Rat Neutrophils Research Articles

Calcium-Sensing Receptor on Neutrophil Promotes Myocardial Apoptosis and Fibrosis After Acute Myocardial Infarction via NLRP3 Inflammasome Activation

BackgroundThe infiltration of neutrophils aggravates inflammatory response in acute myocardial infarction (AMI), and the role of calcium-sensing receptor (CaSR) in neutrophil-associated inflammation is largely unknown. The aim of this study was to evaluate the regulatory effects of CaSR on nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome in neutrophils and to explore its role in AMI-related ventricular remodelling. MethodsThe expression of CaSR, NLRP3 inflammasome, and interleukin 1β (IL-1β) in peripheral blood and infiltrating neutrophils in patients and rats with AMI was detected by western blotting and immunofluorescence. Cardiomyocyte apoptosis was detected by western blotting and transmission electron microscopy. The degree of fibrosis was evaluated by Masson staining and western blotting. ResultsWe found upregulation of CaSR, NLRP3 inflammasome, Caspase-1, and IL-1β in peripheral neutrophils from patients with AMI compared with matched healthy controls, peaking on day 1 and decreasing gradually till 7 days. Peripheral and infiltrating neutrophils from rats with AMI showed the same trend. Calindol enhanced NLRP3 inflammasome activation and IL-1β release in neutrophils from healthy volunteers, which was blocked by inhibitors of the PLC-IP3 pathway and ER-Ca2+ release. Calhex-231 decreased NLRP3 inflammasome activation and IL-1β release in neutrophils from patients with AMI. The calindol-stimulated neutrophils from healthy rats promoted cardiomyocyte apoptosis and fibrosis of cardiac fibroblasts from healthy rats, which were inhibited by calhex-231. ConclusionThe results suggest that CaSR activates NLRP3 inflammasome in neutrophils, contributing to ventricular remodelling after AMI. CaSR inhibition may be a potential therapeutic target for heart failure in AMI.

Abstract 3905: Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor

Abstract Protein arginine methyltransferase 5 (PRMT5) is an epigenetic enzyme with oncogenic properties. JNJ-64619178 (JNJ178) is a potent, selective, structurally unique PRMT5 inhibitor with good preclinical efficacy in inhibiting the growth of hematologic and solid tumor cell lines. Toxicology studies showed that JNJ178 decreased reticulocytes and neutrophils in rats and dogs. The objectives of translational modeling and simulation were to understand the exposure-response relationship of both safety and efficacy and provide guidance to the first-in-human clinical development of JNJ178. Experimental data for the PK/PD (Pharmacokinetics/Pharmacodynamics) modeling included: plasma concentration after single dose of JNJ178 in non-tumor bearing mice, plasma concentration and PD markers of dimethylation of arginine (%SDMA in plasma and %SMD1/3-Me2 in tumor, respectively) after multiple doses (1 to 10 mg/kg) QD (once daily) of JNJ178 in H1048 (human small cell lung carcinoma) xenografts, and tumor volume in four xenograft mouse models (A427, human lung carcinoma; H441, human lung adenocarcinoma; H520, human squamous cell lung carcinoma; and H1048). Plasma PK were first described by a standard two-compartment model and used as a driver of PD and tumor volume (efficacy). Plasma and tumor PD were modeled using an indirect response model. A hybrid tumor growth coupled with transit compartment mediated tumor killing model was used to fit the tumor volume data. To predict the safety profile of JNJ178, lifespan based indirect response model for erythropoiesis and Friberg myelosuppression model were used to simulate hemoglobin and neutrophil kinetics in human. The PK/PD model described the data well and validated the hypothesis that PD is driven by trough concentration. Based on the exposure-response relationship from the four xenograft models, the trough concentration needed to achieve tumor stasis for mouse was determined. In addition, the level of inhibition in tumor and plasma PD marker that was associated with tumor stasis was identified. Together with human PK parameters scaled using allometry, the dose range needed to achieve target therapeutic exposure for a typical human subject was predicted. Simulation results from erythropoiesis and Friberg myelosuppression models informed the optimal doing schedules for certain dose levels that would allow hematological toxicity to be manageable with <40% reduction in hemoglobin and >1.0 x 109/L neutrophil counts at all times. Overall, a translational modeling and simulation approach that considers safety and efficacy has been instrumental in the design of the first-in-human clinical development of PRMT5 inhibitor JNJ178 regarding selection of dose and schedule. Citation Format: Yue Guo, Nahor Haddish-Berhane, Hillary J. Millar, Tinne Verhulst, Tony Greway, Junguo Zhou, Loeckie DeZwart, Dana Gaffney, Joseph Portale, Dirk Brehmer, An Boeckx, Erika Van Heerde, Daniele Ouellet. Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3905.

Gender-Specific Differences of Oxidative Processes in the Population of Circulating Neutrophils of Rats in a Setting of Prolonged Administration of Monosodium Glutamate

AbstractBackground and aims:Monosodium salt of glutamic acid (MSG) is one of the most common food additives. The aim of study was to assess, in gender-specific terms, how prolonged administration of MSG effects on reactive oxygen and nitrogen species formation and the apoptotic/necrotic processes in the population of rats circulating neutrophils.Material and methods:Experimental studies were conducted on 32 mature white rats. MSG was administered intragastrical at a dose of 30 mg/kg body weight for 30 days. The analysis of cell samples to determine neutrophils with overproduction of reactive oxygen species (ROS) and signs of apoptosis\necrosis was evaluated with flow laser cytometry method. The total nitric oxide synthase (NOS) activity was determined by monitoring the rate of conversion of L-arginine into citrulline. The total quantity of NO metabolites was assessed by evaluating of nitrite and nitrate ions.Results:We found a significant increase in generation of ROS, intensification of nitroxydergic processes, an increase in the percentage of apoptotic neutrophils and no changes in the percentage of necrotic neutrophils.Conclusions:We observed activation of oxidative and nitroxydergic processes in rats with prolonged administration of MSG, which initiate apoptosis. In gender-specific terms, a more pronounced changes were seen in male rats.

LXA4 ameliorates cerebrovascular endothelial dysfunction by reducing acute inflammation after subarachnoid hemorrhage in rats

Lipoxin A4 (LXA4) has been reported to reduce inflammation in experimental subarachnoid hemorrhage (SAH), but the mechanism remains unclear. In this study, we investigated the role of LXA4 in inflammation-mediated cerebrovascular endothelial dysfunction and the potential mechanism after SAH. SAH was induced by endovascular perforation in male Sprague–Dawley rats, and recombinant LXA4 was injected intracerebroventricularly 1.5 h after the operation. The expression changes in the markers of endothelial dysfunction (endothelial microparticles and nitric oxide) were analyzed by flow cytometry or Nitric Oxide (NO) assay kit. Microflow in the cerebral cortex was assayed by laser speckle contrast imaging. Neutrophil infiltration was observed by a marker of leukocyte activity (myeloperoxidase, MPO) that colocalized with a specific marker of endothelial cells (von Willebrand factor, VWF). The expression of LXA4 and its downstream molecules, formyl peptide receptor 2 (FPR2), extracellular signal-regulated kinase (ERK1/2), nuclear factor-κB (NF-κB), matrix metalloproteinase-9 (MMP9), and the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), leukocyte adhesion molecule (intercellular adhesion molecule-1, ICAM-1) and MPO were measured either by Western blot or enzyme-linked immunosorbent assay (ELISA). SAH resulted in endothelial dysfunction and a reduction in microflow in the cerebral cortex. The expression of LXA4 was decreased, and the expression of pro-inflammatory factors (NF-κB, MMP9, ICAM-1, MPO) and cytokines (TNF-α, IL-1β, IL-6) was increased after SAH. The administration of LXA4 significantly ameliorated endothelial dysfunction, recovered microflow, and suppressed the inflammation and infiltration of neutrophils in SAH rats. The underlying mechanism of this outcome may involve the LXA4/FPR2/ERK1/2 pathway. LXA4 might be a promising candidate for acute SAH treatment.

Functional Activity of Circulating Phagocytes as Potential Pretreatment Marker of Tumor Drug Resistance

The aim of this work was a study of the functional activity of neutrophils and peripheral blood monocytes in rats with the transplanted Walker carcinosarcoma as potential predictors of the sensitivity of this tumor to doxorubicin treatment. This study provides an evidence that such indices of the functional activity of circulating phagocytes of the tumor-bearing rats as the quantity and the phagocytosis intensity of monocytes, as well as the intensity of ROS production by monocytes and neutrophils, may reflect the degree of sensitivity of the tumor to doxorubicin. So it was shown that the growth of the resistant tumor caused a significant increase of the number of circulating phagocytic cells and the intensity of phagocytosis by more than 100% (p < 0.001) compared with the corresponding indices of intact rats and rats with the parental variant of the tumor. The ability of blood mono-cytes and neutrophils of rats with a resistant tumor to produce ROS was also significantly different from that in intact rats and animals with the parental carcinosarcoma variant. The predictive value of these indices is especially important in the dynamic monitoring of the development of tumor drug resistance during long-term cancer chemotherapy. Considering the standard 2 - 3 week interval between the courses of cancer therapy and the short lifetime of circulating phagocytes, an assessment of the indices of their functional activity before each subsequent course can be considered as a pretreatment assessment. Meanwhile, further studies are needed to determine the spectrum of malignant neoplasms for which the degree of tumor drug resistance correlates with the functional activity of circulating phagocytes.

Effect of patchouli alcohol on Helicobacter pylori-induced neutrophil recruitment and activation

Neutrophil infiltration typically occurs in Helicobacter pylori (H. pylori)-induced acute gastritis; however, this immune response fails to eradicate H. pylori in vivo. Moreover, reactive oxygen species (ROS), which are generated by neutrophils, cause severe damage to gastric mucosa. Patchouli alcohol (PA) has been reported to have effective anti-oxidative and anti-H. pylori activities, and we investigated its effects on H. pylori-induced neutrophil recruitment and activation in this research. In neutrophil recruitment experiment, H. pylori was injected into rat air pouch to explore the effects of PA (10, 20 and 40 mg/kg) on acute inflammatory response. The results revealed that PA significantly reduced the weight of exudate and the number of neutrophils in the air pouch. Meanwhile, remarkable decrements in TNF-α and IL-8 levels in exudates were observed. In neutrophil activation experiment, rat neutrophils were isolated and activated by using 50 μg/mL H. pylori water-soluble surface protein with or without the treatment of PA (5, 10 or 20 μmol/L). Results indicated that PA not only significantly inhibited the production of ROS, but also reduced the gene and protein expressions of p22/p47-phoxes, and the binding of p22/p47-phoxes. Furthermore, the influence of PA on the neutrophil activation genes of H. pylori (h-nap and sabA) was investigated, and the results showed that expressions of h-nap and sabA were remarkably decreased after PA treatment. In conclusion, PA reduced the recruitment and activation of neutrophils induced by H. pylori, as shown by its inhibition of pro-inflammatory factor generation, p22/p47-phoxes function and H. pylori neutrophil activation-related gene expression.

Rutin and curcumin reduce inflammation, triglyceride levels and ADA activity in serum and immune cells in a model of hyperlipidemia

Hyperlipidemia is associated with endothelial dysfunction and inflammatory disorders. Adenosine and adenosine deaminase (ADA) modulate immune responses and lipid metabolism. Curcumin and rutin are polyphenols with antioxidant, anti-inflammatory, and hypolipidemic effects. We evaluated the action of rutin and curcumin in the lipid levels and inflammation, as well as their effect on ADA activity in serum, lymphocytes, platelets, and neutrophils of hyperlipidemic rats. Adult male Wistar rats pretreated with curcumin and/or rutin for 30 days were submitted to Poloxamer-407- induced hyperlipidemia. Biochemical, hematological, and oxidative stress parameters, as well as serum and extracellular ADA activity, were performed 36h post-induction. Hyperlipidemia was confirmed by the increase in total cholesterol (TC) and triglycerides (TG). Hematological alterations, elevated reactive oxygen species (ROS) levels, and increased myeloperoxidase (MPO) and ADA activities were observed in hyperlipidemic rats. Curcumin and the curcumin/rutin association decreased TG and increased high-density lipids (HDL) levels. The pretreatments prevented changes in the hematological parameters, decreased the activities of MPO in plasma and ADA in serum and cells. Cholesterol and ROS levels were not altered by the pretreatments. Our results show that pretreatments with rutin and/or curcumin prevent the hyperlipidemia-induced inflammation. Pretreatments with curcumin and/or rutin are potential complementary therapies in the prevention of hypertriglyceridemia and inflammation.

Obesity and Type 2 Diabetes mellitus induce lipopolysaccharide tolerance in rat neutrophils

Obesity and diabetes implicate in various health complications and increased mortality caused by infection. Innate immune system is broadly affected by these diseases, leading the patients to an immunosuppressive state. A mechanism that leads innate immune cells to a less capacity of killing microorganism is the impaired TLR4 activation. TLR4 recognizes a component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS), and when activated increases the production of inflammatory substances. Neutrophils are components of the innate immune system and are the first responders to an invading agent. The correct activation of TLR4 in these cells is required for the initiation of the inflammatory process and elimination of the microorganisms. The aim of this study was to evaluate the influence of type 2 diabetes and obesity in the TLR4 pathway in rat neutrophils. Two experimental models were used: Goto-Kakizaki rats and high-fat-diet induced obese Wistar rats. To evaluate neutrophil response to LPS, intratracheal LPS instillation was used. Neutrophils from obese and diabetic animals exhibited tolerance to LPS, mainly by the impaired production of cytokines and chemokines and the low content of phospho-NFκB and phospho-IKBα. Neutrophils from both experimental models had increased cell death, impaired in vivo migration and myeloperoxidase activity.

Analysis of the Effect of Intestinal Ischemia and Reperfusion on the Rat Neutrophils Proteome.

Intestinal ischemia and reperfusion injury is a model system of possible consequences of severe trauma and surgery, which might result into tissue dysfunction and organ failure. Neutrophils contribute to the injuries preceded by ischemia and reperfusion. However, the mechanisms by which intestinal ischemia and reperfusion stimulate and activate circulating neutrophils is still not clear. In this work, we used proteomics approach to explore the underlying regulated mechanisms in Wistar rat neutrophils after ischemia and reperfusion. We isolated neutrophils from three different biological groups; control, sham laparotomy, and intestinal ischemia/reperfusion. In the workflow, we included iTRAQ-labeling quantification and peptide fractionation using HILIC prior to LC-MS/MS analysis. From proteomic analysis, we identified 2,045 proteins in total that were grouped into five different clusters based on their regulation trend between the experimental groups. A total of 417 proteins were found as significantly regulated in at least one of the analyzed conditions. Interestingly, the enzyme prediction analysis revealed that ischemia/reperfusion significantly reduced the relative abundance of most of the antioxidant and pro-survival molecules to cause more tissue damage and ROS production whereas some of the significantly up regulated enzymes were involved in cytoskeletal rearrangement, adhesion and migration. Clusters based KEGG pathways analysis revealed high motility, phagocytosis, directional migration, and activation of the cytoskeletal machinery in neutrophils after ischemia and reperfusion. Increased ROS production and decreased phagocytosis were experimentally validated by microscopy assays. Taken together, our findings provide a characterization of the rat neutrophil response to intestinal ischemia and reperfusion and the possible mechanisms involved in the tissue injury by neutrophils after intestinal ischemia and reperfusion.

Transplantation of Endothelial Progenitor Cells Attenuates Lipopolysaccharide-Induced Lung Injury via Inhibiting the Inflammatory Secretion of Neutrophils in Rats

BackgroundEndothelial progenitor cells (EPCs) are special types of stem cells and are a potential novel therapeutic approach in acute lung injury (ALI). Transplantation of EPCs can ameliorate the inflammatory state by reducing adhesion and exudation of inflammatory cells. However, the mechanism underlying the effect of EPCs on inflammatory response modulation remains unclear. The aim of the present study was to investigate the effect of EPCs on the modulation of neutrophils in vitro and in vivo. Materials and MethodsEPCs were cocultured with neutrophils after lipopolysaccharide stimulation in vitro or transplanted into ALI rats, and neutrophil inflammatory mediators including tumor necrosis factor-α, interleukin-1β, neutrophil elastase, myeloperoxidase and matrix metalloproteinases-9 were detected by enzyme-linked immunosorbent assay, an myeloperoxidase detection kits, reverse transcription-polymerase chain reaction and western blotting. ResultsThe results showed that EPCs significantly downregulated the expression of inflammatory mediators when cocultured with neutrophils in vitro or in vivo. ConclusionsThese findings demonstrated that EPCs contributed to lung injury in ALI rats by downregulating neutrophil inflammatory mediators.

The Heme Transporter HtsABC of Group A Streptococcus Contributes to Virulence and Innate Immune Evasion in Murine Skin Infections.

Group A Streptococcus (GAS) requires iron for growth, and heme is an important source of iron for GAS. Streptococcus heme transporter A (HtsA) is the lipoprotein component of the GAS heme-specific ABC transporter (HtsABC). The objective of this study is to examine the contribution of HtsABC to virulence and host interaction of hypervirulent M1T1 GAS using an isogenic htsA deletion mutant (ΔhtsA). The htsA deletion exhibited a significantly increased survival rate, reduced skin lesion size, and reduced systemic GAS dissemination in comparison to the wild type strain. The htsA deletion also decreased the GAS adhesion rate to Hep-2 cells, the survival in human blood and rat neutrophils, and increased the production of cytokine IL-1β, IL-6, and TNF-α levels in air pouch exudate of a mouse model of subcutaneous infection. Complementation of ΔhtsA restored the wild type phenotype. These findings support that the htsA gene is required for GAS virulence and that the htsA deletion augments host innate immune responses.

P-176 - Redox signaling processes in neutrophils are modified in monocrotaline-induced pulmonary arterial hypertension

The mechanisms of reactive oxygen species (ROS) generation in neutrophils in monocrotoline-induced pulmonary arterial hypertension (MCT-PAH) were studied. MCT was injected subcutaneously to rats (60 mg/kg). FMLP-stimulated ROS generation and myeloperoxidase (MPO) secretion from neutrophils was measured using chemiluminescence. The involvement of the signal pathway components in the redox activation of cells was assessed using specific inhibitors of MPO, NADPH oxidase, phosphatidylinositol 3-kinase (PI3K) and cyclooxygenase (CO). It has been shown that initially (up to 28 days of MCT-PAH) ROS generation in rat neutrophils was decreased, which is associated with the decrease of NADPH oxidase activity and the increase of MPO secretion into the extracellular medium. However, at the last stage of MCT-PAH (60 days), ROS production was increased, which is due to MPO and NADPH oxidase activation and the decrease of CO and PI3K pathways contribution. To sum up, the intracellular signaling pathways involved in ROS generation in neutrophils are changing in depends of the pulmonary arterial hypertension stage.

JUS SEMANGKA MENURUNKAN NEUTROFIL TIKUS JANTAN GALUR WISTAR YANG TERPAPAR ASAP ROKOK

Cigarette smoke contains many hazardous substances and free radical which affect respiratory system. Watermelon “Sugar Baby” juice contain lycopene as antiinfl ammation. The aim of this research was to analyze the effect of watermelon as antiinfl ammation toward neutrophils count in wistar strain male rats exposed by cigarette smoke. This study was an experimental laboratory research with pre and post test control group design. Sample consisted of 24 wistar strain male rats with ±150-230 grams weight and 3 months old which were divided into four groups. Control group was given placebo (aquadest) and treatment groups were given w atermelon juice with terraced quantity (1.5 ml, 2.5 ml, and 3.5 ml). Every group was given two sticks of cigarette smoked per day during two weeks then smoked with treatment during one week. Neutrophil from peripheral smear was observed by manual method and statistically analyzed using Anova Mixed Design and continued with One Way Anova. The result showed signifi cant increased of neutrophil at second observation on control group (p=0.002) and treatment groups (p=0.000). Signifi cance decreased also found in the treatment groups’s at third observation p=0.000. Signifi cant between each group p= 0.017. There was signifi cant different between control and treatment groups but there was no signifi cant different between each treatment groups. This study concluded that there was decreased of neutrophil in wistar strain male rats which were given w atermelon juice 1.5 ml, 2.5 ml, and 3.5 ml during infl ammation. 3.5 ml w atermelon juice which were given showed better result as antiinfl ammation..

Codonopsis lanceolata extract prevents hypertension in rats

BackgroundCodonopsis lanceolata, a plant with antioxidant, anti-cancer, anti-inflammatory and blood lipid improving effects, has been widely used as a therapeutic agent in traditional medicine. PurposeThe present study investigated the ability of an ethanol extract of Codonopsis lanceolata (ECL) to prevent hypertension in hypertensive rats. MethodsRats were orally administered daily doses of 0 mg/kg, 200 mg/kg and 400 mg/kg ECL for 3 weeks. As a positive control, rats were orally administered 10 mg/kg/day nifedipine. Hypertension was induced by immobilization stress for 2 h/day and by administration of 0.8 mg/kg/day nicotine for 3 weeks, followed by injection of 3 mg/kg nicotine on the day of sacrifice. Blood pressure and heart rate were measured using a volume pressure recording system. Vasoconstriction and vasodilation of aortic cross sections were measured with a physiological recorder. Neutrophil counts in bronchoalveolar lavage fluid were estimated with an automated cell counter. ResultsTreatment with both dosages of ECL significantly reduced systolic blood pressure (SBP) in hypertensive rats. Both doses of ECL tended to increase ACh- and SNP-induced vascular relaxation in hypertensive rats. Treatment with 200 mg/kg ECL significantly reduced neutrophil in hypertensive rats. ConclusionsThese results suggest that ECL is effective in reducing SBP and inflammation in hypertensive conditions.

Characterization of bactericidal efficiency, cell selectivity, and mechanism of short interspecific hybrid peptides

Facing rising global antibiotics resistance, physical membrane-damaging antimicrobial peptides (AMPs) represent promising antimicrobial agents. Various strategies to design effective hybrid peptides offer many advantages in overcoming the adverse effects of natural AMPs. In this study, hybrid peptides from different species were investigated, and three hybrid antimicrobial peptides, LI, LN, and LC, were designed by combining the typical fragment of human cathelicidin-derived LL37 with either indolicidin, pig nematode cecropin P1 (CP-1) or rat neutrophil peptide-1 (NP-1). In an aqueous solution, all hybrid peptides had an unordered conformation. In simulated membrane conditions, the hybridpeptideLI displayed more β-turn and β-hairpin structures, whereas LN and LC folded into α-helix structures. The three interspecific hybrid peptides LI, LN, and LC exhibited different levels of antimicrobial activity against Gram-positive and Gram-negative bacteria. LI demonstrated the highest antimicrobial activity and cell selectivity. The results of the swimming motility indicated that LI repressed bacterial motility in a concentration-dependent method. Endotoxin binding assay demonstrated that hybrid peptideLI conserved the binding ability to LPS (polyanionic lipopolysaccharides) of its parental peptides. Fluorescence assays, flow cytometry, and SEM further revealed that hybrid peptide LI acted through different bacteriostatic mechanisms than LL37 and indolicidin and that LI killed bacterial cells via membrane damage. In summary, this study demonstrated that hybrid peptide LI produced by interspecific hybrid synthesis possessed strong cell selectivity and is a promising therapeutic candidate for drug-resistant bacteria infection.

Fatty Acid Profile and Anti-Inflammatory Activity of Fixed Plant Oils

Background: Many seed oils have been used as anti-inflammatory agents, administred by ingestion or topical application in traditional medicine. The objective of this research was to perform a chemical analysis of fatty profile and a pharmacological study through a topical experiment of TPA-induced ear edema test and an internal assay - acetic acid-induced vascular permeability in Swiss mice of some fixed oils popularly used for inflammatory problems, trying to confirm their action.Materials, Methods &amp; Results: Fixed lipids of Ouratea fieldingiana (batiputá), Caryocar coreaceum (pequi), Annacardium occidentale (cashew-nuts), Cocos nucifera (coco-da-bahia), Byrsonima crassifolia (murici) e Elaeis guineenses (palm) were selected for the identification of fatty acids profile by gas chromatography coupled to mass spectrometry (GC/MS) analysis and evaluation of anti-inflammatory activity by TPA-induced ear edema test and acetic acid-induced vascular permeability in Swiss male mice. The oils were purchased in local markets or extracted in Soxhlet apparatus with hexane. The oils of cashew nut, murici fruit, and pequi nut presented a high percentage of unsaturated fatty acids (81.80, 74.46 and 60.72 %, respectively). In the oils of batiputá and murici, linoleic acid was the main unsaturated fatty acid (45.06% and 74.66%, respectively) and oleic acid was main constituent in cashew nut, pequi and palm seed oils. Batiputá and palm oils exibit approximately equivalent content of saturated and unsaturated fatty acid and coconut oil more saturated fatty acids (80.72%) with predominance of lauric acid. The result of TPA-induced ear edema test revealed that all oils presented similar anti-inflammatory activity. In the acetic acid-induced vascular permeability model, the oil of O. fieldingiana was the only one who showed anti-inflammatory activity, while C. coreaceum and B. crassifolia oils showed pro-inflammatory activities. The presence of phenols and flavonoids was evaluated in the O. fieldingiana oil by spectrophometric methods.Discussion: All the oils showed anti-inflammatory action in the TPA-induced ear edema, probably the action of unsaturated fatty acids was more important in topical application, nevertheless in internal inflammation process the presence of antioxidant phenolic compounds could contribute to the higher activity of the oil from O. fieldingiana. The effect of linoleic and oleic acids was demonstrated on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in a prevoius study using sunflower oil. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. This pro-inflammatory effect of oleic and linoleic acids may contribute to the wound healing process. In this study with six plant oils, some of them have higher content in linoleic acid and others oleic acid is the major constituent so the antiinflamatory action on ear edema can be associated to these two unsaturaded fatty acids mechanism of action. In the internal model, probably other chemical constituents revealed in Ouratea fieldingiana as phenols, condensed tannins, flavones and flavanones, could contribute to the anti-inflammatory activity.

Preclinical hematological profile studies of an ayurvedic medicine Siddha Makardhwaja after chronic administration to male sprague-dawley rats

Background: Siddha Makardhwaja (SMD) is a classical Ayurvedic formulation markedly used as a traditional medicine in the rural population for various purposes such as stimulant, tonic, and rejuvenator.Methods: The present study is conducted to evaluate the effect of conventionally prepared SMD on the hematological parameters in experimental animals, for providing scientific data base for its logical use in clinical practice. Acute toxicity tests were conducted to determine the LD50 of the drug. To find out the effect of chronic administration of SMD on hematological parameters it was administered chronically to the male Sprague-Dawley rats at a dose of 40mg/kg for 28 days.Results: In this experiment the TC, DC, various erythrocytic parameters, platelet parameters, ESR were determined. The results of the studies are given below. There is an (13.41%) increase in the number of white blood cell count of the male rat, the increase though not significant yet it was prominent (p=0.257). There is an (15.87%) increase in the absolute count of Neutrophils of the male rat, the increase though not significant yet it was prominent (p=0.371). There is an (12.29%) increase in the absolute count of Lymphocytes of the male rat, the increase though not significant yet it was prominent (p=0.388). There is a statistically significant (p=0.035) increase in the number of platelet count of the male rat (11.13% increase). There is a (2.03%) decrease in the platelet volume distribution width of the male rat, the decrease though not significant yet it was noticeable (p=0.094). There is a statistically insignificant (p=0.619) (10.0%) increase in Erythrocyte sedimentation rate in blood from the male rat.Conclusions: As SMD decreases and increases abnormally on the hematological parameters in body of treated rats, so it should not be administered chronically at a higher dose. Further studies should be done by reducing the administered dose.

Streptococcal heme binding protein (Shp) promotes virulence and contributes to the pathogenesis of group A Streptococcus infection.

Streptococcal heme binding protein (Shp) is involved in the process of heme acquisition in group A Streptococcus (GAS). However, no research thus far has examined the contribution of Shp to the virulence of GAS. To this end, we generated an isogenic strain lacking the shp gene (Δshp) and its complemented strain (Δshp-c) using the parent strain MGAS5005 (WT). Deletion of shp increased survival rates and neutrophil recruitment and reduced skin lesion sizes and GAS loads in the blood and the liver, lung, kidney and spleen in subcutaneous infections of mice. These results indicate that Shp significantly contributes to the skin and systemic invasion of GAS. The growth of the Δshp mutant was significantly slower than MGAS5005 and Δshp-c than in non-immune human blood and in incubation with isolated rat neutrophils. Microarray transcriptional analyses found no alteration in expression of virulence genes, indicating that the phenotype of the Δshp mutant was directly linked to the lack of Shp. The findings indicate that Shp significantly contributes to GAS skin invasion, systemic infection and virulence and that these contributions of Shp are mediated by the effects of Shp on systemic GAS growth and neutrophil responses.

Effect of rapamycin on early stage apoptosis of neutrophils in Sprague-Dawley rats with acute lung injury.

Transfusion-related acute lung injury (TRALI) is a serious complication characterized by the acute onset of non-cardiogenic pulmonary edema following transfusion of blood products. It is not known whether rapamycin has an effect on TRALI that is caused by blood transfusion. The aim of the present study was to determine the effect of rapamycin on early stage apoptosis of neutrophils in Sprague-Dawley rats with acute lung injury. Animal models of TRALI and acute pancreatitis-associated lung injury (APALI) were prepared using Sprague-Dawley rats and histopathological examination of lung tissues was used to validate acute lung injury models. Peripheral blood neutrophils were isolated and early stage apoptosis of neutrophils was detected by flow cytometry. The animal models of TRALI and APALI were constructed successfully. Early stage apoptosis of neutrophils increased in the TRALI group and decreased in the APALI group (P<0.05), while the apoptosis rates in rapamycin intervention TRALI and APALI groups were not significantly different compared to the rate in the control group (P>0.05). In conclusion, early stage apoptosis of neutrophils in TRALI was different from that in APALI, and rapamycin had a limited protective effect on TRALI and APALI in Sprague-Dawley rats.

Molecular mechanisms of metabolic disorders in the organs of rats of different ages affected by sodium nitrite in the context of tobacco intoxication

Among pollutants a significant threat is posed by heavy metals, nitrate fertilizers and industrial waste. However, people also contribute to this threat through bad habits such as smoking and alcohol. All these factors affect health and can cause death. The aim of this study was to investigate the content of reactive oxygen species, markers of cytolysis of hepatocytes, cardoza and the degree of endogenous intoxication in the organism of rats affected by nitrite of sodium on the background of tobacco intoxication. The experiments were carried out on white rats, which were divided into three age categories: immature, mature and old, who for 30 days were exposed to tobacco smoke. The research animals were divided into three groups. One of them for 24 hours before the end of the experiment was given sodium nitrite, the second group was given sodium nitrite 72 hours before the end of the experiment. 30 days after poisoning we tested the contents of reactive oxygen species in neutrophils, cytolysis of erythrocyte membrane was investigated by erythrocytic index of intoxication, hepatocytes and cardiocytes – aminotransferase activity. The degree of endogenous intoxication was evaluated by the content of average weight molecules in blood serum. Poisoning of rats with sodium nitrite on the background of the 30 day intoxication by tobacco smoke led to a significant formation of ROS in neutrophils of rats of different age groups. We investigated the activity of aminotransferases in the blood serum and the above organs of rats after exposing them to sodium nitrite and 30 day tobacco smoke. In the blood serum of adult and aged rats, the activity of alanine aminotransferase increased in these conditions by 2.1 and 1.6 times, respectively. Sodium nitrite, which was an additional factor in the intoxication of the rats of all age groups by smoke, caused more pronounced changes in the activity of the studied enzyme in the blood serum. In the liver and myocardium of rats of different ages a reliable decrease of this index was observed in all experimental groups. There was a significant increase in the serum of rats at different ages of aspartate aminotransferase activity. Aspartate aminotransferase activity in the myocardium of all experimental animals showed a reduction by 1.2–1.3 times after smoke inhalation and 1.5–1.6 times with simultaneous intoxication of the rats by sodium nitrite and tobacco smoke. Sodium nitrite, as an additional toxin raised the penetration capacity of erythrocytes among rats poisoned with smoke.By the end of the experiment, the erythrocyte index of intoxication increased in all age groups: immature rats by 31.7%, mature – by 36.0%, in old rats – by 23.6%. The next step of our research was to investigate the markers of endogenous intoxication – average molecular weight. After the simultaneous intoxication of rats by sodium nitrate and tobacco smoke, at the end of the experiment the content of average weight molecules was λ = 254 and of average mass λ=280 inthe blood serum of rats of all age groups increased by 3.1–3.3 times compared with the intact control group. Immature rats proved most sensitive to the action of sodium nitrite and tobacco.