spleen and liver and cytokine titration in serum were performed on day 5. The survival animals were sacrificed on days 15 and 28 to evaluate clearance of bacteria in the spleen and liver. 2.7. Analysis of early infection In additional experiments, mice were treated with LP fraction (i.p.) and infected by Salmonella 24 h later as described above. The animals from the control and LP groups were sacrificed 4 and 24 after infection and bacteria enumeration was performed in blood, peritoneal fluid, spleen and liver. An aliquot of blood was taken for cytokine titration in serum of animals (24 h). 2.8. Histopathological examination Samples of tissues from the liver and spleen from mice sacrificed on day 5 post-infection were fixed in 10% formaldehyde in PBS and processed to embed them in paraffin. The histopathological sections (5 m) were stained with hematoxylin-eosin. The slides were coded and examined by a single pathologist, who was unaware of the experimental conditions of all the groups. 2.9. Salmonella enumeration The liver and spleen were dissected, weighed and macerated in PBS (1:10 or 1:100, w/v). Samples of blood and peritoneal fluid were used as collected. Serial decimal dilutions were made and 0.1 ml aliquots were plated onto MacConkey agar (Oxoide). The colonies were counted after incubation at 37 ◦ C for 24 h and the results expressed as CFU/g of organs (spleen and liver) and CFU/ml (blood and peritoneal fluid). 2.10. Analysis in macrophage-culture cells activated by LP The method described by Cunha and Ferreira (1986) was followed. Peritoneal macrophages of mice were harvested with RPMI (pH 7.4) 4 days after i.p. injection of 3% thioglycolate (3 ml/cavity) and cultured in plastic tissue culture dishes (24 wells) at 37 ◦ C (5% CO2 ). Non-adherent cells were removed by three washes with 1 ml of RPMI medium and the adherent population (95% macrophages)
Fig. 1. (A) Protective effect of LP on mice after lethal challenge with Salmonella enterica Subsp. enterica serovar Typhimurium. Animals were treated intraperitoneally with LP (30 or 60 mg/kg) 24 h before bacteria inoculums (0.2 ml; 106 CFU/ml). Control animals were given PBS. (B) Protective effect of LP (60 mg/kg) given by different routes 24 h before bacteria inoculums (0.2 ml; 107 CFU/ml). Results were statistically assessed by Mantel–Cox log rank test (*p < 0.05) and are representative of three independent experiments (n = 10). All experimental statistically differed from controls.
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2.12. Statistical analyses The data, excepting survival curves, are reported as mean ± S.E.M. Statistical significance was assessed by analyzing the variance (ANOVA) followed by Bonferroni’s test or the Student’s t-test. The level of significance was determined as p < 0.05. Survival curves of mice after bacterial inoculums were expressed as percentages (%) of survival and statistically assessed by Mantel–Cox log rank test (p < 0.05) and are representative of three independent experiments.
3. Results Laticifer proteins obtained from Calotropis procera were used in all experiments. This material has been previously shown to be a potent anti-inflammatory agent. In this study LP was examined as a protective agent against bacterial lethal infection. LP did not inhibit in vitro growth of Salmonella Typhimurium at the highest concentration of 2 mg/ml. Thus, LP fraction does not act directly on the microorganism. The animals that were experimentally infected with a bacterial inoculums (0.2 ml) corresponding to 106 CFU/ml died within 6 days (Fig. 1A). When pre-treated (i.p.) with LP (30 mg/kg), mortality was significantly (p < 0.05) delayed when compared to control but the mortality rate reached 100% on day 12. Very surprisingly, the laticifer proteins given at 60 mg/kg completely prevented the death of mice (p < 0.05), which exhibited only discrete signs of infection commonly seen in infected animals which disappeared latter. These animals remained apparently healthy throughout the 28 days after challenge when the experiment was finished. It is worth noting that the protective effect of LP (60 mg/kg) was achieved by a single dose administered 24 h before bacterial infection. On the other hand, LP given at 30 mg/kg did not exhibit the same protective effect and the animals presented typical signs of infection such as weight loss and prostration. However, the time of death of these animals was delayed by LP treatment (30 mg/kg) as shown in Fig. 1A. Thus pro-
tective effect mediated by LP seems to be dose-dependent. When assayed orally or by subcutaneous route LP remained protective but at a lesser extent compared to protection observed in animals given LP by intraperitoneal route (Fig. 1B). Survival rate after 7 days of animals infected with 107 CFU/ml was only discretely lower (10%) than that of animals infected with 106 CFU/ml, both receiving i.p. LP injection (Fig. 1A versus Fig. 1B). This was certainly correlated with the inoculums that was higher (107 CFU/ml) in the second experiment. Thus, the protective effect of LP is probably dependent of the initial bacterial population. Animals receiving LP 24 h after bacteria inoculums did not resist and mortality rate was documented to be similar to the control (data not shown). This result and the lack of LP activity on in vitro bacteria growth are consistent with the idea that LP fraction acts by immunomodulatory route. Histopathological analysis of the spleen and liver of mice that were given 60 mg/kg of LP or PBS revealed tissue necrosis and inflammatory infiltrates (Fig. 2C and D). These harmful effects were very similar in both the experimental and control groups (Fig. 2A and B versus Fig. 2C and D) but this pattern was compared only on day 5 after initiating infection, since on day 6 the control reached 100% mortality. The number of necrosis foci together with inflammatory infiltrates on day 5 after infection was comparatively lower in the LP-treated mice. The spleen and liver were also examined to determine population numbers of Salmonella in organs. Samples of tissues corresponding to 4 and 24 h and days 5, 15 and 28 were examined. At the early stages (4 and 24 h) of infection, bacteria were estimated to be at similar levels among the groups. This profile was also observed on day 5 (data not shown). To determine the persistence of bacteria in both organs of survival mice, samples corresponding to days 15 and 28 after initial infection were also examined. As previously shown (Fig. 1A), 60 mg/kg of LP completely prevented the death of the animals. However, bacteria were present in both the spleen and liver on day 15 (Fig. 3). Furthermore, the pathogen was still present in high numbers in the spleen of the remaining experimental group, 28 days after challenge, with approximately 104 CFU/g of organ. On day 28 after infection, the bacteria were
Fig. 2. Histopathological damage of organs hematoxylin-eosin stained. Slides of liver (A and C) and spleen (B and D) of control treated with PBS (A and B) and experimental mice (n = 10) treated with LP (60 mg/kg) (C and D) respectively, after 5 days of the initial infection by Salmonella enterica Subsp. enterica serovar Typhimurium (0.2 ml; 106 CFU/ml). The arrows show inflammatory infiltrates and stars indicate necrosis foci. Original magnification, 50×. Splenic macrophages are in detail (1000×). Bar = 50 m.
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Fig. 3. Population numbers of Salmonella enterica Subsp. enterica serovar Typhimurium on days 15 and 28 after initial infection, measured in the spleen and liver of survival experimental mice (n = 10) previously treated with LP (60 mg/kg). Data were statistically accessed by Student’s t-test (*p < 0.05).
reduced in liver tissues (Fig. 3). As expected, the amount of bacteria in the peritoneal fluid (local of inoculums) was elevated after 4 h. This population was maintained after 24 h (Fig. 4A). This kinetic was not observed on blood. Four hours after intraperitoneal inoculums, bacteria levels in the blood were already diminished and after 24 h drastically reduced and statistically different to the control (Fig. 4B). In liver and spleen, bacterial population was determined to be elevated and similar to the controls (data not shown). In an attempt to investigate immunological aspects of the protective effects of LP, the levels of TNF-␣ and IL-12, two known pro-inflammatory cytokines involved in response triggered after Salmonella infection, were determined in serum. The levels of TNF␣ were similar at 24 h and on day 5 in both groups, when infection reached maximum in the control group (Fig. 5A). IL-12 was unaltered at 24 h and thus LP did not modulate this cytokine in the early stage of infection. However, IL-12 was discretely diminished on day 5 in the serum of animals that had received 60 mg/kg of LP (Fig. 5B). Cultured macrophages were stimulated with LPS and treated with LP (500 g/ml) and used to investigate the possible modulating effect of LP on activated macrophages. The levels of TNF-␣ and IL-1 were measured in the supernatant of culture medium. As expected, macrophages incubated with LPS released TNF-␣. Pretreatment with LP did not modify the level of soluble TNF-␣. This result is in accordance with that seen in the serum of LP-treated animals (Fig. 6A). However, cultured macrophages stimulated by LPS and pre-treated with LP released significantly less IL-1 (Fig. 6B). Inhibition of IL-1 production by in vivo pre-treatment of animals with LP would be therefore determined. 4. Discussion LP fraction comprises almost all soluble proteins found in the latex of Calotropis procera. This protein fraction has been shown to exhibit expressive anti-inflammatory and antinociceptive activities in addition to in vitro anti-cancer properties (Alencar et al., 2004; Soares et al., 2005; Oliveira et al., 2007). LP fraction therefore seems to be a promising source of bioactive proteins and thus, a valuable tool to be used in different inflammatory models in order to better characterize the still unknown action mechanisms. This study was undertaken in order to determine if the anti-inflammatory activity of LP could positively contribute to the protection of experimen-
Fig. 4. Viable bacterial counts in the peritoneal fluid (A) and blood (B) of LP or PBStreated mice following infection with Salmonella enterica serovar Typhimurium. Male Swiss mice (n = 10) were treated with LP (60 mg/kg), or PBS 24 h before the challenge with Salmonella Typhimurium (0.2 ml; 106 CFU/ml). Enumeration of colony forming units was carried out onto MacConkey Agar plates after 4 or 24 h postinfection. The number of the viable bacterial was statistically accessed by Student’s t-test (*p < 0.05).
tal animals in the course of a bacterial infection, here induced by Salmonella. The control of salmonelosis in the murine model is mainly dependent on cell-mediated immunity (Jones, 1996). The clearance of bacteria in the liver and spleen is dependent on CD4+ T cells, especially in the late phase of infection (Nauciel, 1990; Hess and Kaufmann, 1996; Withanage et al., 2005). Thus, Interferon-␥ (IFN-␥), produced by either T cells or natural killer cells is important in early infection by restricting growth of bacteria inside macrophages. However, IFN-␥ has a limited role in clearing bacteria disseminated in organs (Mastroeni et al., 1996; Portillo, 2001). Moreover, IFN-␥ is stimulated by IL-12 derived from macrophages that also promote activation of the NK cell effector functions, T cell citotoxicity via interaction to APCs and macrophage activation (Pie et al., 1997; John et al., 2002). Macrophage activation is also dependent on Tumor Necrosis Factor ␣ (TNF-␣) triggering NADPH-oxidase pathway, important in the late phase of infection and its inhibition augments the susceptibility of mice to salmonelosis (Vasquez-Torres et al., 2001; Sebastiani et al., 2002; Mastroeni and Ménager, 2003; Fidan et al., 2008). On the other hand, after 5–7 days of infection, the bacterial population usually increases in the spleen and liver of mice, which are susceptible to infection by
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Fig. 5. Levels of TNF-␣ (A) and IL-12 (B) measured in the serum of mice (n = 10) previously treated with LP (60 mg/kg) or PBS 24 h before the lethal challenge with Salmonella enterica serovar Typhimurium (0.2 ml; 106 CFU/ml). Data was statistically accessed by Student’s t-test (*p < 0.05).
Salmonella Typhimurium (Pie et al., 1997). Bacterial proliferation in the bloodstream induces a high inflammatory stimulus despite bacterial lipid-A, the main consequence being toxic shock usually due to accumulation of pro-inflammatory cytokines such TNF-␣ and IL-1 (Jones, 1996). We observed that LP attenuated signs of infection and at the higher dose tested protected animals of septic shock. LP did not exhibit direct effect on the bacteria and lost its protective effects when given 24 h after bacteria inoculums. Both observations suggested that LP fraction signalizes immunomodulatory responses instead of directly eliminate bacteria. It was observed that neither TNF-␣ nor IL-12 played a key role in the activation of the protective immune response in LP-treated mice against Salmonella Typhimurium (Fig. 5). This was reinforced by the high population of bacteria in the liver and spleen of the animals on days 1 and 5 after initiating infection, confirming that protection was not due to the increase in the bactericidal capacity of macrophages induced by these cytokines. However, IL-12 was slightly decreased after 5 days of infection in the experimental animals (p < 0.05). Since it is not reasonable to expect LP to remain present in the bloodstream for 28 days, it is likely that LP fraction acts by signaling cellular events involved in the control of inflammation induced by bacteria during infection, preventing septic shock and death of the animals. This suggestion is reinforced by our previously findings that
Fig. 6. Levels of TNF-␣ (A) and IL-1 (B) measured in supernatants of macrophage cultures previously treated with LP (500 g/ml) or RPMI 1 h before incubation with LPS (1 g/ml). Data were statistically accessed by ANOVA followed by Bonferrroni’s test (*p < 0.05).
intravenous injection of LP fraction of Calotropis procera inhibits neutrophil migration induced by dialyzable fraction of Calotropis procera (DL) or carrageenin to rat’s peritoneal cavities by inhibiting TNF-␣ release (Alencar et al., 2006). This effect should be similar to thalidomide, which down-regulates TNF-␣ production. However, in this study the measured serum levels of TNF-␣ were similar for both the control and experimental animals, showing this cytokine was less important on control of systemic inflammatory response syndrome (SIRS) on LP-treated mice (Fig. 5A). The drastic reduction of bacteria in bloodstream observed 24 h after the initial infection in animals receiving LP suggested that the protective pathway of LP was initiated in the initial stages of infection. As cited before, the belief that TNF-␣ was not directly involved in the protective effect of LP was also supported by the lost of the protective effect of LP when it was given 24 h after bacteria inoculums. Recently, another study reported that rats given LP intravenously exhibited increase of nitric oxide (NO) in serum, associated with strong anti-inflammatory activity (Ramos et al., 2009). NO is a multifunctional molecule which concentration and local of production interfere in its effects. As a result it is not certain that intraperitoneal injection of LP modulates NO in serum. More experimental evidence will be necessary to investigate this
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hypothesis. Despite the interesting protective effect of LP on infected mice as reported here, poor information was given on its protective mechanism. The full effectiveness of LP (60 mg/kg) in protecting mice by a single administration before experimental infection with Salmonella produced a number of issues to be considered in order to investigate the protective mechanism of the action. Since prevention of septic shock due to down-regulation of immune responses in LP-treated mice could involve others pro-inflammatory cytokines, the release of IL-1 and TNF-␣ was evaluated in macrophage culture supernatants pre-treated with LP and stimulated by LPS. As observed in the in vivo experiments, titration of TNF-␣ in culture of macrophages showed similar content in both the control and experimental cells after LPS stimulus. On the other hand, the level of soluble IL-1 was decreased in the supernatant of cells treated with LP (Fig. 6B). Mononuclear phagocytes stimulated by bacterial LPS and by other cytokines such as TNF-␣ are the major source of IL-1. This cytokine mediates host inflammatory responses to infections together with TNF-␣ in innate immunity (Jones, 1996; Cavaillon and Annane, 2006). In high amounts it can promote fever, synthesis of acute phase plasma proteins by the liver, and cachexia. However, IL-1 does not cause septic shock or apoptotic death of cells by itself. Despite this observation, in vivo modulation of this pro-inflammatory cytokine in LP-treated animals deserves additional approaches. It is also relevant to cite that the protective effect of LP was preserved when animals were treated orally. In this respect, many studies have already demonstrated anti-inflammatory activity of latex extracts of Calotropis procera (Kumar and Roy, 2007). The latex of Calotropis procera is introduced in folk medicine and used by different ways. It is topically applied to treat skin infections or teeth pain. We have found local usages to control diabetes by oral ingestion after diluting in water. However, there is a prevalence of use on inflammatory-related problems. In this case, the ethnopharmacological use of Calotropis procera does not deserve confirmation, since Kumar and co-workers have largely confirmed this folk usage by different experimental approaches and inflammatory models investigated (Kumar and Roy, 2009; Bharti et al., 2010). It is therefore important to advance in understanding mechanisms underlying anti-inflammation; identifying molecules involved and establishing safe use of latex. Here the potential of latex to treat infections was evaluated. Biochemical and functional studies of LP from Calotropis procera are currently under investigation. This material is a rich source of proteins involved in different metabolic pathways typically related to the plant metabolism, including defensive strategies against infections and predators. However, this protein fraction has become a relevant pharmacological tool due to the cumulative benefic properties recently described. At the present it is not possible to nominate or discuss putative proteins involved in the protective effects observed in this study. Despite such a limitation, the results described in this study appear as a relevant contribution to the search for new sources of biological compounds that could be useful in fighting bacterial infections and modulation of the immune responses. This work opens a new window of research since latex is found in many plants and offers now a new biological material to be investigated as a source of molecules capable of modulating immune responses. Furthermore, new approaches are currently being developed to investigate the involvement of NO in the protective effect of LP reported here. In this study, we report for the first time the protective role of the laticifer proteins of Calotropis procera in the control of a systemic murine infection caused by Salmonella enterica serovar Typhimurium. The protective activity of LP was not associated to the increase in the bactericidal capacity of macrophages and preliminary evidence suggested that the protective activity may be due
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to down-regulation of pro-inflammatory cytokines such IL-12 and IL-1 which prevented animals succumbing to toxic shock. Nevertheless, it is not clear yet the involvement of other inflammatory mediators and the effects of LP on NO in this model. The characterization of the biological properties, especially those cross-related to modulation of the immune system performed by latex proteins of Calotropis procera in the control of infections, is currently being investigated by this research group. Acknowledgements Biochemical, functional and applied studies of the latex from Calotropis procera have been supported by grants from FUNCAP, CNPq, CAPES, PADCT, RENORBIO and IFS (M.V.R.). The authors are in debt with Mr. Brian Stephen Curry who critically reviewed the language of the manuscript. References Alencar, N.M., Figueiredo, I.S., Vale, M.R., Bitencurt, F.S., Oliveira, J.S., Ribeiro, R.A., Ramos, M.V., 2004. Anti-inflammatory effect of the latex from Calotropis procera in three different experimental models: peritonitis, paw edema and hemorrhagic cystitis. Planta Medica 70, 1144–1149. Alencar, N.M., Oliveira, J.S., Mesquita, R.O., Lima, M.W., Vale, M.R., Etchells, J.P., Freitas, C.D.T., Ramos, M.V., 2006. Pro- and anti-inflammatory activities of the latex from Calotropis procera (Ait.) R.Br. are triggered by compounds fractionated by dialysis. Inflammation Research 55, 559–564. Arya, S., Kumar, V.L., 2005. Anti-inflammatory efficacy of extracts of latex of Calotropis procera against different mediators of inflammation. Mediators of Inflammation 2005, 228–232. Biedner, B., Rothkoff, L., Witztum, A., 1977. Calotropis procera (Sodom apple) latex keratoconjunctivitis. Israel Journal of Medical Science 13, 914–916. Bharti, S., Wahane, V.D., Kumar, V.L., 2010. Protective effect of Calotropis procera latex extracts on experimentally induced gastric ulcers in rats. Journal of Ethnopharmacology 127, 440–444. Cavaillon, J.M., Annane, D., 2006. Compartmentalization of the inflammatory response in sepsis and SIRS. Journal of Endotoxin Research 12, 151–170. Choedon, T., Mathan, G., Arya, S., Kumar, V.L., Kumar, V., 2006. Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma. World Journal Gastroenterology 12, 2517–2522. Cunha, F.Q., Ferreira, S.H., 1986. The release of a neutrophil chemotactic factor from peritoneal macrophage by endotoxin: inhibition by glucocorticoids. European Journal of Pharmacology 129, 65–76. El Badwi, Samia, M.A., Adam, S.E., Shigidi, M.T., Hapke, H.J., 1998. Studies on laticiferous plants: toxic effects in goats of Calotropis procera latex given by different routes of administration. Dtsch Tierarztl Wochenschr 105, 425–427. Fidan, I., Yesilyurt, E., Gurelik, F.C., Erdal, B., Imir, T., 2008. Effects of recombinant interferon-␥ on cytokine secretion from monocyte-derived macrophages infected with Salmonella typhi. Comparative Immunology and Microbiology Infectious Diseases 31, 467–475. Freeman, B.D., Natanson, C., 2000. Anti-inflammatory therapy in sepsis and septic shock. Expert Opinion on Investigational Drugs 9, 1651–1663. Groisman, E.A., Ochman, H., 1997. How Salmonella became a pathogen. Trends Microbiology 5, 343–349. Hess, J., Kaufmann, S.H., 1996. Salmonella enterica infection. Research Immunology 147, 581–586. John, B., Rajagopal, D., Pashine, A., Rath, S., George, A., Bal, V., 2002. Role of IL-12independent and IL-12-dependent pathways in regulating generation of the IFNgamma component of T cell responses to Salmonella typhimurium. Journal of Immunology 169, 2545–2552. Jones, B.D., 1996. Salmonellosis: host immune responses and bacterial virulence determinants. Annual Review of Immunology 14, 533–561. Koneman, E.W., Allen, S.D., Janda, W.M., 2001. Diagnóstico microbiológico-texto e atlas colorido, 5th ed. Médica e Científica, Rio de Janeiro, 1465 pp. Koyama, Y., Motobu, M., Hikosaka, K., Yamada, M., Nakamura, K., Saido-Sakanaka, H., Asaoka, A., Yamakawa, M., Sekikawa, K., Kitani, H., Shimura, K., Nakai, Y., Hirota, Y., 2006. Protective effects of antimicrobial peptides derived from the beetle Allomyrina dichotoma defensin on endotoxic shock in mice. International Immunopharmacology 6, 234–240. Kumar, V.L., Shivkar, Y.M., 2004. Involvement of prostaglandins in inflammation induced by latex of Calotropis procera. Mediators of Inflammation 13, 151–155. Kumar, V.L., Sehgal, R., 2007. Calotropis procera latex-induced inflammatory hyperalgesia – effect of bradyzide and morphine. Autonomic and Autacoid Pharmacology 27, 143–149. Kumar, V.L., Roy, S., 2007. Calotropis procera latex extract affords protection against inflammation and oxidative stress in Freund’s complete adjuvant-induced monoarthritis in rats. Mediators of Inflammation 2007, 47523. Kumar, V.L., Roy, S., 2009. Protective effect of latex of Calotropis procera in Freund’s Complete Adjuvant induced monoarthritis. Phytotherapy Research 23, 1–5.
334
J.V. Lima-Filho et al. / Journal of Ethnopharmacology 129 (2010) 327–334
Larhsini, M., Markouk, M., Jaouhari, J.T., Bekkouche, K., Lazrek, H.B., Jana, M., 2002. The antipyretic activity of some Moroccan medicinal plants. Phytotherapy Research 1, S97–S98. Mastroeni, P., Harrison, J.A., Chabalgoity, J.A., Hormaeche, C.E., 1996. Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid. Infection and Immunology 64, 189–196. Mastroeni, P., Ménager, N., 2003. Development of acquired immunity to Salmonella. Journal of Medical Microbiology 52, 453–459. Mossa, J.S., Tariq, M., Mohsin, A., Ageel, A.M., al-Yahya, M.A., al-Said, M.S., Rafatullah, S., 1991. Pharmacological studies on aerial parts of Calotropis procera. The American Journal of Chinese Medicine 19, 223–231. Motobu, M., Amer, S., Koyama, Y., Hikosaka, K., Sameshima, T., Yamada, M., Nakamura, K., Koge, K., Kang, C.B., Hayasidani, H., Hirota, Y., 2006. Protective effects of sugar cane extract on endotoxic shock in mice. Phytotherapy Research 20, 359–363. Nauciel, C., 1990. Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection. Journal of Immunology 145, 1265–1269. Oliveira, J.S., Bezerra, D.P., Freitas, C.D.T., Marinho-Filho, J.D.B., Moraes, O.M., Pessoa, C., Costa-Lotufo, L.V., Ramos, M.V., 2007. In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br. Toxicology In Vitro 21, 1563–1573. Pathak, A.K., Argal, A., 2007. Analgesic activity of Calotropis gigantea flower. Fitoterapia 78, 40–402. Pie, S., Truffa-Bachi, P., Pla, M., Nauciel, C., 1997. Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance. Infection and Immunity 65, 4509–4514. Portillo, F.G., 2001. Salmonella intracellular proliferation: where, when and how? Microbes and Infection 3, 1305–1311. Qureshi, A.A., Prakash, T., Patil, T., Viswanath-Swamy, A.H., Gouda, A.V., Prabhu, K., Setty, S.R., 2007. Hepatoprotective and antioxidant activities of flowers of Calotropis procera (Ait) R. Br. in CCl4 induced hepatic damage. Indian Journal of Experimental Biology 45, 304–310. Ramos, M.V., Oliveira, J.S., Figueiredo, J.G., Figueiredo, I.S., Kumar, V.L., Bitencourt, F.S., Cunha, F.Q., Oliveira, R.S.B., Bomfim, L.R., Lima-Filho, J.V., Alencar, N.M.N.,
2009. Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model. Journal of Ethnopharmacology 125, 387–392. Rasik, A.M., Raghubir, R., Gupta, A., Shukla, A., Dubey, M.P., Srivastava, S., Jain, H.K., Kulshrestha, D.K., 1999. Healing potential of Calotropis procera on dermal wounds in Guinea pigs. Journal of Ethnopharmacology 68, 261–266. Sangraula, H., Dewan, S., Kumar, V.L., 2002. Evaluation of anti-inflammatory activity of latex of Calotropis procera in different models of inflammation. Inflammopharmacology 9, 257–264. Sebastiani, G., Blais, V., Sancho, V., Vogel, S.N., Stevenson, M.M., Gros, P., Lapointe, J.M., Rivest, S., Malo, D., 2002. Host immune response to Salmonella enterica serovar Typhimurium infection in mice derived from wild strains. Infection and Immunity 70, 1997–2009. Shivkar, Y.M., Kumar, V.L., 2003. Histamine mediates the pro-inflammatory effect of latex of Calotropis procera in rats. Mediators of Inflammation 12, 299–302. Soares, P.M., Lima, S.R., Matos, S.G., Andrade, M.M., Patrocínio, M.C., Freitas, C.D.T., Ramos, M.V., Criddle, D.N., Cardi, B.A., Carvalho, K.M., Assreuy, A.M., Vasconcelos, S.M., 2005. Antinociceptive activity of Calotropis procera latex in mice. Journal of Ethnopharmacology 99, 125–129. Van Dien, M., Takahashi, K., Mu, M.M., Koide, N., Sugiyama, T., Mori, I., Yoshida, T., Yokochi, T., 2001. Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine sensitized mice. Microbiology and Immunology 45, 751–756. Vasquez-Torres, A., Fantuzzi, G., Edwards, C.K., Dinarello, C.A., Fang, F.C., 2001. Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagossomes in tumor necrosis factor p55 receptor-deficient macrophages. Proceedings of National Academy of Sciences of USA 98, 2561–2565. Wang, X.M., Liang, M.F., Yuan, Y., Jiang, W., 2008. The different effects of glutamine on macrophage cytokines release in vivo and in vitro. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 20, 456–460. Withanage, G.S.K., Wigley, P., Kaiser, P., Mastroeni, P., Brooks, H., Powers, C., Beal, R., Barrow, P., Maskell, D., McConnell, I., 2005. Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infection and Immunity 73, 5173–5182.