Levels Of Nitric Oxide Production Research Articles

Suppression of Inflammation in Adipocyte-Macrophage Coculture by Passion Fruit Seed Extract: Insights into the p38 and NF-ҡB Pathway.

Obesity, which is characterized by chronic low-grade inflammation, involves the infiltration of immune cells into adipose tissue, leading to the secretion of inflammatory cytokines and subsequent inflammation. Therefore, the aim of this study was to examine the potential of passion fruit seed extract (PSEE) in mitigating lipopolysaccharide (LPS)-induced inflammation in a coculture system comprising macrophages and adipocytes. PSEE demonstrated significant reductions in reactive oxygen species (ROS) and nitric oxide (NO) levels, primarily achieved through the downregulation of inducible nitric oxide synthase (iNOS) protein expression in LPS-induced adipocyte-macrophage cocultures. Furthermore, PSEE effectively suppressed the secretion of TNF-α and IL-1β by attenuating the gene expression of these cytokines, as well as other inflammation-related genes such as MMP-2, IL-6, and MCP-1. Notably, PSEE exhibited potent inhibitory effects on the p38 and NF-κB signaling pathways, thus alleviating inflammation in the LPS-induced adipocyte-macrophage cocultures. Additionally, PSEE led to a decrease in the expression of ACC, HSL, and FaSN, while aP2 and ATGL showed increased expression in LPS-induced cocultured macrophages and adipocytes. These findings suggest that passion fruit seed extract effectively combats inflammation by suppressing the p38 and NF-κB signaling pathways, resulting in reduced levels of proinflammatory cytokines, NO, and ROS production.

NO production of granulocytes associated with antibacterial immune response in Tegillarca granosa

Nitric oxide (NO) is a signaling molecule and immune effector produced by the nitric oxide synthases (NOS), which involved to various physiological processes of animals. In marine bivalves, hemocytes play important roles in antimicrobial innate immune response. Although hemocyte-derived NO has been detected in several bivalves, the immune function of hemocyte-derived NO is not well understood. Here, we investigated the antibacterial response of hemocyte-derived NO in the blood clam Tegillarca granosa. Two types of hemocytes including erythrocytes and granulocytes were isolated by Percoll density gradient centrifugation, their NO production and TgNOS expression level were analyzed. The results showed that NO was mainly produced in granulocytes and almost no detected in erythrocytes. The granulocytes showed significantly higher NO level and TgNOS expression level than the erythrocytes. And the TgNOS expression level was significantly increased in granulocytes after Vibro parahemolyticus challenge. In addition, the NO donor sodium nitroprusside (SNP) significantly increased the NO production of hemocytes to kill pathogenic bacteria. In summary, the results revealed that granulocytes-derived NO play vital roles in the antimicrobial immune response of the blood clam.

Immunomodulatory effects of lotus seed (Nelumbo nucifera Gaertn.) peptides on macrophages in mice

Immunomodulatory effects of lotus seed (Nelumbo nucifera Gaertn.) peptides (LSPs) were studied in this paper. The LSPs were prepared by trypsin enzymatic hydrolysis and were separated by ultrafiltration separation technology. The following four peptide fractions with different molecular weight were obtained: LSP-I (>10 kDa), LSP-II (5–10 kDa), LSP-III (3–5 kDa), and LSP-IV (<3 kDa). Macrophage phagocytosis capacity, NO production and cytokine levels were measured in healthy and immunosuppressed mice to explore the effects of immunomodulatory activity of LSPs. In vitro, among the four peptides, LSP-IV showed the greatest effect on RAW264.7 macrophage phagocytosis. In vivo, LSP-IV had no significant effect on the levels of nitric oxide (NO), interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) in healthy mice in a dose-dependent manner, but significantly increased the levels of NO, IL -6 and TNF-α secreted by peritoneal macrophages (p < 0.01 and p < 0.001, respectively) in immunosuppressed mice. In particular, high doses of LSP-IV (0.10 g/kg body weight) showed a stronger immunomodulatory potential. Our findings shed light on the potential of low-molecular-weight peptides from lotus seeds as novel natural immunoregulatory dietary factors.

The dual-targeting mechanism of an anti-inflammatory diarylheptanoid from Curcuma zedoaria (Christm.) Roscoe with the capacity for β2-adrenoreceptor agonism and NLRP3 inhibition

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by symptoms of shortness of breath and chronic inflammation. Curcuma zedoaria (Christm.) Roscoe is a well-documented traditional medical herb that is frequently used in the treatment of COPD. Previously, we identified a diarylheptanoid compound (1-(4-hydroxy-5-methoxyphenyl)-7-(4,5-dihydroxyphenyl)-3,5-dihydroxyheptane; abbreviated as HMDD) from this herb that exhibited potent agonistic activity on β2-adrenergic receptors (β2 adrenoreceptor) that are present on airway smooth muscle cells. In this work, we used chemically synthesized HMDD compound, and confirmed its bioactivity on β2 adrenoreceptors. Then by a proteomics study and anti-inflammatory evaluation detections, we found that HMDD downregulated the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway and suppressed the NLRP3 receptor expression in RAW264.7 macrophages and in a COPD model in A549 lung carcinoma cells. HMDD also decreased nitric oxide production levels, and impacted other interleukins and the phosphorylation of NF-κB and ERK pathways. We performed molecular docking of HMDD on β2 adrenoreceptor and NLRP3 protein models. This work reports the anti-inflammatory effects of HMDD and suggests a dual-targeting mechanism of β2-adrenoreceptor agonism and NLRP3 inhibition. Such a mechanism scientifically supports the clinical uses of Curcuma zedoaria (Christm.) Roscoe in treating COPD, as it can simultaneously relieve persistent breathlessness and inflammation. HMDD can be considered as a potential non-steroidal anti-inflammatory drug in novel therapy design for the treatment of COPD and other inflammatory diseases.

Molecular properties prediction, anticancer and anti-inflammatory activities of some pyrimido[1,2-b]pyridazin-2-one derivatives.

The anticancer and anti-inflammatory activities of a novel series of eleven pyrimido[1,2-b]pyridazin-2-one analogues substituted at position 7 were assessed in the current study. The physicochemical characteristics were studied using MolSoft software. The antiproliferative activity was investigated by MTT cell viability assay, and cell cycle analysis elucidated the antiproliferative mechanism of action. Western blot analysis examined the expression levels of key pro-apoptotic (Bax, p53) and pro-survival (Bcl-2) proteins. The anti-inflammatory activity was assessed by measuring the production levels of nitric oxide in RAW264.7 cells, and the expression levels of COX-2 enzyme in LPS-activated THP-1 cells. In addition, the gene expression of various pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α) and chemokines (CCL2, CXCL1, CXCL2, CXCL3) was assessed by RT-qPCR. Compound 1 bearing a chlorine substituent displayed the highest cytotoxic activity against HCT-116 and MCF-7 cancer cells where IC50 values of 49.35 ± 2.685 and 69.32 ± 3.186 µM, respectively, were achieved. Compound 1 increased the expression of pro-apoptotic proteins p53 and Bax while reducing the expression of pro-survival protein Bcl-2. Cell cycle analysis revealed that compound 1 arrested cell cycle at the G0/G1 phase. Anti-inflammatory assessments revealed that compound 1 displayed the strongest inhibitory activity on NO production with IC50 of 29.94 ± 2.24 µM, and down-regulated the expression of COX-2. Compound 1 also induced a statistically significant decrease in the gene expression of various cytokines and chemokines. These findings showed that the pyrimidine derivative 1 displayed potent anti-inflammatory and anticancer properties in vitro, and can be selected as a lead compound for further investigation.

Neurotrophic phenolic glycosides from the roots of Armoracia rusticana

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis (1H, 13C, 1H–1H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells.

Synergistic Anti-inflammatory Activity of Ginger Extract and Phlai Oil on Lipopolysaccharide-induced Inflammatory Mediators

Objective: This study aimed to evaluate the anti-inflammatory activity of the combination of ginger extract and phlai oil on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Material and Methods: Cell viability, inhibition activity of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) were measured in vitro on RAW 264.7 cell cultures stimulated with bacterial lipopolysaccharide.Results: The IC50 values of ginger extract and phlai oil on RAW 264.7 cells were 1.37±0.112 mg/ml and 3.79±0.261 mg/ml, respectively. The production levels of NO, TNF-α and PGE2 was induced by LPS. The inhibition percentages of NO increases in the presence of ginger extract (0.313 mg/ml) and phlai oil (0.625 mg/ml) were 38.22±0.236% and 25.00±0.198% in comparison to the control cells. The combination of ginger extract and phlai oil at a ratio 1:1 w/w exhibited statistically significant inhibitory effects on NO production, having an inhibition percentage of 75.98±0.591%. In the TNF-α assay, an inhibition percentage value of ginger extract (58.74±0.136%) was higher than that of phlai oil (52.13±0.238%), while the combination of ginger extract and phlai oil had the highest inhibitory effect (70.03±0.215%). In the PGE2 assay, the inhibition percentage of the ginger extract-phlai oil combination at a ratio of 1:1 w/w (62.34±0.187%) was higher than those of both ginger extract (18.21±0.132%) and phlai oil (34.80±0.279%).Conclusion: This study provides scientific evidence in support of the efficacy of the combined use of ginger and phlai oil to reduce inflammatory processes.

Red blood cell eNOS: a major player in regulating cardiovascular health.

Red blood cells (RBCs) have traditionally been seen as simple carriers of gases and nutrients in the body. One important non-canonical function of RBCs in the cardiovascular system is the regulation of nitric oxide (NO) metabolism. It was shown that RBCs can scavenge NO, transport NO metabolites, and produce NO in hypoxic conditions, thereby inducing hypoxic vasodilation. RBCs also express an endothelial nitric oxide synthase (eNOS). However, its physiological significance has been controversial for many years. This review article provides a comprehensive overview of the experimental research on RBC eNOS signaling in vivo. These data show that RBC eNOS signaling modulates intracellular NO production and NO-heme levels, as well as extracellular paracrine NO metabolite signaling, which contributes to regulating peripheral vascular resistance, blood pressure, and cardioprotection. Additionally, this article explores the potential intracellular molecular mechanisms and the implications of RBC eNOS in cardiovascular health and disease.

Immune-enhancing activity of Astragalus maximus extract for inhibiting the Toxoplasma gondii infection: experimental research.

This study aimed to evaluate the in-vitro anti-Toxoplasma effects and cytotoxicity effects of Astragalus maximus chloroformic extract (AMCE) on the T. gondii Rh strain. In-vitro effects of AMCE (2-64µg/ml) on tachyzoites were measured by MTT assay for 48h. The effects of AMCE on infectivity rate and intracellular parasites into macrophage cells (J774-A1) were evaluated. The Griess reaction assay and quantitative real-time PCR were used to determine the nitric oxide (NO) and the mRNA expression levels of IFN-γ and iNOS in infected J774-A1 macrophage cells. The mortality rate of the parasites significantly (P<0.001) increased in a dose-dependent manner with an IC50 value of 9.85μg/ml. The rate of infection and the mean number of intracellular tachyzoites in macrophage cells were significantly reduced (P<0.001) after exposure of the macrophage cells to AMCE. The mRNA expression levels of IFN-γ, iNOS, and NO production in macrophage cells after treatment with the AMCE were increased, especially at the concentration of ½ IC50 and IC50 (P<0.001). The findings of the current in vitro investigation revealed favorable anti-Toxoplasma effects of AMCE against tachyzoites and intracellular forms of T. gondii. Despite the fact that the accurate anti-Toxoplasma mechanisms of AMCE are not clear, our results showed that triggering NO production and cellular immunity can be considered as the main mechanisms of action of AMCE for controlling and eliminating T. gondii. However, further surveys are mandatory to assess the efficacy and safety of AMCE in an animal model and its accurate mechanisms of action.

A zinc-modified Anemarrhena asphodeloides polysaccharide complex enhances immune activity via the NF-κB and MAPK signaling pathways

Anemarrhena asphodeloides polysaccharide (AAP70-1) was reported to have immunomodulatory effects in our previous report. To further improve the immunomodulatory effects of AAP70–1, an A. asphodeloides polysaccharide-zinc complex (AAP-Zn) was synthesized using a ZnCl2 modification method, and the potential mechanisms by which AAP-Zn activates macrophages were investigated. The results showed that the structural features of AAP-Zn were similar to those of AAP70–1 with a Zn content of 0.2 %, confirming that Zn mainly interacted with AAP70–1 by forming ZnO coordination bonds and Zn…OH bonds. In addition, the administration of AAP70–1 and AAP-Zn effectively improved the immunomodulatory effects by enhancing phagocytosis and upregulating the mRNA expression of cytokines (TNF-α, IL-6, IL-1β, and IL-18), as well as increasing the production levels of nitric oxide (NO) and reactive oxygen species (ROS) in zebrafish embryos. The intracellular mechanism by which AAP-Zn activates macrophages was found to involve activation of the NF-κB and MAPK signaling pathways. Our findings suggested that AAP-Zn may be a potential immunopotentiator in the field of biomedicine or functional foods.

Human Umbilical Cord Blood Endothelial Progenitor Cell-Derived Extracellular Vesicles Control Important Endothelial Cell Functions.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.

Multiple myeloma cell-derived exosomes promote favorable tumor functional performance by polarizing macrophages toward M2-like cells.

It has long been hypothesized that leukemic cells are able to modulate the fate of resident cells in the tumor microenvironment (TME) toward either supporting or immunosuppressive cells for the development of tumors. Exosomes can be a potential culprit in imposing tumor desire. There is evidence about the impact of tumor-derived exosomes on different immune cells in different malignancies. However, findings about macrophages are contradictory. Here, we evaluated the potential influence of multiple myeloma (MM)-cell-derived exosomes on the polarization of macrophages by examining hallmarks of M1 and M2 macrophages. After treatment of M0 macrophages with isolated exosomes (from U266B1), gene expression (Arg-1, IL-10, TNF-α and IL-6), immunophenotyping markers (CD206), cytokine secretion (IL-10 and IL-6), nitric oxide (NO) production, and redox potentiality of target cells were assessed. Our results revealed significantly increased expression of the genes involved in the development of M2-like cells but not M1 cells. The CD 206 marker and IL-10 protein levels were significantly increased at different time points. The expression of IL-6 mRNA and IL-6 protein secretion did not change significantly. MM-cell-derived exosomes induced significant changes in NO production and intracellular ROS levels in M0 cells.

Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions

Abstract Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).

Boswellia serrata inhibits LPS-induced cardiotoxicity in H9c2 cells: Investigating role of anti-inflammatory and antioxidant effects

Sepsis-induced myocardial dysfunction is the main reason for mortality and morbidity. Recent investigations have shown that inflammation and oxidative stress play a central role in lipopolysaccharide (LPS)-induced cardiac injury pathophysiology. Gum-resin extracts of Boswellia serrata have been traditionally used in folk medicine for centuries to treat various chronic inflammatory diseases. The present study aimed to investigate the effects of B. serrata pretreatment on LPS-induced cardiac damage in H9c2 cells. The cells were pretreated with various concentrations of B. serrata (5–45 μg/ml) for 24 h and then stimulated with LPS (10 μg/ml) for another 24 h. Afterward, the levels of cell viability, tumor necrosis factor (TNF)-α, prostaglandin (PGE)-2, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, nitric oxide (NO) and glutathione (GSH) were determined using enzyme-linked immunosorbent assay (ELISA), real time-PCR or appropriated biochemical methods. Our results demonstrated that LPS treatment caused a remarkable decrease in cell viability and GSH, and on the contrary, it led to a significant increase in the levels of gene and protein expression of inflammatory markers and NO. However, pretreatment of B. serrata (5, 15, and 45 μg/ml) decreased the levels of TNF-α, PGE2, IL-1β, COX-2, iNOS, IL-6, and NO production, while cell viability and GSH levels were increased. Taken together, our results demonstrated that B. serrata might be a potential therapeutic agent against LPS and endotoxemia-induced cardiac injury, through its anti-inflammatory and antioxidant properties.

Comparative study on the impacts of visnagin and its methoxy derivative khellin on human lymphocyte proliferation and Th1/Th2 balance.

Visnagin is a phenolic and natural compound in turmeric and fenugreek, and its anti-inflammatory effect has been indicated. Therefore, this study aimed to investigate and compare the anti-inflammatory properties of visnagin and its methoxy derivative khellin on human lymphocytes. Human lymphocytes were treated with khellin, visnagin (10, 30, and 100µM), and dexamethasone (0.1mM) in the presence of phytohemagglutinin (PHA). The levels of cell proliferation, nitric oxide (NO), glutathione (GSH), malondialdehyde (MDA), and MDA/GSH ratio were measured using biochemistry methods. Furthermore, the mRNA levels of interferon-γ (IFN-γ), interleukin (IL)-4, and IL-10 were assessed using real-time PCR, while IFN-γ/IL-4(Th1/Th2), IFN-γ/IL-10(Th1/Treg), and IL-4/IL-10(Th2/Treg) ratios were made by dividing their exact values. In the PHA-stimulated group, GSH and IFN-γ/IL-4 levels were markedly diminished, but other variables were significantly elevated compared to the control group. Khellin and visnagin significantly declined the levels of cell proliferation, MDA, MDA/GSH ratio, and NO production. Khellin and visnagin concentration-dependently diminished IFN-γ and IL-4 levels and increased IL-10 levels compared to the PHA-stimulated group. Two higher concentrations of khellin and visnagin (30 and 100μM) considerably diminished the IFN-γ, IFN-γ/IL-10, and IL-4/IL-10 values compared to the PHA-stimulated group. However, 100µM of khellin and visnagin significantly increased GSH level compared to the PHA-stimulated group. In PHA-stimulated lymphocytes, representing Th2 dominant allergic diseases, khellin and visnagin provides more specific anti-oxidant, anti-inflammatory, and immunomodulatory functions than dexamethasone. In addition, the effects of khellin were more prominent than visnagin.

The level of nitric oxide and arginase activity in patients with arterial hypertension and diabetes mellitus during COVID-19

The aim of this study was to assess the level of nitric oxide production and arginase activity in patients with arterial hypertension and type II diabetes mellitus during infection with SARS-CoV-2. The study groups included patients with arterial hypertension, patients with arterial hypertension combined with a severe course of COVID-19 and patients who, in addition to arterial hypertension and COVID-19, were suffering from type II diabetes mellitus. The volunteers without any clinical signs of diseases and normal blood pressure formed the control group. It has been established that arterial hypertension, combined with COVID-19 occurs along with reduced L-arginine, nitric oxide, superoxide dismutase activity and increased arginase activity. At the same time, the presence of arterial hypertension in patients with diabetes and coronavirus disease is accompanied by a decline in the content of L-arginine and arginase activity. Our study’s results may help scientists find new pharmacological targets in the future treatment of coronavirus disease and comorbid disorders. Keywords: arginase, COVID-19, diabetes mellitus, hypertension, L-arginine, nitric oxide, superoxide dismutase

남해산 사포나리아 알로에로부터 분리한 다당체의 항염증 활성

To identify the physiological activity of Saponaria aloe cultivated in the Namhae-gun, polysaccharides, as the main physiologically active substances, were isolated and their antioxidant and anti-inflammatory activities were confirmed. Hot-air dried saponaria were extracted with hot water (80°C, 24 h) and then the extracts were freeze dried (HWE). HWE was dialyzed using 3.5 kDa dialysis membrane and only intra-dialysis solution was taken and freeze dried (IDS) for experiment. Total polysaccharide content was higher in IDS (195.32 mg/g) than HWE however, anthraquinone content was lower in IDS. Total phenolic compounds (TPC) contents of HWE and IDS were 594.04 and 1,047.23 mg/100 g, respectively. The IC50 values of 2,2-Azinobis-(3-ethylbenzo-thiazoline-6-sulphonate (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities were higher 1.7 and 1.8 folds in HWE than IDS, respectively. The MTT assay showed that the sample concentrations below than 100 μg/mL were sufficient to exert anti-inflammatory activity in lipopolysaccharide (LPS) induced RAW 264.7 cells. The production levels of nitric oxide (NO) and reactive oxygen species (ROS) were significantly decreased in 100 μg/mL of HWE (p⟨0.01) and IDS (p⟨0.001) treated group than those in LPS only treated group. The expression levels of tumor necrosis faction (TNF)-α and interleukin (IL)-6 were significantly lower in all treated samples than in the LPS only treated group. However, there was no significant difference between the HWE and IDS-treated group. The expression activity of IL-1β was no significant difference among all samples.

Rosmarinus Officinalis L, Eriobotrya Japonica and Olea Europaea L Attenuate Adipogenesis in 3T3-L1-Derived Adipocytes and Inflammatory Response in LPS-Induced THP-1-Derived Macrophages

This study aims to in vitro evaluate the impact of three traditionally used anti-obesity medicinal plant extracts on inflammatory mediators in the THP-1 cell line and adipocyte differentiation and fat accumulation in the 3T3-L1 cell line. After extract preparation, an in vitro evaluation of cell viability using MTT on THP-1 and 3T3-L1, NO production levels using Griess reagent and cytokines production in THP-1 cells, and adipogenicity evaluation using Oil Red O on 3T3-L1 cells were performed. O. europaea showed the highest cytostatic on THP-1 cells with IC50 of 290.6 µg/mL, while R. officinalis was the highest on 3T3-L1 cells with IC50 of 486.6 µg/mL. All extracts significantly reduced dose-dependently nitric oxide (NO) production by THP-1-derived macrophages from 80 µM to control levels after treatment with 125 µg/mL. Similarly, all extracts decreased the levels of TNF-α and IL-6 production dose-dependently, with the highest effects reached at 250 µg/mL. all extracts increased the production levels of IL-10 from 32 pg/mL to 106.8 pg/mL, 83.5 pg/mL and 87 pg/mL at 125µg/mL, respectively. After treatment with plant extracts, the adipocyte differentiation and fat accumulation in 3T3-L1 were reduced to 20% of the control values. All three medicinal plants exhibit their traditionally known anti-obesity effects through cytostatic effects, modulation of proinflammatory and anti-inflammatory cytokine production levels, and reduction of adipocyte differentiation and fat accumulation.

CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment

Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.

Identification of a potent NAFLD drug candidate for controlling T2DM-mediated inflammation and secondary damage in vitro and in vivo.

Accumulation of glucose/sugar results in the formation of reactive di-carbonyl compounds such as MGO and GO that interact with several amino acids and proteins to form toxic advanced glycation end products (AGEs). Induction of AGEs breakdown can control symptoms and severity in T2DM and other related complications like NAFLD where AGEs are the key players. Therefore, an AGE cross-link breaker has been suggested for preventing the onset/progression of NAFLD. In this study, we reported novel synthetic naphthalene-2-acyl thiazolium derivatives (KHAGs). Among synthesized KHAG derivatives, we observed that a novel KHAG-04, a 1,4-dimethoxynaphthalen-2-acyl thiazolium salt which is an analog of alagebrium, dramatically cleaves MGO/GO-AGE cross-links, and it also inhibited inflammation by lowering the level of nitric oxide production and IL-1β and TNF-α secretion in LPS and/or MGO-AGE–activated macrophage. Moreover, it also reduced FFA and MGO-AGE–induced lipogenesis in Hep-G2 cells. In mice, KHAG-04 significantly reduced the level of glyoxal in the liver, which was induced by DMC. Furthermore, KHAG-04 treatment significantly reduced blood glucose levels, lipid accumulation, and inflammation in the NAFLD/T2DM animal model. Novel KHAG-04–mediated induction of AGEs breakdown could be the possible reason for its anti-inflammatory, antihyperglycemic, and anti-lipidemic effects in cells and NAFLD in the T2DM animal model, respectively. Further research might explore the pharmacological efficacy and usefulness and consider the ability of this compound in the treatment strategy against various models of NAFLD in T2DM where MGO/GO-AGEs play a key role in the pathogenesis.