Spondias mombin L. attenuates ventricular remodelling after myocardial infarction associated with oxidative stress and inflammatory modulation.

The objective of this study was to evaluate Spondias mombin L. (SM) pulp and its influence on cardiac remodelling after myocardial infarction (MI). Male Wistar rats were assigned to four groups: a sham group (animals underwent simulated surgery) that received standard chow (S; n = 20), an infarcted group that received standard chow (MI; n = 24), an infarcted group supplemented with 100 mg of SM/kg bodyweight/d, (MIS100; n = 23) and an infarcted group supplemented with 250 mg of SM/kg bodyweight/d (MIS250; n = 22). After 3 months of treatment, morphological, functional and biochemical analyses were performed. MI induced structural and functional changes in the left ventricle with worsening systolic and diastolic function, and SM supplementation at different doses did not influence these variables as analysed by echocardiography and an isolated heart study (P > .05). However, SM supplementation attenuated cardiac remodelling after MI, reducing fibrosis (P = .047) and hypertrophy (P = .006). Biomarkers of oxidative stress, inflammatory processes and energy metabolism were further investigated in the myocardial tissue. SM supplementation improved the efficiency of energy metabolism and decreased lipid hydroperoxide in the myocardium [group S (n = 8): 267.26 ± 20.7; group MI (n = 8): 330.14 ± 47.3; group MIS100 (n = 8): 313.8 ± 46.2; group MIS250: 294.3 ± 38.0 nmol/mg tissue; P = .032], as well as decreased the activation of the inflammatory pathway after MI. In conclusion, SM supplementation attenuated cardiac remodelling processes after MI. We also found that energy metabolism, oxidative stress and inflammation are associated with this effect. In addition, SM supplementation at the highest dose is more effective.

Protein Carbonyl, But Not Malondialdehyde, Is Associated With ICU Mortality in Patients With Septic Shock.

BACKGROUND: The objective of our study was to evaluate the association of serum malondialdehyde (MDA) and protein carbonyl concentration with intensive care unit (ICU) mortality in patients with septic shock. METHODS: We prospectively evaluated 175 patients aged over 18 years with septic shock upon ICU admission. However, 16 patients were excluded. Thus, 159 patients were enrolled in the study. In addition, we evaluated 16 control patients. At the time of the patients' enrollment, demographic information was recorded. Blood samples were taken within the first 24 hours of the patient's admission to determine serum MDA and protein carbonyl concentrations. RESULTS: The mean age was 67.3 ± 15.9 years, 44% were males, and the ICU mortality rate was 67.9%. Median MDA concentration was 1.53 (0.83-2.22) µmol/L, and median protein carbonyl concentration was 24.0 (12.7-32.8) nmol/mL. Patients who died during ICU stay had higher protein carbonyl concentration. However, there was no difference in MDA levels between these patients. Receiver operating characteristic curve analysis showed that higher levels of protein carbonyl were associated with ICU mortality (area under the curve: 0.955; 95% confidence interval [CI]: 0.918-0.992; P < .001) at the cutoff of >22.83 nmol/mL (sensibility: 80.4% and specificity: 98.1%). In the logistic regression models, protein carbonyl concentrations (odds ratio [OR]: 1.424; 95% CI: 1.268-1.600; P < .001), but not MDA concentrations (OR: 1.087; 95% CI: 0.805-1.467; P = .59), were associated with ICU mortality when adjusted for age, gender, and Acute Physiology and Chronic Health Evaluation (APACHE) II score; and when adjusted by APACHE II score, lactate, and urea; protein carbonyl concentrations (OR: 1.394; 95% CI: 1.242-1.564; P < .001); and MDA (OR: 1.054; 95% CI: 0.776-1.432; P = .73). CONCLUSION: In conclusion, protein carbonyl, but not MDA, concentration is associated with ICU mortality in patients with septic shock.

Lipid damage is the best marker of oxidative injury during the cardiac remodeling process induced by tobacco smoke.

BACKGROUND: Oxidative stress is one potential mechanism that explain the direct effects of smoking on cardiac remodeling process. However, no study has compared different myocardial products of macromolecule oxidation after tobacco smoke exposure. Thus, the aim of this study was to investigate the lipid hydroperoxide (LH) levels, protein carbonyl concentrations and DNA damage in cardiac tissue of rats exposed to tobacco smoke. METHODS: Male Wistar rats were divided into two groups: group C (control, n = 14) composed of animals not exposed to cigarette smoke; group ETS (exposed to tobacco smoke, n = 14) composed by animals exposed to cigarette smoke. The animals were exposed to 2 month of ETS and morphological, biochemical and functional analyses were performed. RESULTS: Cardiac cotinine levels were elevated in the ETS group. In addition, the myocyte cross-sectional area was higher in the ETS group. (C = 266.6 ± 23.2 µm2 and ETS = 347.5 ± 15.1 µm2, p <  0.001). Cardiac LH was higher in the ETS group than in group C (C = 196.4 ± 51.5 nmol/g and ETS = 331.9 ± 52.9 nmol/g, p <  0.001). However, there were no between-group differences in cardiac protein carbonyl concentration or DNA damage. CONCLUSIONS: Therefore, our results suggest that, in this model, lipid damage is a good marker of oxidative damage during the cardiac remodeling process induced by 2 months of exposure to tobacco smoke.

Spondias mombin supplementation attenuated cardiac remodelling process induced by tobacco smoke.

The objective of this study was to investigate the influence of Spondias mombin (SM) supplementation on the cardiac remodelling process induced by exposure to tobacco smoke (ETS) in rats. Male Wistar rats were divided into 4 groups: group C (control, n = 20) comprised animals not exposed to cigarette smoke and received standard chow; group ETS (n = 20) comprised animals exposed to cigarette smoke and received standard chow; group ETS100 (n = 20) received standard chow supplemented with 100 mg/kg body weight/d of SM; and group ETS250 (n = 20) received standard chow supplemented with 250 mg/kg body weight/d of SM. The observation period was 2 months. The ETS animals had higher values of left cardiac chamber diameters and of left ventricular mass index. SM supplementation attenuated these changes. In addition, the myocyte cross-sectional area (CSA) was lower in group C compared with the ETS groups; however, the ETS250 group had lower values of CSA compared with the ETS group. The ETS group also showed higher cardiac levels of lipid hydroperoxide (LH) compared with group C; and, groups ETS100 and ETS250 had lower concentrations of LH compared with the ETS group. Regarding energy metabolism, SM supplementation decreased glycolysis and increased the ß-oxidation and the oxidative phosphorylation. There were no differences in the expression of Nrf-2, SIRT-1, NF-κB, interferon-gamma and interleukin 10. In conclusion, our results suggest that ETS induced the cardiac remodelling process. In addition, SM supplementation attenuated this process, along with oxidative stress reduction and energy metabolism modulation.

Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: Role on extracellular killing.

Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.

Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro.

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.

Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages.

Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.

Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis.

Paracoccidiodomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), which is endemic in Latin America. The host innate immune response against the fungus has been well characterized and several studies have shown the important role played by phagocytic cells. Our laboratory has studied the relationship between human neutrophils (PMNs)/Pb, focusing the effector mechanisms of these cells against the fungus. However, in last years, studies have shown that in addition to their phagocytic and killer functions, PMNs can modulate and instruct the immune response, since these cells have been shown to produce and release several cytokines. Thus, we evaluated whether PMNs stimulated with Pb can modulate the immune response to a Th1 phenotype through the production of IFN-γ, as well as the role of "pattern-recognition receptors" (PRRs) such as TLR2, TLR4 and Dectin-1 in this production. Furthermore, we asked whether activation of the cells with the cytokines IL-12, IL-15 and IL-18 could result in increased levels of this cytokine. Peripheral blood PMNs obtained from 20 healthy donors were nonactivated or activated with IL-12, IL-15 or IL-18 in different concentrations and challenged with strain 18 Pb (Pb18) for 2 h, 4 h, 12 h, 24 h and 48 h and evaluated for IFN-γ production, by ELISA. In other experiments, PMNs were treated with monoclonal antibodies anti-TLR2, TLR4 and Dectin-1, challenged with Pb and evaluated for IFN-γ production. We found that Pb induces human PMNs to produce IFN-γ, probably by binding to TLR4 and Dectin-1 receptors expressed by these cells. Moreover, IFN-γ levels were significantly increased when cells were activated with each of the tested cytokines or a combination of two of them, being the association IL-12 plus IL-15 the most effective. The results support our hypothesis that during infection by Pb, human PMNs modulate the adaptive immune response to a Th1 response pattern, via IFN-γ production.

Clove and eugenol in noncytotoxic concentrations exert immunomodulatory/anti-inflammatory action on cytokine production by murine macrophages.

OBJECTIVES: The extract and essential oil of clove (Syzygium aromaticum) are widely used because of their medicinal properties. Eugenol is the most important component of clove, showing several biological properties. Herein we have analysed the immunomodulatory/anti-inflammatory effect of clove and eugenol on cytokine production (interleukin (IL)-1ß, IL-6 and IL-10) in vitro. METHODS: Macrophages were incubated with clove or eugenol (5, 10, 25, 50 or 100µg/well) for 24h. Concentrations that inhibited the production of cytokines were used before or after incubation with lipopolysaccharide (LPS), to verify a preventive or therapeutic effect. Culture supernatants were harvested for measurement of cytokines by enzyme-linked immunosorbent assay. KEY FINDINGS: Clove (100µg/well) inhibited IL-1ß, IL-6 and IL-10 production and exerted an efficient action either before or after LPS challenge for all cytokines. Eugenol did not affect IL-1ß production but inhibited IL-6 and IL-10 production. The action of eugenol (50 or 100µg/well) on IL-6 production prevented efficiently effects of LPS either before or after its addition, whereas on IL-10 production it counteracted significantly LPS action when added after LPS incubation. CONCLUSIONS: Clove exerted immunomodulatory/anti-inflammatory effects by inhibiting LPS action. A possible mechanism of action probably involved the suppression of the nuclear factor-κB pathway by eugenol, since it was the major compound found in clove extract.

Lemongrass and citral effect on cytokines production by murine macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (DC) Stapf (Poaceae-Gramineae), an herb commonly known as lemongrass (LG), is an important source of ethnomedicines as well as citral, the major constituent of Cymbopogon citratus, used in perfumery, cosmetic and pharmaceutical industries for controlling pathogens. Thus, the goal of this work was to analyze the effect of LG and citral on cytokines production (IL-1ß, IL-6 and IL-10) in vitro, as well as before or after LPS incubation. MATERIALS AND METHODS: Peritoneal macrophages from BALB/c mice were treated with LG or citral in different concentrations for 24h. The concentrations that inhibited cytokines production were tested before or after macrophages challenge with LPS, in order to evaluate a possible anti-inflammatory action. Supernatants of cell cultures were used for cytokines determination by ELISA. RESULTS: As to IL-1ß, only citral inhibited its release, exerting an efficient action before LPS challenge. LG and citral inhibited IL-6 release. Cymbopogon citratus showed inhibitory effects only after LPS challenge, whereas citral prevented efficiently LPS effects before and after LPS addition. Citral inhibited IL-10 production and although LG did not inhibit its production, the concentration of 100 µg/well was tested in the LPS-challenge protocol, because it inhibited IL-6 production. LG inhibited LPS action after macrophages incubation with LPS, while citral counteracted LPS action when added before or after LPS incubation. CONCLUSION: LG exerted an anti-inflammatory action and citral may be involved in its inhibitory effects on cytokines production. We suggest that a possible mechanism involved in such results could be the inhibition of the transcription factor NF-κB.

Th1/Th2 cytokine production by clove-treated mice.

Although clove possesses several biological and therapeutic properties, its immunomodulatory action has not been fully investigated. The goal of this work was to investigate the effect of administration of the water extract of clove over a short-term to BALB/c mice on Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokine production. After treatment, spleen cells were aseptically removed and cells were stimulated with concanavalin A. Supernatants of cell cultures were used for cytokine determination by ELISA. The chemical composition of the extract was also carried out, revealing that eugenol(4-allyl-2-methoxyphenol) was the major component in our sample. Although the anti-inflammatory action of clove has been mentioned, our data showed that clove administration to mice did not influence the Th1/Th2 cytokine balance. Further studies dealing with cytokine expression and production will provide a better understanding of clove's immunomodulatory and anti-inflammatory actions, using different extract concentrations and different intake periods.

Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice.

INTRODUCTION: Propolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1beta and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells. METHODS: BALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator. RESULTS: Stressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1beta and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1beta and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress. CONCLUSION: Propolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions.

Propolis effect on Th1/Th2 cytokines production by acutely stressed mice.

AIM OF THE STUDY: Propolis has gained special attention due to its biological properties, however, little is known about its immunomodulatory effects in stress conditions. The purpose of this study was to investigate propolis effect on Th1/Th2 cytokines production by spleen cells of acutely stressed mice. Serum corticosterone concentration was determined as a stress indicator. MATERIALS AND METHODS: Male BALB/c mice were submitted to restraint stress and treated with propolis (200mg/kg) for 3 days. Supernatants of splenocytes cultures were assessed for Th1/Th2 cytokines determination. RESULTS: Regarding Th1 cytokines production, no alterations were seen in IL-2 production; however, IFN-gamma production was inhibited in stressed mice, even when treated with propolis. As to Th2 cytokines, IL-4 was inhibited in stressed mice, but normal levels were seen when these animals were treated with propolis. No significant differences were found in IL-10 production between the experimental groups. Stressed groups (treated or not with propolis) showed higher corticosterone concentrations in comparison to control group. CONCLUSIONS: Data suggest that propolis treatment was not able to counteract the stress-induced immunosuppressive effect on IFN-gamma production; however, propolis showed an immunorestorative role, increasing IL-4 production in stressed mice, favoring humoral immune response during stress. Since the exact mechanisms of this natural product on immune system are still unclear, further studies are still required for a better comprehension of propolis use as a therapeutic alternative against the stress-induced negative effects that could lead to the development of various diseases.