LPS-induced Nitric Oxide Release Research Articles

Rubus fruticosus leaf extract inhibits vascular dementia-induced memory impairment and neuronal loss by attenuating neuroinflammation.

Vascular dementia (VaD) is characterized by progressive memory impairment, which is associated with microglia-mediated neuroinflammation. Polyphenol-rich natural plants, which possess anti-inflammatory activities, have attracted scientific interest worldwide. This study investigated whether Rubus fruticosus leaf extract (RFLE) can attenuate VaD. Sprague-Dawley rats were separated into five groups: SO, sham-operated and treated with vehicle; OP, operated and treated with vehicle; RFLE-L, operated and treated with low dose (30 mg/kg) of RFLE; RFLE-M, operated and treated with medium dose (60 mg/kg) of RFLE; and RFLE-H, operated and treated with high dose (90 mg/kg) of RFLE. Bilateral common carotid artery and hypotension were used as a modeling procedure, and the RFLE were intraorally administered for 5 days (preoperative 2 and postoperative 3 days). The rats then underwent memory tests including the novel object recognition, Y-maze, Barnes maze, and passive avoidance tests, and neuronal viability and neuroinflammation were quantified in their hippocampi. The results showed that the OP group exhibited VaD-associated memory deficits, neuronal death, and microglial activation in hippocampi, while the RFLE-treated groups showed significant attenuation in all above parameters. Next, using BV-2 microglial cells challenged with lipopolysaccharide (LPS), we evaluated the effects of RFLE in dynamics of proinflammatory mediators and the upstream signaling pathway. RFLE pretreatment significantly inhibited the LPS-induced release of nitric oxide, TNF-α, and IL-6 and upregulation of the MAPKs/NF-κB/iNOS pathway. Collectively, we suggest that RFLE can attenuate the histologic alterations and memory deficits accompanied by VaD, and these roles are, partly due to the attenuation of microglial activation.

Antioxidant and Antineuroinflammatory Mechanisms of Kaempferol-3-O-β-d-Glucuronate on Lipopolysaccharide-Stimulated BV2 Microglial Cells through the Nrf2/HO-1 Signaling Cascade and MAPK/NF-κB Pathway.

Aglycone- and glycoside-derived forms of flavonoids exist broadly in plants and foods such as fruits, vegetables, and peanuts. However, most studies focus on the bioavailability of flavonoid aglycone rather than its glycosylated form. Kaempferol-3-O-β-d-glucuronate (K3G) is a natural flavonoid glycoside obtained from various plants that have several biological activities, including antioxidant and anti-inflammatory effects. However, the molecular mechanism related to the antioxidant and antineuroinflammatory activity of K3G has not yet been demonstrated. The present study was designed to demonstrate the antioxidant and antineuroinflammatory effect of K3G against lipopolysaccharide (LPS)-stimulated BV2 microglial cells and to evaluate the underlying mechanism. Cell viability was determined by MTT assay. The inhibition rate of reactive oxygen species (ROS) and the production of pro-inflammatory mediators and cytokines were measured by DCF-DA assay, Griess assay, enzyme-linked immunosorbent assay (ELISA), and western blotting. K3G inhibited the LPS-induced release of nitric oxide, interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) as well as the expression of prostaglandin E synthase 2. Additionally, K3G reduced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB) related proteins. Mechanistic studies found that K3G downregulated phosphorylated mitogen-activated protein kinases (MAPKs) and upregulated the Nrf2/HO-1 signaling cascade. In this study, we demonstrated the effects of K3G on antineuroinflammation by inactivating phosphorylation of MPAKs and on antioxidants by upregulating the Nrf2/HO-1 signaling pathway through decreasing ROS in LPS-stimulated BV2 cells.

Isolation, structure elucidation and in-vitro evaluation of new trisubstituted Tetrahydrofuran Lignans from Rabdosia lophanthoides var. gerardiana as anti-inflammatory agents

Four new trisubstituted tetrahydrofuran lignans, named (−)-sesaminone-rutinoside ( 1 ), (+)-episesaminone-rutinoside ( 2 ) and rabdosiacosides B ( 3 ) and C ( 4 ) were isolated from the air-dried whole herbs of Rabdosia lophanthoides var. gerardiana together with two known compounds, (+)− 1-hydroxypinoresinol-1-β- d -glucoside ( 5 ) and rosmarinic acid ( 6 ). The structures of these new lignans were elucidated by a combination of mass spectrometry, 1D and 2D NMR experiments including distortionless enhancement by polarization transfer, 1 H– 1 H correlation, heteronuclear single quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser effect spectroscopies. Both the cell viability and their anti-inflammatory effects of the lignans were further studied. Among them, no significant cytotoxic activity was exhibited. Compound 4 dramatically suppressed LPS-induced nitric oxide release and reduced the release of the pro-inflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Overall, our findings indicate that compound 4 was noteworthy in reducing LPS-induced inflammatory responses in RAW264.7 macrophages. • Four new lignans were isolated and identified from Rabdosia lophanthoides. • All compounds exhibited no cytotoxic activity. • Rabdosiacoside C ( 4 ) suppressed LPS-induced NO release. • The release of pro-inflammatory cytokines was inhibited by rabdosiacoside C ( 4 ).

Silymarin-Functionalized Selenium Nanoparticles Prevent LPS-Induced Inflammatory Response in RAW264.7 Cells through Downregulation of the PI3K/Akt/NF-κB Pathway.

Silymarin exhibits an anti-inflammatory property in various cancers and inflammatory diseases. In our previous work, silymarin-mediated selenium nanoparticles (SeNPs) (Si-SeNPs) were developed using a green synthesis technique, and its potential as an anticancer agent was confirmed. In order to further examine the extended comprehensive potential of Si-SeNPs, this investigation focuses on studying the enhanced anti-inflammatory effect of Si-SeNPs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction were used to evaluate the expression of pro-inflammatory mediators and cytokines. Western blotting and immunofluorescence assays were conducted to assess the protein expression of p-PI3K, p-Akt, p-NF-κB, and p-IκBα. Compared to silymarin, Si-SeNPs exhibited a significantly increased inhibitory effect on LPS-induced release of nitric oxide and the expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β) in RAW264.7 cells. A western blot assay indicated that Si-SeNPs downregulated the PI3K/Akt and NF-κB signaling pathways. The immunofluorescence assay suggested that Si-SeNPs inhibited the nuclear translocation and the activation of NF-κB. In addition, 740 Y-P (PI3K agonist) was used to demonstrate that activating the PI3K/Akt signal could partially reverse the inflammatory response, suggesting a causal role of the PI3K/Akt signaling pathway in the anti-inflammatory effect of Si-SeNPs. Consequently, these findings indicate that Si-SeNPs could be a functional agent of the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages through inhibiting the PI3K/Akt/NF-κB signaling pathway. In addition, biosynthesized Si-SeNPs could be more effective at reducing inflammation than only silymarin extracts. Thus, this study lays an experimental foundation for the clinical application of using biosynthesized SeNPs as a novel candidate in the field of inflammation-associated diseases.

Anti-inflammatory withajardins from the leaves of Athenaea velutina

Withajardins, uncommon modified withanolide-type steroids, have been isolated exclusively from plants of the Solanaceae family so far. Two undescribed withajardins and the known tuboanosigenin were isolated from the hexane/EtOAc 1:1 extract from Athenaea velutina leaves. Their structures were established by an extensive analysis of 1D and 2D-NMR and HRMS data. The absolute configuration was determined by X-ray diffraction (withajardin L and tuboanosigenin) and circular dichroism (CD) analyses (withajardin M). The anti-inflammatory activity of compounds was evaluated through the inhibition of the lipopolysaccharide (LPS)-induced nitric oxide (NO), TNF-α, and IL-6 release in RAW264.7 cells. The cell viability effects to RAW 264.7 cells showed IC50 values of 74.4–354.4 μM. The compounds attenuated LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 in RAW264.7 cells.

Hypopurolides A - G, Labdane Diterpenoids from Hypoestes purpurea and Their Nitric Oxide Inhibitory Activity.

Seven new labdane diterpenoids, hypopurolides A-G (1-7) were discovered from the aerial part of Hypoestes purpurea, along with one known analog, hypopurin D (8). The structures of 1-7 were characterized based on 1 H-, 13 C-, and 2D-NMR, and HR-ESI-MS spectra. The absolute configurations of 1-7 were defined by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data. Compounds 1-8 were tested for their nitric oxide (NO) inhibitory and cytotoxic effects. Compound 6 displayed moderate inhibitory effect toward LPS-induced NO release in RAW 264.7 cells with an IC50 value of 41.50 μM.

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

BackgroundThe brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types.MethodsMouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function.ResultsHerein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function.ConclusionsAgo2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Polymethoxyflavones in Citrus Regulate Lipopolysaccharide-Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression.

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1β, ASC, and Caspase1, while other flavonoids only affected IL-1β and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

Two methoxy derivatives of resveratrol, 3,3\u2032,4,5\u2032-tetramethoxy-trans-stilbene and 3,4\u2032,5-trimethoxy-trans-stilbene, suppress lipopolysaccharide-induced inflammation through inactivation of MAPK and NF-\u03baB pathways in RAW 264.7 cells

Background3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and 3,4′,5-trimethoxy-trans-stilbene (3,4′,5-TMS) are two methoxy derivatives of resveratrol. Previous researches have proved that resveratrol and its analogues have anti-inflammatory effect through suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. This study aims to study whether 3,3′,4,5′-TMS and 3,4′,5-TMS alleviate inflammation and the underlying mechanism.MethodsRAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS) to induce inflammation and pretreated with 3,3′,4,5′-TMS or 3,4′,5-TMS. Cell viability was measured with the 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) release was detected by Griess reagent. The secretions of pro-inflammatory cytokines were assessed by ELISA kits. Protein expressions of signaling molecules were determined by Western blotting. Reactive oxygen species (ROS) production was detected by fluorescence staining and malondialdehyde (MDA) assay.Results3,3′,4,5′-TMS and 3,4′,5-TMS suppressed LPS-induced NO release and pro-inflammatory cytokines (IL-6 and TNF-α) secretions in a dose-dependent manner in RAW 264.7 cells. 3,3′,4,5′-TMS and 3,4′,5-TMS significantly down-regulated the LPS-induced expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and partially suppressed the activation of MAPK (phosphorylation of p38, JNK, ERK), and NF-κB (phosphorylation of IKKα/β, p65 and IκBα) signaling pathways; where phosphorylation of ERK and p65 was mildly but not significantly decreased by 3,3′,4,5′-TMS. LPS-induced NF-κB/p65 nuclear translocation was inhibited by both 3,3′,4,5′-TMS and 3,4′,5-TMS. Moreover, both resveratrol derivatives decreased the ROS levels.Conclusions3,3′,4,5′-TMS and 3,4′,5-TMS significantly suppress LPS-induced inflammation in RAW 264.7 cells through inhibition of MAPK and NF-κB signaling pathways and also provide anti-oxidative effect. This study reveals potential therapeutic applications of 3,3′,4,5′-TMS and 3,4′,5-TMS for inflammatory diseases.

Anti-Inflammatory Effect of Alpha7 Nicotinic Acetylcholine Receptor Modulators on BV2 Cells

Objective: The development of neuroinflammation shares numerous risk factors and involves many complex interactions which contribute to disease pathology. An important cell type in neuroinflammation is the active microglia cell – the resident immune cell of the CNS. There is increasing need to understand how these pathways related to neuroinflammation work and how they can be regulated. Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated receptors and widely distributed in the brain. The α7 nAChR is a penta-homomeric receptor and is one of the nAChRs expressed in microglia. This study was first designed to characterize the effects of lipopolysaccharide (LPS) on BV2 culture cells, a cell line of murine microglia origin, on release of inflammatory markers and to characterize the inhibitory effects of α7 nAChR modulators in these cells. Methods: First, the BV2 cell cultures were functionally validated by exposing them to LPS for 4–24 h and then examining the release of tumor necrosis factor-alpha (TNF-α) using ELISA and nitric oxide (NO) release using the Griess assay, respectively. Next, α7 nAChR modulators with different pharmacological profiles were applied dose-dependently to study their effects on LPS-induced release of NO and TNF-α. Results: The time-course and dose-response curve revealed that LPS dose-dependently activated (EC50 = 2.5 ng/mL) BV2 cells releasing TNF-α at 4 h, followed by release of NO that occurred first at 8-h time point. The α7 nAChR subunit mRNA was identified in the BV2 cells. The pharmacology studies showed the α7 nAChR selective modulators NS6740 and TQS reduced NO and cytokine release from BV2 cell cultures. Conclusion: We here identified the dose- and time-dependent effects of LPS in BV2 cell cultures on several inflammatory readouts and showed that α7 nAChR modulators with little or no ion channel activity inhibited this anti-inflammatory mechanism.

Dp44mT regulates the levels of inflammatory mediators through blocking NF-κB nuclear translocation in LPS-stimulated RAW 264.7 macrophages.

Inflammation is increased by infection with pathogens such as viruses, bacteria, and parasites. High levels of inflammatory mediators and infiltration of macrophages into inflammatory lesions were reported in severe inflammatory diseases. Here, the aim of this study was to evaluate an anti-inflammatory activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Dp44mT (1–100 ng/mL) had no effect on viability of RAW 264.7 macrophages. Dp44mT (100 ng/mL) significantly reduced LPS-induced release of nitric oxide and expression of inducible nitric oxide synthase and cyclooxygenase-2. A significant upregulation of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by LPS stimulation was downregulated by treatment with Dp44mT. Dp44mT blocked activation of nuclear factor-κB by the interruption of IκBα phosphorylation. Dp44mT suppressed the phagocytosis. Furthermore, administration of Dp44mT significantly reduced the serum levels of TNF-α and IL-6 in LPS-treated mice without side effects. In conclusion, these results indicate that Dp44mT has an anti-inflammatory activity and may be of therapeutic significant for the prevention and treatment of inflammatory diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11626-021-00552-y.

Anti-inflammatory effects of Lonomia obliqua caterpillar protein rlosac on macrophages and chondrocytes

Purpose: Ostheoarthritis (OA) is a disease characterized by the degeneration of articular cartilage. The role of activated macrophages in the synovial inflammation has been reported to contribute with the knee OA. Activated macrophages increase inflammatory mediators in the synovial fluid, such as IL-1β, TNF-α and nitric oxide (NO), acting on chondrocytes which in turn produce an excess of matrix metalloproteinases (MMPs) and aggrecanases ), which result in the degradation of extracellular matrix (ECM) components such as collagen II (Col-2) and aggrecan. Chondrocytes respond to these stimuli by secreting cytokines like IL-1β, TNFα, IL-6 and IL-8, and altering gene expression leading to a feedback activation of inflammation. Studies using the bristle extract of the Lonomia obliqua caterpillar (LOCBE) allowed the identification of the protein LOSAC, a cytoprotective molecule with effect on serum-deprived cells including HUVECs, fibroblasts, and neurons. Moreover, rLOSAC was reported to promote wound healing (in vivo) by increasing expression of ECM involved in tissue remodeling (as collagen), preservation of substrate attachment and ECM integrity in fibroblast, and prevention of ROS generation, suggesting an anti-oxidative activity. The aim of this work was to evaluate the cytotoxic effects of rLOSAC in chondrocytes and macrophages and to establish if this protein has anti-inflammatory activity on these types of cells. Methods: For this study, rLOSAC was obtained in bacteria system as previously reported. Normal Human Articular Primary human chondrocytes cells (NHAC-Kn) were obtained from LONZA and used until passage 6 for all the assays. Expansion and subculture of chondrocytes were performed according the supplier. Peritoneal macrophage were obtained from male Wistar rats (I. Butantan Ethical Committee CEUA n° 9139050517. For chondrocytes and macrophages inflammation was induced by treatment with 1 ng/mL IL-1β or 1 μg/mL LPS, respectively. Under these conditions, rLOSAC was added and the cytoprotective effect assessed by the MTT assay. Expression of metalloprotease-1 (MMP-1) and NF-kB-p65 translocation were analyzed by the ImageXpress Micro-Confocal High-Content Screening (HCS) with specific antibodies. Cytokines present in the supernatants, after 24h of treatment, were assayed using a Milliplex MAP Cytokine/Chemokine Magnetic Bead Panel with detection and analysis using the Multiplex system (Millipore). The kit was performed according to the manufacturer’s specification. Range of detection was in pg/mL and designed for 3 analytes for chondrocytes (IL-6, IL-8 and TNF-α), and 10 cytokines for macrophages (IL-1α, MIP-1α, IL-4, IL-6, IL-10, IL-17, GRO/KC/CINC-1, LIX, TNF-α, Rantes). Results: To verify the toxic effect of this protein, several concentrations of rLOSAC, in the presence or absence of LPS (macrophages) or IL-1β (chondrocytes) were added to the cells. None concentrations assayed were toxic to those cells. Therefore, rLOSAC showed cytoprotective effect at the nanomolar concentration, while under inflammatory conditions (in the presence of 1ng/mL IL-1β), rLOSAC was cytoprotective at the concentrations of 10 and 20 nM. Regarding chondrocytes, the expression of MMP-1 and NF-kB translocation were analyzed by HCS using IL-1β at 1ng/ml and 24h of incubation for cytokines and MMP-1 release, and 30 min for NF-κB-p65, as well as, the nuclei count. rLOSAC was able to reduce the release of IL-6, IL-8 and TNF-α in a statistically significant manner. However, rLOSAC was no able to affect the LPS-induced NO release in macrophages. rLOSAC did not induced directly NF-κB-p65 translocation. However, in the presence of 1ng/mL IL-1β, some concentrations of rLOSAC and LOCBE increased the translocation of NF-κB in chondrocytes. MMP-1 release was reduced in the presence of rLOSAC in chondrocytes. In macrophages, rLOSAC was also no toxic to the cells. Ten cytokines/chemokines were analyzed in LPS-treated or not-treated macrophages supernatants in the presence or absence of rLOSAC. rLOSAC alone was able to reduce the release of IL-4, IL-1α and TNF-α. Both, in the presence or absence of LPS, rLOSAC was able to induce the release of IL-10, a potent anti-inflammatory cytokine. Conclusions: The data presented in this report showed that the rLOSAC shows a cytoprotective and anti-inflammatory activity. Further studies will be performed with co-culture of chondrocytes and activated macrophages in order to confirm the anti-inflammatory effect of rLOSAC. This makes it clear that rLOSAC is a potential molecule to be explored in order to seek new molecular targets that may be related to osteoarthritis. The combination of HCS and cytokines release analysis had shown to be effective for the screening of new compounds or molecular targets/pathways for OA. Supported by Grant 2015/50040-4, São Paulo Research Foundation (FAPESP), GlaxoSmithKline and Instituto Butantan; FAPESP Grant 2018/04467-4, FAPESP Grant 2013/07467-1 (CETICs).

Anti-Helicobacter pylori, Anti-Inflammatory, Cytotoxic, and Antioxidant Activities of Mace Extracts from Myristica fragrans.

The aril (mace) of Myristica fragrans, known as Dok-Chan, is a spice that has long been used for treating stomach discomfort, peptic ulcer, and nausea. It is an ingredient in many remedies in Thai traditional medicine, e.g., Ya-Hom-Thep-Bha-Jit, Ya-Hom-Nao-Wa-Kot, and Ya-That-Bun-Job, which are used to treat dyspepsia and other gastrointestinal tract symptoms. The aqueous and ethanolic extracts of mace were used for all tests. Anti-H. pylori activities were determined by the disc diffusion method and agar dilution. Anti-inflammatory activity was determined by the LPS-induced nitric oxide (NO) inhibition in a RAW264.7 cell line, and cytotoxicity was determined against gastric cancer cell lines (Kato III) using the sulphorhodamine B (SRB) assay. The DPPH radical scavenging and ABTS radical cation decolorization assays were used to determine the antioxidant activities. The result found that the ethanolic extract of mace exhibited antimicrobial activity against H. pylori ATCC 43504 and six clinical strains with MIC values of 125–250 μg/ml. The aqueous extract MICs against H. pylori ATCC reference strain and six clinical strains were 500 μg/ml compared with 0.5 μg/ml for the positive control, clarithromycin. The inhibitory effect of LPS-induced NO release and cytotoxic activity of the ethanolic extract had IC50 values of 82.19 μg/ml and 26.06 μg/ml, respectively, and the EC50 values for the DPPH and ABTS antioxidant assays were 13.41 μg/ml and 12.44 μg/ml, respectively. The mace extract also had anticancer properties. In conclusion, the ethanolic mace extract had anti-H. pylori, anti-inflammatory, antioxidant, and anticancer activities. These data support further preclinical and clinical investigation to see if the mace extract could have a role in treating patients with dyspepsia, peptic ulcers, and possibly gastric cancer.

Three new triterpenoids from Mallotus macrostachyus

Three new triterpenoids, mallomacrostins A-C (1–3), and 11 known ones (4–14) were obtained from the twigs and leaves of Mallotus macrostachyus. Mallomacrostin A possessed a new trinor-D:B-friedobaccharane skeleton. The structures of the new compounds were elucidated on the basis of extensive spectroscopic techniques including HR-ESIMS and NMR and the structure of 1 was confirmed by single crystal X-ray diffraction analysis. Spectroscopic data of the known compound 4 were provided for the first time. Compounds 2 and 10 exhibited significant anti-inflammatory activity by inhibiting LPS-induced release of nitric oxide with IC50 of 70.0 μM and 14.0 μM, respectively.

Green synthesis of gold nanoparticles using Euphrasia officinalisleaf extract to inhibit lipopolysaccharide-induced inflammation through NF-κB and JAK/STAT pathways in RAW 264.7 macrophages.

BackgroundGold nanoparticles (AuNPs) have potential applications in the treatment and diagnosis process, which are attributed to their biocompatibility and high efficiency of drug delivery. In the current study, we utilized an extract of Euphrasia officinalis, a traditional folk medicine, to synthesize gold nanoparticles (EO-AuNPs), and investigated their anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Materials and methodsThe AuNPs were synthesized from an ethanol extract of E. officinalis leaves and characterized using several analytical techniques. Anti-inflammatory activities of EO-AuNPs were detected by a model of LPS-induced upregulation of inflammatory mediators and cytokines including nitric oxide (NO), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 in RAW 264.7 cells. The activation of nuclear factor (NF)-κB and Janus kinase/signal transducer and activators of transcription (JAK/STAT) signaling pathways was investigated by Western blot.ResultsThe results confirmed the successful synthesis of AuNPs by E. officinalis. Transmission electron microscopy images showed obvious uptake of EO-AuNPs and internalization into intracellular membrane–bound compartments, resembling endosomes and lysosomes by RAW 264.7 cells. Cell viability assays showed that EO-AuNPs exhibited little cytotoxicity in RAW 264.7 cells at 100 µg/mL concentration after 24 hours. EO-AuNPs significantly suppressed the LPS-induced release of NO, TNF-α, IL-1β, and IL-6 as well as the expression of the iNOS gene and protein in RAW 264.7 cells. Further experiments demonstrated that pretreatment with EO-AuNPs significantly reduced the phosphorylation and degradation of inhibitor kappa B-alpha and inhibited the nuclear translocation of NF-κB p65. In addition, EO-AuNPs suppressed LPS-stimulated inflammation by blocking the activation of JAK/STAT pathway.ConclusionThe synthesized EO-AuNPs showed anti-inflammatory activity in LPS-induced RAW 264.7 cells, suggesting they may be potential candidates for treating inflammatory-mediated diseases.

Pterostilbene and 4'-Methoxyresveratrol Inhibited Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages.

Pterostilbene (Pte) and 4′-Methoxyresveratrol (4MR) are methylated derivatives of resveratrol. We investigated the anti-inflammatory effect of Pte and 4MR in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. Both Pte and 4MR significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression. Moreover, both of them inhibited LPS-induced mRNA expression of inflammatory cytokines including monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6 and IL-1β, and tumor necrosis factor α (TNF-α), and attenuated LPS-induced nuclear factor-κB (NF-κB) activation by decreasing p65 phosphorylation. In addition, 4MR but not Pte inhibited LPS-induced the activator protein (AP)-1 pathway in RAW 264.7 macrophages. Further study suggested that Pte had an inhibitory effect on extracellular regulated protein kinases (ERK) and p38 activation, but not on c-Jun N-terminal kinase (JNK), while 4MR had an inhibitory effect on JNK and p38 activation, but not on ERK. Taken together, our data suggested that Pte induced anti-inflammatory activity by blocking mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, while 4MR showed anti-inflammatory activity through suppression of MAPK, AP-1, and NF-κB signaling pathways in LPS-treated RAW 264.7 macrophages.

Suppression of Proinflamatroy cytokine gene expression with Aqueous Extract of Berberin

Based on knowledge from traditional Iranian herbal medicine, this ex vitro study aims to examine the anti-inflammatory mechanism of Berberis vulgaris L by measuring the expression and release of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), cyclooxygenase-1 (COX-1), and interleukine-18 (IL-6), IL-1β and inducible nitric oxide synthase (iNOS) in THP-1. The effects were assessed by measuring the levels of secretory proteins and mRNA of TNF-α and IL-6, the levels of nitric oxide (NO) secretion and the expression of iNOS in THP-1 cells. Cells were treated with 20 ng lipopolysaccharide/ml (LPS) in the presence and absence of concentrations of extracts from the Berberis vulgaris L. During the entire experimental period, we used extract concentrations (0.3 μg/mL) that had no cytotoxic effects, as measured with MTT, MTT and LDH assays. Berberis vulgaris extracts remarkably suppressed the LPS-induced NO release, significantly attenuated the LPS-induced transcription of iNOS and inhibited in a dose-dependent manner the expression and release of TNF-α. significant effects were observed on the release of IL-6. Taken together, these results suggest that Berberis vulgaris probably exerts anti-inflammatory effects through the suppression of TNF-α and iNOS expressions.

Stachaegyptin A-C: Neo-clerodane diterpenes from Stachys aegyptiaca

Phytochemical investigation of Stachys aegyptiaca resulted in the characterization of three new diterpenes (1-3) together with eleven known compounds including four neo-clerodane diterpenes and seven flavonoid aglycones. Structure elucidation was performed by spectroscopic analysis by HRFABMS, 1D and 2D NMR and X-ray. Isolated compounds were screened for anti-inflammatory activity using a lipopolysaccharide-induced nitric oxide inhibition assay employing murine macrophage cells. Among the assayed compounds, 13 (calycopterin) showed a concentration-dependent inhibition of LPS-induced nitric oxide release with a IC50 of 62.5μM.

Effects of hypnotic bromovalerylurea on microglial BV2 cells

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

Anti-Inflammatory Strategy for M2 Microglial Polarization Using Retinoic Acid-Loaded Nanoparticles.

Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP) as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2). RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state), promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases.