Inducible Nitric Oxide Synthase Research Articles

Histopathology and changes in the expression of metallothioneins, heat shock proteins and inducible nitric oxide synthase in Prochilodus costatus from a neotropical river contaminated by heavy metals

The most recent dam rupture in Brazil released tons of mining tailings into the upper course of the Paraopeba River, affecting this river in an unprecedented way. The present study aimed to evaluate the influence of heavy metals on Prochilodus costatus, an important commercial species in Brazil, four years after the dam colapse. To this end, biomarkers of heavy metals, oxidative stress, and environmental stress were analyzed, and histological analyses of target organs were performed. The results demonstrated critical contamination of fish from the Paraopeba River. Increased expression of Metallothioneins - MTs, Heat Shock Protein - HSP70, and inducible nitric oxide synthase - iNOS, as well as greater rates of histological changes in the liver, spleen, and gonads, were observed in P. costatus. These findings demonstrate that, despite past contamination, the metals present in mining tailings have significantly increased the contamination of the Paraopeba River basin.

Expression profile and immunomodulatory roles of methionine-enkephalin and delta opioid receptor in Octopus ocellatus

In this study, the expressions and distributions of methionine-enkephalin (Met-enk) and δ opioid receptor in the nervous system of Octopus ocellatus, and the immune regulatory mechanisms of Met-enk on O. ocellatus were explored. The distributions and expressions of Met-enk and δ opioid receptor were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. UV-spectrophotometer, microplate reader, and flow cytometer were used to examine the effects of different concentrations of Met-enk on phagocytosis, antioxidant effects, and body surface mucus immunity of O. ocellatus hemocytes. The data were used to study the mechanisms of Met-enk immunity regulation in O. ocellatus. According to the results, the expression levels of Met-enk and δ opioid receptor in O. ocellatus lymphocytes were higher than those in hemocytes. The expression levels of Met-enk in the ganglia of O. ocellatus decreased in the following order: pedal ganglia > cerebral ganglia > visceral ganglia > optic ganglia > stellate ganglia. Moreover, the phagocytic activity of O. ocellatus hemocytes was enhanced with increasing Met-enk concentration. With increasing Met-enk concentration, the expressions of nitric oxide, total nitric oxide synthase, inducible nitric oxide synthase, catalase, hydrogen peroxide, myeloperoxidase, reduced glutathione, α-naphthy acetate esterase, and methionine aminopeptidases decreased in serums of O. ocellatus in the experimental group compared to the blank group. Similarly, the content of reduced glutathione in the hemocytes of O. ocellatus was also lower in the experimental group than in the blank group; however, the expressions of other substances were higher compared to the blank group. Furthermore, α-naphthy acetate esterase, myeloperoxidase, and hydrogen peroxide expressions in mucus immunity trials of the body surface were lower in the experimental group compared to the blank group. These results indicate that the distributions and expressions of Met-enk and δ opioid receptor in the nervous system of O. ocellatus were related to axoplasmic transport and immune regulation mechanisms. Met-enk participates in cellular immunity, humoral immunity, and mucus immunity in the form of neurotransmitters, thereby regulating the immune response of O. ocellatus.

Screening Antioxidant Components in Different Parts of Dandelion Using Online Gradient Pressure Liquid Extraction Coupled with High-Performance Liquid Chromatography Antioxidant Analysis System and Molecular Simulations.

Utilizing online gradient pressure liquid extraction (OGPLE) coupled with a high-performance liquid chromatography antioxidant analysis system, we examined the antioxidative active components present in both the aerial parts and roots of dandelion. By optimizing the chromatographic conditions, we identified the ferric reducing-antioxidant power system as the most suitable for online antioxidant reactions in dandelion. Compared to offline ultrasonic extraction, the OGPLE method demonstrated superior efficiency in extracting chemical components with varying polarities from the samples. Liquid chromatography-mass spectrometry revealed twelve compounds within the dandelion samples, with nine demonstrating considerable antioxidant efficacy. Of these, the aerial parts and roots of dandelion contained nine and four antioxidant constituents, respectively. Additionally, molecular docking studies were carried out to investigate the interaction between these nine antioxidants and four proteins associated with oxidative stress (glutathione peroxidase, inducible nitric oxide synthase, superoxide dismutase, and xanthine oxidase). The nine antioxidant compounds displayed notable binding affinities below -5.0 kcal/mol with the selected proteins, suggesting potential receptor-ligand interactions. These findings contribute to enhancing our understanding of dandelion and provide a comprehensive methodology for screening the natural antioxidant components from herbs.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line by impairing mTOR-mediated autophagy

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy.

Potential Role and Mechanism of Mulberry Extract in Immune Modulation: Focus on Chemical Compositions, Mechanistic Insights, and Extraction Techniques.

Mulberry is a rapidly growing plant that thrives in diverse climatic, topographical, and soil types, spanning temperature and temperate countries. Mulberry plants are valued as functional foods for their abundant chemical composition, serving as a significant reservoir of bioactive compounds like proteins, polysaccharides, phenolics, and flavonoids. Moreover, these compounds displayed potent antioxidant activity by scavenging free radicals, inhibiting reactive oxygen species generation, and restoring elevated nitric oxide production induced by LPS stimulation through the downregulation of inducible NO synthase expression. Active components like oxyresveratrol found in Morus demonstrated anti-inflammatory effects by inhibiting leukocyte migration through the MEK/ERK signaling pathway. Gallic and chlorogenic acids in mulberry leaves (ML) powder-modulated TNF, IL-6, and IRS1 proteins, improving various inflammatory conditions by immune system modulation. As we delve deeper into understanding its anti-inflammatory potential and how it works therapeutically, it is crucial to refine the extraction process to enhance the effectiveness of its bioactive elements. Recent advancements in extraction techniques, such as solid-liquid extraction, pressurized liquid extraction, superficial fluid extraction, microwave-assisted extraction, and ultrasonic-assisted extraction, are being explored. Among the extraction methods tested, including Soxhlet extraction, maceration, and ultrasound-assisted extraction (UAE), UAE demonstrated superior efficiency in extracting bioactive compounds from mulberry leaves. Overall, this comprehensive review sheds light on the potential of mulberry as a natural immunomodulatory agent and provides insights into its mechanisms of action for future research and therapeutic applications.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Effect of Lactiplantibacillus plantatum HFY11 on Colitis in Mice.

This study aimed to examine the potential impact of the intervention of Lactiplantibacillus plantatum HFY11 (LP-HFY11) on colitis using in vivo animal trials. The impact of LP-HFY11 intervention on colitis was determined by measuring the levels of relevant indicators in the intestine, colon, and blood after oxazolone-induced colitis in BALB/c mice. The results of the trial show that LP-HFY11 improved the colon weight-to-length ratio, reduced the colitis-induced colon length shortening, and reduced colonic abstinence. Furthermore, it decreased the levels of myeloperoxidase, nitric oxide, and malondialdehyde activities while increasing the glutathione content in the colon tissue of colitis-affected animals. LP-HFY11 lowered the interleukin-10 (IL-10) level and increased the IL-2 level in the serum of colitis mice. LP-HFY11 also upregulated the expression of neuronal nitric oxide synthase, endothelial nitric oxide synthase, c-Kit, and stem cell factor (SCF), and downregulated the expression of IL-8, C-X-C chemokine receptor type 2 (CXCR2), and inducible nitric oxide synthase (iNOS) in the colon tissue of mice with colitis. LP-HFY11 decreased the expression of Firmicutes in the gut while increasing the expression of Bacteroidetes, Bifidobacteria, and Lactobacillus. This indicates that LP-HFY11 could control physiological alterations in the serum and colon tissue, as well as the expression of gut microorganism.

Fatty acids in Plantago asiatica seeds are responsible for the production of the pro-inflammatory mediator nitric oxide

Background: Plantago asiatica L. (Plantaginaceae) commonly grows in East Asia, with its seeds (Shazenshi), having been used as diuretic and anti-inflammatory drugs in traditional Japanese medicine. It is not known which constituents of P. asiatica seeds elicit the anti-inflammatory effects, such as reduced expression of the inducible nitric oxide synthase (iNOS) in interleukin (IL)-1β-treated hepatocytes which leads to a reduction in the pro-inflammatory mediator nitric oxide (NO). Objective: To identify the anti-inflammatory constituents of P. asiatica seeds, the anti-inflammatory activity of purified constituents was determined by assaying NO production in IL-1β-treated hepatocytes. Methods: P. asiatica seeds were extracted with 50% methanol and successively fractionated into three crude fractions with ethyl acetate (EtOAc) and n-butanol. The compounds were methylated and analyzed by gas chromatography–mass spectrometry (GC–MS). Primary cultured rat hepatocytes were prepared by collagenase perfusion, and P. asiatica seed extract (PASE), a fraction, or a compound was added to the culture medium with IL-1β and incubated at 37 °C. Potency of each fraction was determined by the Griess method for measuring the levels of nitrite in the medium. Results: PASE suppressed IL-1β-induced NO production without showing cytotoxicity, and an EtOAc-soluble fraction of PASE significantly inhibited NO production. GC–MS analysis detected 26 distinct fatty acids as their methyl esters in this fraction. Among them, three unsaturated fatty acids (linoleic, oleic, and α-linolenic acids) and palmitic acid were abundant. These unsaturated fatty acids are known to reduce NO levels. In contrast, acteoside and aucubin, which are thought to be present in the n-butanol-soluble fraction, showed only a low level of NO production suppression. Conclusion: The EtOAc-soluble fraction of PASE included many fatty acids, which may suppress the production of NO. The results imply that the anti-inflammatory activity of P. asiatica seeds may be produced by three unsaturated fatty acids. Because the fatty acids are abundant in the seeds of medicinal plants, they are likely to contribute to anti-inflammatory activity of the seeds. Keywords: Plantain seed, polyunsaturated fatty acid, Kampo medicine, hepatocyte, nitric oxide.

Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties

Food-derived soybean peptides possess inflammation-preventive effects beyond basic nutrition due to their structural configuration. However, the relationship between sequence characteristics and their anti-inflammatory properties remains poorly understood. The lack of targeted peptide production methods limits their use in functional foods. Hydrophobic and basic amino acids enhance peptide anti-inflammatory activity. Alcalase and Papain facilitate hydrophobic and basic amino acid preparation, while Bromelain lacks specific cleavage sites. Endopeptidases selectively break peptide bonds within proteins. We subsequently used Flavourzyme, lacking specific cleavage sites, to sequentially shorten peptide chains from their terminal ends. This sequential use of endo- and exopeptidases enables efficient peptide preparation. However, the proteases' ability to selectively produce peptides with specific sequence characteristics and their contribution to the soybean peptide mixture remain unknown. This study examined three soybean peptide groups prepared using distinct methods (Alcalase + Flavourzyme: hydrophobic amino acid-rich peptides, Papain + Flavourzyme: basic amino acid-rich peptides, and Bromelain + Flavourzyme: balanced amino acid compositions) with different sequence characterizations linked to endonuclease cleavage sites. All groups inhibited nitric oxide (NO) and inducible nitric oxide synthase (iNOS) secretion, suppressing protein phosphorylation in the Phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (Akt)/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (PI3K/Akt/NF-κB) signaling pathway. PF peptides showed the strongest anti-inflammatory effect, targeting iNOS, inhibiting TLR4, and reducing Akt and IκB phosphorylation. Mass spectrometry revealed correlations between regulatory mechanisms and the abundance of branched amino acids at PF peptides' N-terminals and significant Arg presence at their C-terminals. This study validates optimal enzymatic processes, introduces sequence characterization analysis, and identifies key features influencing anti-inflammatory properties, providing a theoretical basis for soybean peptide applications in inflammation-preventing functional foods.

MiR-122 overexpression alleviates oxygen-glucose deprivation-induced neuronal injury by targeting sPLA2-IIA.

Ischemic stroke (IS) is a neurological disease with significant disability and mortality. MicroRNAs were proven to be associated with cerebral ischemia. Previous studies have demonstrated miR-122 downregulation in both animal models of IS and the blood of IS patients. Nonetheless, the role and mechanism of miR-122-5p in IS remain unclear. We established primary human and mouse astrocytes, along with HT22 mouse hippocampal neuronal cells, through oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. To assess the impact of miR-122, we employed CCK8 assays, flow cytometry, RT-qPCR, western blotting, and ELISA to evaluate cell viability, apoptosis, reactive oxygen species (ROS) generation, and cytokine expression. A dual-luciferase reporter gene assay was employed to investigate the interaction between miR-122 and sPLA2-IIA. Overexpression of miR-122 resulted in decreased apoptosis, reduced cleaved caspase-3 expression, and increased cell viability in astrocytes and HT22 cells subjected to OGD/R. RT-qPCR and ELISA analyses demonstrated a decrease in mRNA and cytokine levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in both astrocytes and HT22 cells following miR-122 overexpression. Moreover, miR-122 overexpression reversed OGD/R-induced ROS levels and 8-OHdG formation in astrocytes. Additionally, miR-122 overexpression decreased the mRNA and protein expression of inducible nitric oxide synthase (iNOS). Furthermore, we found that miR-122 attaches to the 3'-UTR of sPLA2-IIA, thereby downregulate its expression. Our study demonstrates that miR-122-mediated inhibition of sPLA2-IIA attenuates OGD/R-induced neuronal injury by suppressing apoptosis, alleviating post-ischemic inflammation, and reducing ROS production. Thus, the miR-122/sPLA2-IIA axis may represent a promising target for IS treatment.

Increasing the radiation-induced cytotoxicity by silver nanoparticles and docetaxel in prostate cancer cells.

Radiation therapy is utilized for treatment of localized prostate cancer. Nevertheless, cancerous cells frequently develop radiation resistance. While higher radiation doses have not always been effective, radiosensitizers have been extensively studied for their ability to enhance the cytotoxic effects of radiation.So, this study aims to evaluate the possible radiosensitization effects of docetaxel (DTX) and silver nanoparticles (SNP) in LNCaP cells. The cytotoxic effects of DTX, SNP and2Gy of X-Ray radiation treatments were assessed in human LNCaP cell line using the MTT test after 24 h. Moreover, the effects of DTX, SNP and radiation on Epidermal growth factor (EGF), Caspase 3, inducible nitric oxide synthase and E-cadherin gene expression were analyzed using the Real-time PCR method. The level of Hydrogen peroxide (H2O2), an oxidative stress marker, was also detected 24h after various single and combined treatments. The combinations of SNP (in low toxic concentration) and/or DTX (0.25× IC50 and 0.5 × IC50 concentrations for triple and double combinations respectively) with radiation induced significant cytotoxicity in LNCaP cells in comparison to monotherapies. These cytotoxic effects were associated with the downregulation of EGF mRNA. Additionally, H2O2 levels increased after Radiation + SNP + DTX triple combination and double combinations including Radiation + SNP and Radiation + DTX versus single treatments. The triple combination treatment also increased Caspase 3 and and E-cadherin mRNA levels in compared to single treatments in LNCaP cells. Our results indicate that the combination of SNP and DTX with radiation induces significant anti-cancer effects. Upregulation of Caspase 3 and E-cadherin gene expression, and decreased mRNA expression level of EGF may be exerted specifically by use of this combination versus single treatments.

High nitrate levels in skeletal muscle contribute to nitric oxide generation via a nitrate/nitrite reductive pathway in mice that lack the nNOS enzyme.

Nitric oxide (NO) is a vasodilator gas that plays a critical role in mitochondrial respiration and skeletal muscle function. NO is endogenously generated by NO synthases: neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), or inducible NO synthase (iNOS). NO in skeletal muscle is partly generated by nNOS, and nNOS deficiency can contribute to muscular dystrophic diseases. However, we and others discovered an alternative nitrate/nitrite reductive pathway for NO generation: nitrate to nitrite to NO. We hypothesized that nitrate supplementation would increase nitrate accumulation in skeletal muscle and promote a nitrate/nitrite reductive pathway for NO production to compensate for the loss of nNOS in skeletal muscle. Wild-type (WT) and genetic nNOS knockout (nNOS-/-) mice were fed normal chow (386.9nmol/g nitrate) and subjected to three treatments: high-nitrate water (1g/L sodium nitrate for 7days), low-nitrate diet (46.8nmol/g nitrate for 7days), and low-nitrate diet followed by high-nitrate water for 7days each. High-nitrate water supplementation exhibited a greater and more significant increase in nitrate levels in skeletal muscle and blood in nNOS-/- mice than in WT mice. A low-nitrate diet decreased blood nitrate and nitrite levels in both WT and nNOS-/- mice. WT and nNOS-/- mice, treated with low-nitrate diet, followed by high-nitrate water supplementation, showed a significant increase in nitrate levels in skeletal muscle and blood, analogous to the increases observed in nNOS-/- mice supplemented with high-nitrate water. In skeletal muscle of nNOS-/- mice on high-nitrate water supplementation, on low-nitrate diet, and in low-high nitrate treatment, the loss of nNOS resulted in a corresponding increase in the expression of nitrate/nitrite reductive pathway-associated nitrate transporters [sialin and chloride channel 1 (CLC1)] and nitrate/nitrite reductase [xanthine oxidoreductase (XOR)] but did not show a compensatory increase in iNOS or eNOS protein and eNOS activation activity [p-eNOS (Ser1177)]. These findings suggest that a greater increase in nitrate levels in skeletal muscle of nNOS-/- mice on nitrate supplementation results from reductive processes to increase NO production with the loss of nNOS in skeletal muscle.

Evaluation of Anti-Inflammatory Activity in Methanolic Seed Extracts of International Sorghum bicolor L. Resources

Sorghum is an important cereal with high value as a health food ingredient because it contains various phenolic compounds. Anti-inflammatory activity was assessed using 12 sorghum resources collected from various countries to explore their potential as medicinal resources. The findings revealed that, at extract concentrations of 25 µg/mL and 50 µg/mL, cell survival rates were observed to be between 70 and 80% for most varieties, with the exception of K159081. In the analysis of anti-inflammatory activity, measured by the rate of nitric oxide (NO) production, sorghum varieties K159041 and K159081 exhibited NO production rates of 0.46 ± 0.38% and 2.58 ± 0.20%, respectively, indicating significant anti-inflammatory properties. The investigation into anti-inflammatory effects also included examining the expression of the inducible nitric oxide synthase (iNOS) gene, which is related to the inflammatory response triggered by LPS in macrophages. Varieties K159041, K159048, K159077, K159078, K159081, K159089, and K159096 were analyzed for this purpose. Further, an expression test of the cyclooxygenase 2 (COX-2) gene revealed values less than 0.4 in K159077, K159081, and K159089, suggesting these sorghum lines possess higher anti-inflammatory activity compared to others. Additionally, the expression analysis of tumor necrosis factor alpha (TNF-α), a gene identified as an inflammatory cytokine, showed that the mRNA levels in the lines K159048, K159077, K159078, K159088, K159089, K159093, and K159096 were expressed at lower levels relative to other sorghum resources, categorizing them as having high anti-inflammatory activity. Notably, the K159081 line exhibited the lowest expression level of all genes associated with inflammation, marking it as a valuable medicinal resource with potential development as an anti-inflammatory agent.

Characterization, In Silico Antimalarial, Antiinflammatory, Antioxidant, and ADMET Assessment of Neonauclea excelsa Merr.

In our study, we identified the phytoconstituents and carried out antimalarial, anti-inflammatory, antioxidant, and ADMET assessments of Neonauclea excelsa. The phytochemicals were detected and quantified followed by identification via GC-MS. The antimalaria, anti-inflammatory, and antioxidant assessments were done by molecular docking (MD) and molecular dynamics simulation (MDS) while ADMET by ADMET predictions. Saponins (27.33% ±1.20) and terpenes (8.33% ±0.73) were detected while alkaloids, steroids, glycosides, and flavonoids were absent. Exactly 29 compounds were identified with squalene being the most abundant (32.41%). Compound II exhibited the lowest BA (-6.4 kcal/mol) and Ki (20.12 µM), interacting with dihydrofolate reductase-thymidylate synthase. IV exhibited the lowest respective BA and Ki interacting with Plasmodium falciparum hexose transporter protein 1 (-6.2 kcal/mol and 28.20 µM), cyclo-oxygenase-2 (-7.2 kcal/mol and 5.21 µM), and myeloperoxidase (-7.4 kcal/mol and 3.71 µM). Compound VII had the lowest respective BA and Ki interacting with inducible nitric oxide synthase (-8.0 kcal/mol and 1.35 µM), xanthine oxidase (-7.2 kcal/mol and 5.21 µM), and cytochrome p450 21A2 (-7.0 kcal/mol and 7.30 µM). The MDS showed various cluster mobilities and residue fluctuations up to 5.26, 2.96, 5.10, 3.51, 5.02, 4.65, and 6.18 Å for dihydrofolate reductase-thymidylate synthase, Plasmodium falciparum hexose transporter protein 1, inducible nitric oxide synthase (INOS), cyclo-oxygenase-2 (COX2), xanthine oxidase (XO), cytochrome p450 21A2, and myeloperoxidase, respectively. Additionally, these compounds demonstrated good pharmacological properties with minimal toxicity. Conclusively, the identified compounds might be significant contributors to the antimalarial, anti-inflammatory, and antioxidant activity of N. excelsa and are good sources of novel antimalarial, anti-inflammatory, and antioxidant drugs.

Absence of PD-L1 signaling hinders macrophage defense against Mycobacterium tuberculosis via upregulating STAT3/IL-6 pathway

The blockade of programmed death-ligand 1 (PD-L1) pathway has been clinically used in cancer immunotherapy, while its effects on infectious diseases remain elusive. Roles of PD-L1 signaling in the macrophage-mediated innate immune defense against M.tb is unclear. In this study, the outcomes of tuberculosis (TB) in wild-type (WT) mice treated with anti-PD-1/PD-L1 therapy and macrophage-specific Pdl1-knockout (Pdl1ΔΜΦ) mice were compared. Treatment with anti-PD-L1 or anti-PD-1 benefited protection against M.tb infection in WT mice, while Pdl1ΔΜΦ mice exhibited the increased susceptibility to M.tb infection. Mechanistically, the absence of PD-L1 signaling impaired M.tb killing by macrophages. Furthermore, elevated STAT3 activation was found in PD-L1-deficient macrophages, leading to increased interleukin (IL)-6 production and reduced inducible nitric oxide synthase (iNOS) expression. Inhibiting STAT3 phosphorylation partially impeded the increase in IL-6 production and restored iNOS expression in these PD-L1-deficient cells. These findings provide valuable insights into the complexity and mechanisms underlying anti-PD-L1 therapy in the context of tuberculosis.

Impact of Inducible Nitric Oxide Synthase Activation on Endothelial Behavior under Magnesium Deficiency.

Endothelial dysfunction is a crucial event in the early pathogenesis of cardiovascular diseases and is linked to magnesium (Mg) deficiency. Indeed, in endothelial cells, low Mg levels promote the acquisition of a pro-inflammatory and pro-atherogenic phenotype. This paper investigates the mechanisms by which Mg deficiency promotes oxidative stress and affects endothelial behavior in human umbilical vascular endothelial cells (HUVECs). Our data show that low Mg levels trigger oxidative stress initially by increasing NAPDH oxidase activity and then by upregulating the pro-oxidant thioredoxin-interacting protein TXNIP. The overproduction of reactive oxygen species (ROS) activates NF-κB, leading to its increased binding to the inducible nitric oxide synthase (iNOS) promoter, with the consequent increase in iNOS expression. The increased levels of nitric oxide (NO) generated by upregulated iNOS contribute to disrupting endothelial cell function by inhibiting growth and increasing permeability. In conclusion, we provide evidence that multiple mechanisms contribute to generate a pro-oxidant state under low-Mg conditions, ultimately affecting endothelial physiology. These data add support to the notion that adequate Mg levels play a significant role in preserving cardiovascular health and may suggest new approaches to prevent or manage cardiovascular diseases.

Ziziphus jujuba Miller Ethanol Extract Restores Disrupted Intestinal Barrier Function via Tight Junction Recovery and Reduces Inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by the disruption of the intestinal barrier. The intestinal barrier is maintained by tight junctions (TJs), which sustain intestinal homeostasis and prevent pathogens from entering the microbiome and mucosal tissues. Ziziphus jujuba Miller (Z. jujuba) is a natural substance that has been used in traditional medicine as a therapy for a variety of diseases. However, in IBD, the efficacy of Z. jujuba is unknown. Therefore, we evaluated ZJB in Caco2 cells and a dextran sodium sulfate (DSS)-induced mouse model to demonstrate its efficacy in IBD. Z. jujuba extracts were prepared using 70% ethanol and were named ZJB. ZJB was found to be non-cytotoxic and to have excellent antioxidant effects. We confirmed its anti-inflammatory properties via the down-regulation of inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). To evaluate the effects of ZJB on intestinal barrier function and TJ improvement, the trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran 4 kDa (FITC-Dextran 4) permeability were assessed. The TEER value increased by 61.389% and permeability decreased by 27.348% in the 200 μg/mL ZJB group compared with the 50 ng/mL IL-6 group after 24 h. Additionally, ZJB alleviated body weight loss, reduced the disease activity index (DAI) score, and induced colon shortening in 5% DSS-induced mice; inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were down-regulated in the serum. TJ proteins, such as Zonula occludens (ZO)-1 and occludin, were up-regulated by ZJB in an impaired Caco2 mouse model. Additionally, according to the liquid chromatography results, in tandem with mass spectrometry (LC-MS/MS) analysis, seven active ingredients were detected in ZJB. In conclusion, ZJB down-regulated inflammatory factors, protected intestinal barrier function, and increased TJ proteins. It is thus a safe, natural substance with the potential to be used as a therapeutic agent in IBD treatment.

Protective Effects of an Oligo-Fucoidan-Based Formula against Osteoarthritis Development via iNOS and COX-2 Suppression following Monosodium Iodoacetate Injection.

Osteoarthritis (OA) is a debilitating joint disorder characterized by cartilage degradation and chronic inflammation, accompanied by high oxidative stress. In this study, we utilized the monosodium iodoacetate (MIA)-induced OA model to investigate the efficacy of oligo-fucoidan-based formula (FF) intervention in mitigating OA progression. Through its capacity to alleviate joint bearing function and inflammation, improvements in cartilage integrity following oligo-fucoidan-based formula intervention were observed, highlighting its protective effects against cartilage degeneration and structural damage. Furthermore, the oligo-fucoidan-based formula modulated the p38 signaling pathway, along with downregulating cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, contributing to its beneficial effects. Our study provides valuable insights into targeted interventions for OA management and calls for further clinical investigations to validate these preclinical findings and to explore the translational potential of an oligo-fucoidan-based formula in human OA patients.

Fermenting kale (Brassica oleracea L.) enhances its functional food properties by increasing accessibility of key phytochemicals and reducing antinutritional factors.

The properties of kale as a functional food are well established. We sought to determine how fermentation further enhances these properties. We tested different fermentation conditions: (i) spontaneous fermentation with naturally occurring bacteria, (ii) spontaneous fermentation with 2% salt, (iii) Lactococcus lactis, (iv) Lactobacillus acidophilus, (v) mixture of L. lactis and L. acidophilus, (vi) mixture of L. lactis, L. acidophilus, and Clostridium butyricum. We quantified selected bioactive components using high-performance liquid chromatography (HPLC) and antinutritional factors using a gravimetric method and spectrophotometry. We then determined (i) the antioxidant capacity of the vegetable, (ii) anti-inflammation capacity, and (iii) the surface microbiota composition by 16S sequencing. All fermentation methods imparted some benefits. However, fermentation with mixed culture of L. lactis and L. acidophilus was most effective in increasing polyphenols and sulforaphane accessibility, increasing antioxidant activity, and reducing antinutritional factors. Specifically, fermentation with L. lactis and L. acidophilus increased total polyphenols from 8.5 to 10.7 mgGAE/g (milligrams of gallium acid equivalent per gram) and sulforaphane from 960.8 to 1777 μg/g (microgram per gram) but decreased the antinutritional factors oxalate and tannin. Total oxalate was reduced by 49%, while tannin was reduced by 55%-65%. The antioxidant capacity was enhanced but not the anti-inflammation potential. Both unfermented and fermented kale protected equally against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and prevented increases in inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 messenger RNA (IL-6 mRNA) expression by 84.3%, 62%, 68%, and 85.5%, respectively. Unfermented and naturally fermented kale had high proportions of sulfur reducing Desulfubrio and Proteobacteria usually associated with inflammation. Fermenting with L. lactis and/or L. acidophilus changed the bacterial proportions, reducing the Proteobacteria while increasing the genera Lactobacilli and Lactococcus. In summary, fermentation enhances the well-known beneficial impacts of kale. Fermentation with mixed cultures of L. lactis and L. acidophilus imparts higher benefits compared to the single cultures or fermentation with native bacteria present in the vegetable.

Development of novel nitric oxide production inhibitors based on the 7H-pyrrolo[2,3-d]pyrimidine scaffold.

Nitric oxide (NO), the smallest signaling molecule known, can be excessively produced by overexpressed inducible nitric oxide synthase (iNOS), and eventually leads to multiple inflammatory related diseases. Thus, reducing the overexpression of NO represents as very potential anti-inflammatory strategy. In current study, a series of compounds were designed and synthesized based on the hybridization of 7H-pyrrolo[2,3-d]pyrimidine and cinnamamide fragments in order to develop novel NO production inhibitors. Among them, compound S2h displayed a vigorous inhibitory activity on NO production with an IC50 value of 3.21 ± 0.67 µM, which was much lower than that of the positive control Nω-nitro-L-arginine (L-NNA, IC50 = 28.36 ± 3.13 µM). Due to its obeying Lipinski's and Veber's rules that guarantee compounds with good oral bioavailability, S2h effectively suppressed the paw swelling in carrageenan-induced mice. Additionally, compound S2h formed clear interactions with iNOS protein according to the docking analysis. Therefore, compounds S2h is a promising lead compound for further development of potent iNOS inhibitors or anti-inflammatory agents.