Murine Peritoneal Cells Research Articles

Gender- and Age-Based Characterization and Comparison of the Murine Primary Peritoneal Mesothelial Cell Proteome.

Organs in the abdominal cavity are covered by a peritoneal membrane, which is comprised of a monolayer of mesothelial cells (MC). Diseases involving the peritoneal membrane include peritonitis, primary cancer (mesothelioma), and metastatic cancers (ovarian, pancreatic, colorectal). These diseases have gender- and/or age-related pathologies; however, the impact of gender and age on the peritoneal MC is not well evaluated. To address this, we identified and characterized gender- and age-related differences in the proteomes of murine primary peritoneal MC. Primary peritoneal MC were isolated from young female (FY) or male (MY) mice (3-6 months) and aged female (FA) or male (MA) mice (20-23 months), lysed, trypsin digested using S-Traps, then subjected to bottom-up proteomics using an LC-Orbitrap mass spectrometer. In each cohort, we identified >1000 protein groups. Proteins were categorized using Gene Ontology and pairwise comparisons between gender and age cohorts were conducted. This study establishes baseline information for studies on peritoneal MC in health and disease at two physiologic age/gender points. Segregation of the data by gender and age could reveal novel factors to specific disease states involving the peritoneum. [This in vitro primary cell model has utility for future studies on the interaction between the mesothelium and foreign materials.].

Antibacterial Activity and Proposed Mode of Action of Extracts from Selected Zimbabwean Medicinal Plants against Acinetobacter baumannii.

Acinetobacter baumannii was identified by the WHO as a priority pathogen in which the research and development of new antibiotics is urgently needed. Plant phytochemicals have potential as sources of new antimicrobials. The objective of the study was to determine the antibacterial activity of extracts of selected Zimbabwean medicinal plants against A. baumannii and determine their possible mode of action. Extracts were prepared from the leaves of the eight plants including the bark of Erythrina abyssinica using solvents of different polarities. Antibacterial activity was evaluated using the microbroth dilution method coupled with the in vitro iodonitrotetrazolium colorimetric assay. The effect of the extracts on membrane integrity was determined by quantifying the amount of protein and nucleic acid leaked from the cells after exposure to the extracts. The effects of the extracts on biofilms were investigated. Toxicity studies were carried out using sheep erythrocytes and murine peritoneal cells. Seven out of eight evaluated plant extracts were found to have antibacterial activity. The Combretum apiculatum acetonie (CAA) extract showed the highest inhibitory activity against A. baumannii with a minimal inhibitory concentration of 125 µg/mL. The minimum inhibitory concentration (MIC) of the CAA extract caused a protein leakage of 32 µg/mL from A. baumannii. The Combretum apiculatum acetonie (CAA), C. apiculatum methanolic (CAM), Combretum zeyheri methanolic (CZM), and Erythrina abyssinica methanolic (EAM) extracts inhibited A. baumannii biofilm formation. The EAM extract was shown to disrupt mature biofilms. The potent extracts were nontoxic to sheep erythrocytes and mouse peritoneal cells. The activities shown by the extracts indicate that the plants have potential as sources of effective antibacterial and antibiofilm formation agents against A. baumannii.

Tumor suppressor p73 induces apoptosis of murine peritoneal cell after exposure to hydatid cyst antigens; a possibly survival mechanism of cystic echinococcosis in vivo mice model.

Cystic echinococcosis (CE) is a life-threatening helminthic disease caused by the Echinococcus granulosus sensulato complex. Previous evidence indicates that the host's innate immune responses against CE can combat and regulate the growth rate and mortality of hydatid cyst in the host's internal organs. However, the survival mechanisms of CE are not yet fully elucidated in the human body. In the present study, the apoptotic effects of fertile and infertile hydatid fluid (HF) were tested on murine peritoneal cells in vivo mice model. Mice were divided into five groups including; control group, fertile HF-treated peritoneal cells, infertile HF-treated peritoneal cells, protoscolices (PSCs)-treated peritoneal cells and HF+PSCs-treated peritoneal cells group. Mice groups were intraperitoneally inoculated with PBS, HF, and/or PSCs. Afterwards, peritoneal cells were isolated and mRNA expression of STAT3, caspase-3, p73 and Smac genes were evaluated by quantitative Real-time PCR. After 48 hours of exposure, the protein levels of Smac and STAT3 was determined by western blotting technique. After 6 hours of exposure, Caspase-3 activity was also measured by fluorometric assay. The intracellular reactive oxygen species (ROS) production was examined in all groups. The mRNA expression levels of p73, caspase-3 and also Caspase-3 activity in HF+PSCs-treated peritoneal cells were higher than in the test and control groups (Pv<0.05), while the mRNA expression level of anti-apoptotic STAT3 and Smac genes in HF+PSC-treated peritoneal cells were lower than in the other groups (Pv<0.05). As well, the level of intracellular ROS in the fertile HCF-treated peritoneal cells, infertile HCF-treated peritoneal cells, PSC-treated peritoneal cells and HF+PSC-treated peritoneal cells groups were significantly higher than in the control group (Pv<0.05).Current findings indicates that oxidative stress and p73 can trigger the apoptosis of murine peritoneal cells through modulator of HF-treated PSCs that is likely one of the hydatid cyst survival mechanisms in vivo mice model.

In-vitro and in-vivo anti-leishmanial activity of the hanging sedge flavonoids based on bio- guided fractionation assay.

As a major public health issue, cutaneous leishmaniasis (CL) has a number of complications, including drug resistance and poor response to conventional treatments. Over the last decade, research on natural sources for finding new anti-leishmanial agents has been a critical part of tropical disease research. Natural products also should be regarded as one of the most valuable applications for CL infection drug development. In this study, we assessed the in-vitro and in-vivo anti-leishmanial potential of Carex pendula Huds. (hanging sedge) methanolic extract and its fractions against Leishmania major produced cutaneous infection. Although the methanolic extract and its fractions exhibited suitable activity, the ethyl acetate fraction showed the best activity (with the half maximal inhibitory concentration IC50 = 1.627±0.211 mg/mL). The toxicity and selectivity indices (SI) of all samples were determined in murine peritoneal macrophage cells (J774A.1) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The flavonoid components of the ethyl acetate fraction were identified using liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS). Nine chemical compounds were identified in this fraction, including three flavonols, four flavanonols, and two flavan derivatives. L. major infected mice were used as an in-vivo model because the methanolic extract was effective against L. major promastigotes in the mammalian cell line J774A.1 with SI=2.514 (tail lesion size model). In-silico analysis of identified compounds also revealed a favorable interaction between compounds 2-5 and L. major protein targets (3UIB, 4JZX, 4JZB, 5L4N, 5L42). According to the findings of this study, the ethyl acetate fraction (as flavonoid fraction) exhibited considerable in-vitro anti-leishmanial activity.

Characterization of the immunomodulatory protein isolated from guduchi (Tinospora cordifolia) churna, an ayurvedic rejuvenator

Objective: Guduchi [Tinospora cordifolia (Willd.) f. and Thoms.], a rasayana (rejuvenator) in the traditional system of Indian medicine (Ayurveda), is known for its diverse bioactivities. The aim of this study was to isolate the immunomodulatory protein (ImP) from commercial guduchi churna (stem powder) and characterize it. Methods: Guduchi churna extract following 80% ammonium sulfate precipitation was chromatographed on Q-Sepharose. Purified guduchi ImP was evaluated for its lymphoproliferative and macrophage activation properties, structural relatedness to certain plant lectins, amino acid composition, stability to trypsin, and N-terminal sequencing. Results: The purified protein, obtained in a yield of 140[Formula: see text]mg/100[Formula: see text]g guduchi churna, showed a molecular mass of [Formula: see text] kDa by SDS-PAGE. Guduchi ImP (10 [Formula: see text]g) showed seven-fold increase over control in mitogenic activity of murine splenocytes, and a four-fold increase over control in nitric oxide (NO) release from murine peritoneal exudates cells (PECs). These activities were abolished in pronase-treated guduchi ImP. Guduchi ImP does not agglutinate rabbit erythrocytes. Rabbit antiserum to guduchi stem ImP strongly reacted with purified guduchi ImP, and did not recognize common plant lectins (concanavalin A (Con A), poke weed mitogen (PWM) and garlic lectin). Amino acid composition of guduchi ImP shows high content of acidic amino acids, serine (12.4%) and cysteine (10.2%); guduchi ImP is highly stable to trypsin and has a blocked N-terminus. Conclusion: Guduchi ImP is an acidic, non-lectin ImP with a high content of serine and cysteine, and appears to have an immense potential for future structural, immunological, and therapeutic studies.

Protoscolicidal activity of Atriplex halimus leaves extract against Echinococcus granulosus protoscoleces

Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 μg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 μg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.

Protective Role of Mytilus edulis Hydrolysate in Lipopolysaccharide-Galactosamine Acute Liver Injury.

Acute liver injury in its terminal phase trigger systemic inflammatory response syndrome with multiple organ failure. An uncontrolled inflammatory reaction is difficult to treat and contributes to high mortality. Therefore, to solve this problem a search for new therapeutic approaches remains urgent. This study aimed to explore the protective effects of M. edulis hydrolysate (N2-01) against Lipopolysaccharide-D-Galactosamine (LPS/D-GalN)-induced murine acute liver injure and the underlying mechanisms. N2-01 analysis, using Liquid Chromatography Mass Spectrometry (LCMS) metabolomic and proteomic platforms, confirmed composition, molecular-weight distribution, and high reproducibility between M. edulis hydrolysate manufactured batches. N2-01 efficiently protected mice against LPS/D-GalN-induced acute liver injury. The most prominent result (100% survival rate) was obtained by the constant subcutaneous administration of small doses of the drug. N2-01 decreased Vascular Cell Adhesion Molecule-1 (VCAM-1) expression from 4.648 ± 0.445 to 1.503 ± 0.091 Mean Fluorescence Intensity (MFI) and Interleukin-6 (IL-6) production in activated Human Umbilical Vein Endothelial Cells (HUVECs) from 7.473 ± 0.666 to 2.980 ± 0.130 ng/ml in vitro. The drug increased Nitric Oxide (NO) production by HUVECs from 27.203 ± 2.890 to 69.200 ± 4.716 MFI but significantly decreased inducible Nitric Oxide Synthase (iNOS) expression from 24.030 ± 2.776 to 15.300 ± 1.290 MFI and NO production by murine peritoneal lavage cells from 6.777 ± 0.373 µm to 2.175 ± 0.279 µm. The capability of the preparation to enhance the endothelium barrier function and to reduce vascular permeability was confirmed in Electrical Cell-substrate Impedance Sensor (ECIS) test in vitro and Miles assay in vivo. These results suggest N2-01 as a promising agent for treating a wide range of conditions associated with uncontrolled inflammation and endothelial dysfunction.

A group of cationic amphiphilic drugs activates MRGPRX2 and induces scratching behavior in mice

Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.

SGLT2 Inhibition by Intraperitoneal Dapagliflozin Mitigates Peritoneal Fibrosis and Ultrafiltration Failure in a Mouse Model of Chronic Peritoneal Exposure to High-Glucose Dialysate.

Peritoneal dialysis (PD) is limited by glucose-mediated peritoneal membrane (PM) fibrosis, angiogenesis, and ultrafiltration failure. Influencing PM integrity by pharmacologically targeting sodium-dependent glucose transporter (SGLT)-mediated glucose uptake has not been studied. In this study, wildtype C57Bl/6N mice were treated with high-glucose dialysate via an intraperitoneal catheter, with or without addition of selective SGLT2 inhibitor dapagliflozin. PM structural changes, ultrafiltration capacity, and peritoneal equilibration testing (PET) status for glucose, urea, and creatinine were analyzed. Expression of SGLT and facilitative glucose transporters (GLUT) was analyzed by real-time PCR, immunofluorescence, and immunohistochemistry. Peritoneal effluents were analyzed for cellular and cytokine composition. We found that peritoneal SGLT2 was expressed in mesothelial cells and in skeletal muscle. Dapagliflozin significantly reduced effluent transforming growth factor (TGF-β) concentrations, peritoneal thickening, and fibrosis, as well as microvessel density, resulting in improved ultrafiltration, despite the fact that it did not affect development of high-glucose transporter status. In vitro, dapagliflozin reduced monocyte chemoattractant protein-1 release under high-glucose conditions in human and murine peritoneal mesothelial cells. Proinflammatory cytokine release in macrophages was reduced only when cultured in high-glucose conditions with an additional inflammatory stimulus. In summary, dapagliflozin improved structural and functional peritoneal health in the context of high-glucose PD.

Heat killed Dietzia maris reduces lipopolysaccharide-induced inflammatory responses in murine adherent peritoneal cells

Heat killed Dietzia maris reduces lipopolysaccharide-induced inflammatory responses in murine adherent peritoneal cells

Direct Inhibition of the Allergic Effector Response by Raw Cow's Milk-An Extensive In Vitro Assessment.

The mechanisms underlying the allergy-protective effects of raw cow’s milk are poorly understood. The current focus is mainly on the modulation of T cell responses. In the present study, we investigated whether raw cow’s milk can also directly inhibit mast cells, the key effector cells in IgE-mediated allergic responses. Primary murine bone marrow-derived mast cells (BMMC) and peritoneal mast cells (PMC), were incubated with raw milk, heated raw milk, or shop milk, prior to IgE-mediated activation. The effects on mast cell activation and underlying signaling events were assessed. Raw milk was furthermore fractionated based on molecular size and obtained fractions were tested for their capacity to reduce IgE-mediated mast cell activation. Coincubation of BMMC and PMC with raw milk prior to activation reduced β-hexosaminidase release and IL-6 and IL-13 production, while heated raw milk or shop milk had no effect. The reduced mast cell activation coincided with a reduced intracellular calcium influx. In addition, SYK and ERK phosphorylation levels, both downstream signaling events of the FcεRI, were lower in raw milk-treated BMMC compared to control BMMC, although differences did not reach full significance. Raw milk-treated BMMC furthermore retained membrane-bound IgE expression after allergen stimulation. Raw milk fractionation showed that the heat-sensitive raw milk components responsible for the reduced mast cell activation are likely to have a molecular weight of > 37 kDa. The present study demonstrates that raw cow’s milk can also directly affect mast cell activation. These results extend the current knowledge on mechanisms via which raw cow’s milk prevents allergic diseases, which is crucial for the development of new, microbiologically safe, nutritional strategies to reduce allergic diseases.

Imiquimod-related dermatitis is mainly mediated by mast cell degranulation via Mas-related G-protein coupled receptor B2

While imiquimod (IMQ) has been widely used in dermatology, its side effect manifested as dermatitis couldn't be ignored. However, the underlying mechanism has not been fully understood. Considering the clinical features of IMQ-related dermatitis similar to pseudo-allergic reaction and the presence of large numbers of mast cell in tissues treated with IMQ, the possibility that IMQ-related dermatitis mediated by mast cell-specific Mas-related G protein-coupled receptor X2 (MRGPRX2) should be addressed. To investigate the role of MRGPRX2 in vivo, MrgprB2, the mice homology of human MRGPRX2, was detected in IMQ-induced dermatitis mouse model. Histopathological changes including mast cell degranulation and footpad swelling were assayed in wild-type and MrgprB2−/− mice. The results showed that IMQ application induced dermatitis and footpad swelling with inflammatory cells infiltration plus mast cell activation in the skin of wild-type mice but reduced significantly in MrgprB2−/− mice. Further, compared to wild-type mice, serum histamine and inflammatory cytokine levels were compromised in MrgprB2−/− mice treated with IMQ, while the serum IgE level didn’t change significantly. In vitro studies, levels of mediators released from murine peritoneal mast cells (MPMCs) after IMQ treatment were increased in a dose-dependent manner, which were much mild in MPMCs from MrgprB2−/− mice. Intracellular Ca2+ concentration was increased in a dose dependent manner after IMQ treatment both in MrgprB2-HEK293 and MRGPRX2-HEK293 cells. Moreover, β-hexosaminidase released after IMQ treatment was blocked by siRNA directed at the MRGPRX2 receptor in LAD2 cells. In summary, MrgprB2 /MRGPRX2 mediate mast cell activation and participate in IMQ-related dermatitis.

Estandarización de las técnicas de citometría de flujo para evaluar las células fagocíticas de Corynebacterium pseudotuberculosis murinas

The understanding of bioprocesses has been allowed the increase of techniques to study cellular interactions, such as flow cytometry (FC). This technic is an important tool for counting and classifying microscopic particles suspended in flowing liquid medium, analyzing parameters from cells diverse origins, including bacteria. Thus, the aim of this study was to standardize protocols by flow cytometry to evaluate the absolute count and physiological aspects of Corynebacterium pseudotuberculosis in murine phagocytic cells. C. pseudotuberculosis was grown in 0.5% BHI –Tween80 medium, followed by the labeling of this microorganism with fluorescent dyes, at different periods and different physiological stages. In the phagocytosis assay, murine peritoneal cells from two strains were used after sodium thioglycolate-induced peritonitis. After cell adhesion, the challenge with stained bacteria was done and after 1h 30 min the adherent cells were trypsinized, washed and centrifuged for the (FC) assay. A growth period similar to a standard microbial growth curve was observed, and allowed to distinguish among different physiological stages. Data obtained from (FC) show phagocytic cell populations containing bacteria in the cytoplasm. A population with low internal complexity, similar to that presented by lymphocytes, and a population with moderate internal complexity similar to that presented by cells of monocytic origin were observed. The (FC) technique with flowering marking was effective to evaluate the growth and viability of C. pseudotuberculosis, as well as the interaction of bacterial cells with murine peritoneal phagocytic cells. Therefore, FC is an efficient technique to evaluating the growth curve of this bacterium, distinguishing different physiological stages, and may even indicate a mechanism of escape from phagocytosis.

IFN-γ stimulates CpG-induced IL-10 production in Bcells via p38 and JNK signalling pathways.

The production of IL-10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN-γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL-10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN-γ on Toll-like receptor (TLR)-induced IL-10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN-γ inhibited IL-10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL-10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN-γ on TLR9-induced IL-10 was restricted to Bcells. In line with the increased IL-10, Bcells stimulated with CpG and IFN-γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen-activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 - an inhibitor of p38 and JNK activity - is downregulated after combined stimulation with IFN-γ and CpG. Our data may represent a novel immunoregulatory role of IFN-γ in Bcells after triggering of TLR9, by stimulating IL-10 production.

P-195 - Oxidative stress parameters as markers of the rate of aging and longevity in mice

Given the different rate of aging of each individual, it is necessary to find valid biomarkers for measuring biological age. In this sense parameters of oxidative stress could be useful. Moreover, it has been shown that there is an age-related decrease in antioxidant as well as an increase in oxidant compounds. Due to the difficulty of carrying out longitudinal studies in humans, all parameters suitable as markers of biological age, have to be validated using experimental animals. Therefore, antioxidant (Catalase, Gluthatione Peroxidase and Gluthatione Reductase activities and GSH levels), oxidant (GSSG and superoxide anion) and malondialdehyde levels were studied in murine peritoneal immune cells at the adult, mature, old, very old and long-lived ages, in a longitudinal study. The results show decreased antioxidant defenses and increased oxidant compounds in old mice. However, long-lived mice show similar values to those of adults. In addition, the values of these parameters in adult prematurely aging mice were similar to those in chronologically old animals, and they died before their non-prematurely aging mice counterparts. Thus, the parameters studied are proposed as markers of the rate of aging.

A new role for mast cells as scavengers for clearance of erythrocytes damaged due to oxidative stress

Anemia, inflammation, and oxidative stress are interconnected. Erythrocytes are continuously exposed to oxidative stress, normally and during inflammatory diseases. Systemic mastocytosis and genetic depletion of mast cells affect anemia. In the present study, a direct role for mast cells in clearance of erythrocytes was explored. We show, for the first time, direct phagocytosis of opsonized as well as oxidatively damaged erythrocytes in vitro by mast cell lines, bone marrow derived mast cells (BMMCs) and in vivo by murine peritoneal mast cells. Also, activated mast cells, as may be present in inflammatory conditions, showed a significantly higher uptake of oxidatively damaged erythrocytes than resting mast cells. This suggests the involvement of mast cells in erythrocyte clearance during oxidative stress or inflammatory disorders. Partial inhibition of phagocytosis by various inhibitors indicated that this process may be controlled by several pathways. Our study provides important evidence for a scavenging role for mast cells in anemia due to inflammation and oxidative stress.

Honokiol attenuates diet‐induced non‐alcoholic steatohepatitis by regulating macrophage polarization through activating peroxisome proliferator‐activated receptor γ

Non-alcoholic steatohepatitis (NASH) may develop into hepatic cirrhosis. This study aimed to investigate whether honokiol could prevent NASH induced by high-cholesterol and high-fat (CL) diet in mice and the possible mechanism involved. Mice were fed with CL diet for 12weeks to establish a NASH model; honokiol (0.02% w/w in diet) was added to evaluate its effect on NASH. Murine peritoneal macrophages, RAW264.7 and ANA-1 cells, were used to explore the possible mechanisms of honokiol on macrophage polarization. Mice developed NASH after fed with CL diet for 12weeks. Honokiol supplementation alleviated insulin resistance, hepatic steatosis, inflammation, and fibrosis induced by CL diet. Immunohistochemistry showed that honokiol induced more M2 macrophages in livers compared with CL diet alone. Honokiol decreased M1 marker genes (TNFα and MCP-1) and increased M2 marker gene (YM-1, IL-10, IL-4R and IL-13) expression in mice liver compared with CL diet. Moreover, treatment with honokiol lowered alanine aminotransferase and aspartate aminotransferase in serum and preserved liver from lipid peroxidation, evidenced by lowered hepatic malondialdehyde level. Honokiol has antioxidant function, as honokiol upregulated hepatic glutathione and superoxide dismutase level and downregulated hepatic CYP2E1 protein level. Hepatic peroxisome proliferator-activated receptor γ (PPARγ) and its target genes were upregulated by honokiol. Furthermore, honokiol (10μM) treatment in mouse peritoneal cells, RAW264.7 cells and ANA-1 cells, led to M2 macrophage polarization, whereas a PPARγ antagonist, GW9662, abolished this effect of honokiol. Honokiol can attenuate CL diet-induced NASH and the mechanism in which possibly is polarizing macrophages to M2 phenotype via PPARγ activation.

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca2+ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca2+ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca2+-dependent exocytosis of inflammatory mediators. We show that the Ca2+ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca2+ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2−/− PMCs at high (2mM) extracellular Ca2+ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1mM) Ca2+ concentration. Likewise, the density of CRAC currents, Ca2+-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca2+ transients, store-operated Ca2+ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca2+ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.

A trypsin inhibitor from Moringa oleifera flower extract is cytotoxic to Trypanosoma cruzi with high selectivity over mammalian cells

In this study, Moringa oleifera flower extract and a trypsin inhibitor (MoFTI) isolated from it were evaluated for anti-protozoal activity against Trypanosoma cruzi and cytotoxicity to mammalian cells. The presence of flavonoids was remarkable in the HPLC fingerprints of the extract at 254 and 360 nm. Amino acid sequences of peptides derived from in-gel digestion of MoFTI were determined. Both the extract and MoFTI caused lysis of T. cruzi trypomastigotes with LC50/24 h of 54.18 ± 6.62 and 41.20 ± 4.28 μg/mL, respectively. High selectivity indices (7.9 to >12) for T. cruzi cells over murine peritoneal macrophages and Vero cells were found for the extract and MoFTI. The results show that MoFTI is a trypanocidal principle of the flower extract.

Microparticles prepared with 50–190 kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190–310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50–190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50–190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation.