Reactive Oxygen Species In RAW Research Articles

Facile Preparation of Multifunctional Hydrogels with Sustained Resveratrol Release Ability for Bone Tissue Regeneration.

Injectable hydrogels show great promise for bone tissue engineering applications due to their high biocompatibility and drug delivery capabilities. The bone defects in osteoporosis are usually characterized by an oxidative and inflammatory microenvironment that impairs the regeneration capability of bone tissues. To attenuate the reactive oxygen species (ROS) and promote bone regeneration, an anti-oxidative hydrogel with osteogenic capacity was developed in this study. The poorly water soluble, natural antioxidant, resveratrol, was encapsulated in thiolated Pluronic F-127 micelles with over 50-times-enhanced solubility. The injectable hydrogel was facilely formed because of the new thioester bond between the free thiol group in modified F-127 and the arylate group in hyaluronic acid (HA)-acrylate. The resveratrol-loaded hydrogel showed good viscoelastic properties and in vitro stability and was cyto-compatible with bone-marrow-derived mesenchymal stem cells (BMSCs). The hydrogel allowed for a sustained release of resveratrol for at least two weeks and effectively enhanced the osteogenic differentiation of BMSCs by the up-regulation of osteogenic markers, including ALP, OCN, RUNX-2, and COL1. Moreover, the hydrogel exhibited anti-oxidative and anti-inflammatory abilities through the scavenging of intracellular ROS in RAW264.7 cells and inhibiting the gene expression and secretion of pro-inflammatory cytokines TNF-α and IL-1β under LPS exposure. In summary, the results suggest that our multifunctional hydrogel loaded with resveratrol bearing osteogenic, anti-oxidative, and anti-inflammatory actions is easily prepared and represents a promising resveratrol delivery platform for the repair of osteoporotic bone defects.

Structural Characterization and Anti-Inflammatory Activity of Polysaccharides from Tremella fuciformis on Monosodium Urate-Stimulated RAW264.7 Macrophages.

The structural characteristics and anti-inflammatory activity of Tremella fuciformis polysaccharides (TFPs) were investigated. The study showed that TFPs were mainly composed of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose. TFPs significantly inhibited monosodium urate (MSU)-induced inflammation of RAW264.7 cells, as well as the secretion levels of TNF-α, IL-1β, and IL-18 cytokines. The concentrations of malondialdehyde and reactive oxygen species in RAW264.7 macrophages were reduced, but superoxide dismutase activity was increased. RNA-Seq technology was applied to explore the mechanisms of TFPs ameliorating MSU-induced inflammation of RAW264.7 macrophages. Results revealed that TFPs significantly reduce MSU-stimulated inflammatory damage in RAW 264.7 cells by inhibiting signaling pathways like the hypoxia inducible factor-1 (HIF-1) signaling pathway and erythroblastic oncogene B (ErbB) signaling pathway. This study provides a foundation for TFPs to be developed as novel anti-inflammatory drugs.

Structural Characterization and Immunomodulatory Effects of Polysaccharide PCP‐1 from Pleurotus citrinopileatus

AbstractThe use of fungal polysaccharides as immunomodulators has garnered significant interest. Here, the polysaccharide PCP‐1 is isolated and purified from the fruiting bodies of Pleurotus citrinopileatus. PCP‐1 is identified as a glucan with a molecular weight of 1670 kDa. Methylation analysis reveals that PCP‐1 consists of T‐Glcp, T‐Rhap, 1,3‐Glcp, 1,4‐Glcp, 1,4,6‐Glcp, and 1,3,6‐Glcp. PCP‐1 exhibits exceptional immunomodulatory effects, as it can dose‐dependently stimulate phagocytosis and promote the secretion of nitric oxide and reactive oxygen species in RAW264.7 cells. Additionally, PCP‐1 significantly enhances the secretion of cytokines such as TNF‐α, IL‐1β, and IL‐6 at both the protein and mRNA levels, with concentrations ranging from 2.5 to 40 µg mL−1. At a concentration of 40 µg mL−1, the immunomodulatory effects are comparable to those of the positive control. Furthermore, PCP‐1 promotes the nuclear translocation of the p65 protein, indicating activation of the NF‐κB signaling pathway. The results demonstrate that PCP‐1 has the potential as a natural immunomodulator, with possible applications in the food, pharmaceutical, and other industries.

Immunomodulatory Effects of Nervonic Acid in RAW264.7 Macrophages

The present work studies the immunomodulatory potential of nervonic acid (NA) on the growth and proliferation of murine RAW264.7 macrophages. The immunomodulatory effects of NA were assessed through the evaluation of nitric oxide (NO) levels, cytokine secretion, phagocytic activity, reactive oxygen species (ROS) generation, and nuclear factor-κB (NF-κB) signaling pathway of RAW264.7 macrophages. The methodology used included a cell counting kit-8 (CCK-8) technique, enzyme-linked immunosorbent assay (ELISA), neutral red phagocytosis test, flow cytometry, western blotting, and immunofluorescence, respectively. Obtained outcomes pointed to NA as a potential promoter of the production of NO, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and ROS in RAW264.7 cells. Meanwhile, the expression of iNOS, TNF-α, IL-1β, and IL-6 mRNA was upregulated. Besides, the expression of TLR4 protein and the phosphorylation level of NF-κB were increased in RAW264.7 cell lines in a concentration-dependent manner when compared against the control. As a conclusion, NA can activate the NF-κB signaling pathway through the TLR4 signaling pathway, which has immunomodulatory potential.

Effect of antioxidant and anti-inflammatory on bioactive components of carrot (Daucus carota L.) leaves from Jeju Island

The present study evaluated the potential of carrot (Daucus carota L.) leaf, a non-edible part of carrots, which are among the most consumed vegetables worldwide. The antioxidant activities of 70% ethanol extract (EEC) and hot water extract (HEC) of carrot leaves were compared. The results revealed that the total polyphenol content, total flavonoid content, and DPPH and ABTS radical scavenging activities were higher in EEC than in HEC. Both extracts protected the cells against H2O2-induced toxicity and markedly reduced the levels of reactive oxygen species in RAW 264.7 cells. Moreover, pretreatment of RAW 264.7 cells with EEC and HEC prior to H2O2 (500 μM) exposure increased superoxide dismutase and glutathione peroxidase activities in these cells. Notably, EEC and HEC increased intracellular catalase activity by 36.77 and 6.39 times, respectively. Compared to HEC, EEC remarkably inhibited the production of lipopolysaccharide-induced nitric oxide and reduced the gene expression of IL-6, IL-1β, iNOS, COX-2, and TNF-α. Comparative analysis of the composition of the extracts using HPLC–UV suggested notably higher contents of catechin, chlorogenic acid, caffeic acid, rutin, quercetin, and cynaroside in EEC than in HEC. Collectively, these results imply that carrot leaves are a potentially beneficial natural source of antioxidants and anti-inflammatory compounds in functional foods.

Human TFF2-Fc fusion protein alleviates DSS-induced ulcerative colitis in C57BL/6 mice by promoting intestinal epithelial cells repair and inhibiting macrophage inflammation.

The clinical drugs for ulcerative colitis mainly affect the inflammatory symposiums with limited outcomes and various side effects. Repairing the damaged intestinal mucosa is a promising and alternative strategy to treat ulcerative colitis. Trefoil factor family 2 (TFF2) could repair the intestinal mucosa, however, it has a short half-life in vivo. To improve the stability of TFF2, we have prepared a new fusion protein TFF2-Fc with much stability, investigated the therapeutic effect of TFF2-Fc on ulcerative colitis, and further illustrated the related mechanisms. We found that intrarectally administered TFF2-Fc alleviated the weight loss, the colon shortening, the disease activity index, the intestinal tissue injury, and the lymphocyte infiltration in dextran sulfate sodium (DSS)-induced colitis mice. In vitro, TFF2-Fc inhibited Caco2 cells injury and apoptosis, promoted cellular migration, and increased the expression of Occludin and ZO-1 by activating P-ERK in the presence of H2O2 or inflammatory conditioned medium (LPS-RAW264.7/CM). Moreover, TFF2-Fc could reduce lipopolysaccharide (LPS)-induced production of inflammation cytokines and reactive oxygen species in RAW264.7 cells, and also inhibits the polarization of RAW264.7 cells to M1 phenotype by reducing glucose consumption and lactate production. Taken together, in this work, we have prepared a novel fusion protein TFF2-Fc, which could alleviate ulcerative colitis in vivo via promoting intestinal epithelial cells repair and inhibiting macrophage inflammation, and TFF2-Fc might serve as a promising ulcerative colitis therapeutic agent.

Effect of selenized Athyrium multidentatum (Doll.) Ching polysaccharides on TNF / MAPKs / NF‐κB signaling pathways in RAW 264.7 cells

Polysaccharides from Athyrium multidentatum (Doll.) Ching (AMC) were fractionated and selenylated by HNO3-Na2SeO3 method. Four selenized polysaccharides were synthesized and fully characterized by ICP-MS, UV, FT-IR, 1H NMR, high-performance gel permeation chromatography, high-performance liquid chromatography (HPLC), and SEM. Chemical analysis demonstrated that the selenized polysaccharides were composed of mannose (Man), rhamnose (Rha), glucuronic acid (Glc A), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc) with different molar ratios and molecular weights. The immunoactivity results showed that the selenized polysaccharides elevated cell proliferation, phagocytic activity, and promoted the production of TNF-α, IL-6, IL-1β, IFN-γ, IL-10, NO, and reactive oxygen species in RAW 264.7 cells, indicating marked immune enhancement activities. RT-qPCR and Western blot assays revealed that the selenized AMC polysaccharides upregulated the expression levels of many key factors involved in TNF, MAPKs, and NF-κB signaling pathways. TNF/MAPKs/NF-κB signaling pathways might play crucial roles in the immune function of selenized AMC polysaccharides. Practical applications Polysaccharide is an essential nutrient in Athyrium multidentatum (Doll.) Ching. The selenized AMC polysaccharide possesses the activities of organic selenium and polysaccharide, making it abroad application prospects in the fields of medicine, food, and cosmetics.

Cinnamaldehyde protects against ligature-induced periodontitis through the inhibition of microbial accumulation and inflammatory responses of host immune cells.

Cinnamaldehyde (CA), the main active ingredient in cinnamon, has been proved to be a potential candidate for controlling inflammation; however, there has been little evidence demonstrating its role in alleviating periodontitis. The aim of this study was to investigate the effect of orally administered CA on ligature-induced periodontitis in mice and the administration of CA on the Porphyromonas gingivalis (Pg) supernatant-induced inflammatory responses of murine macrophages and human periodontal ligament cells (HPDLCs). In vivo experiments showed that the oral administration of CA significantly inhibited bone resorption, the accumulation of anaerobic bacteria and host immuno-inflammatory responses in ligature-induced periodontitis in mice. In vitro, CA inhibited the expression of Pg supernatant-induced IL6, IL8, TNFA and IL1B and reactive oxygen species in RAW 264.7 and HPDLCs, involving the inactivation of the NFKB signaling pathway, which was activated by the Pg supernatant. Also, the expression of adherent and chemotactic-related cytokines was inhibited by CA, accompanied with a reduction in adherent HPDLCs. Moreover, CA ameliorated the cellular senescence of HPDLCs induced by H2O2, together with a decrease in senescence-associated-β-galactosidase positive cells and decrease in the expression of P53, P21 and P16. Furthermore, CA promoted the osteogenic differentiation of HPDLCs with an increase in alkaline phosphatase expression and activity, formation of more mineralization nodules, and increased the expression of bone sialoprotein and osteopontin. Conclusions: Daily diet-added CA may be beneficial for oral health care, especially for the control of periodontic disease by suppressing the dysbiosis of biofilms and inhibiting the immunoinflammatory responses of migrated macrophages and local resident periodontal ligament cells to specific pathogen irritations.

The human milk oligosaccharide 2′-fucosyllactose attenuates β-lactoglobulin–induced food allergy through the miR-146a–mediated toll-like receptor 4/nuclear factor-κB signaling pathway

Our previous experiments have confirmed that human milk oligosaccharides (HMO) and its main component 2'-fucosyllactose (2'-FL), as prebiotics, could effectively alleviate cow milk allergy by regulating the intestinal microecology. This study intended to further explore the molecular mechanism of HMO regulating intestinal immunity. The results of the allergic mouse model showed that oral administration of 2'-FL or HMO reduced β-lactoglobulin (β-LG)-induced serum-specific IgE secretion and mast cell degranulation, while reducing the inflammatory cytokines, TNF-α, IL-4, and IL-6 production and promoting the miR-146a expression. In vitro results further confirmed that 2'-FL or HMO treatment reduced allergen-induced secretion of iNOS, NO, pro-inflammatory cytokines and reactive oxygen species in RAW264.7 cells. At the same time, in contrast to the β-LG group, 2'-FL dose-dependently inhibited the TLR4/NF-κB inflammatory pathway and upregulated miR-146a expression, and the effect of the 2'-FL mid-dose group was similar to that of the HMO intervention group. In particular, adding miR-146a inhibitors to macrophages attenuated the inhibitory effect of 2'-FL on the expression of TRAF6 and IRAKI in the TLR4 pathway, suggesting that miR-146a might be involved in the immune regulation of 2'-FL. The above results indicated that 2'-FL had a similar effect to HMOs, and its effect of reducing β-LG allergy might be related to the regulation of miR-146a to inhibit TLR4/NF-κB signaling pathway.

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.

Tuna blood inhibits lipopolysaccharide-induced inflammatory mediators in RAW264.7 macrophages

Background: This study aimed to evaluate the anti-inflammation effects of the freeze-dried tuna whole blood (FTB), and freeze-dried tuna blood cell (FTC) in LPS-induced RAW264.7 cells.Methods: The RAW264.7 cells were pre-administered with FTB and FTC at different concentrations for 2 h and then stimulated with lipopolysaccharide (LPS) for 24 h. The production of reactive oxygen species (ROS), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) in RAW 264.7 cells were then determined.Results: The results showed that only FTB remarkably abolished LPS-induced ROS in RAW264.7 cells. FTB and FTC significantly decreased LPS-induced NO which IC50 values of FTB and FTC after 24 h were 78.58 and 22.47 μg/mL, respectively. TNF-α and IL-1β secretion were abolished by FTB and FTC in LPS-stimulated macrophages which IC50 values of both FTB and FTC after 24 h were more than 25 μg/mL, respectively. However, the efficacy of FTC against inflammatory mediators was due to cytotoxic effects on RAW 264.7 cells.Conclusion: Tuna whole blood potentially inhibits inflammation through modulating the synthesis of several mediators and cytokines associated with the development of inflammation. These findings suggest a role of tuna blood on anti-inflammatory activity.Keywords: Anti-inflammatory activity, RAW 264.7 cells, red blood cell, tuna blood, waste utilization

Ferumoxytol-β-glucan Inhibits Melanoma Growth via Interacting with Dectin-1 to Polarize Macrophages into M1 Phenotype.

Background: Regulating the polarization of macrophages to antitumor M1 macrophages is a promising strategy for overcoming the immunosuppression of the tumor microenvironment for cancer therapy. Ferumoxytol (FMT) can not only serve as a drug deliver agent but also exerts anti-tumor activity. β-glucan has immuno-modulating properties to prevent tumor growth. Thus, a nanocomposite of FMT surface-coated with β-glucan (FMT-β-glucan) was prepared to explore its effect on tumor suppression.Methods: Male B16F10 melanoma mouse model was established to explore the antitumor effect of FMT-β-glucan. The viability and apoptotic rates of B16F10 cells were detected by cell counting kit-8 and Annexin-V/PI experiments. The levels of M1 markers were quantified by quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Phagocytic activity and intracellular reactive oxygen species (ROS) in macrophages were evaluated by the neutral red uptake assay and flow cytometry, respectively. Small interfering RNA (siRNA) transfection was applied to knock down the Dectin-1 gene in RAW 264.7 cells.Results: FMT-β-glucan suppressed tumor growth to a greater extent and induced higher infiltration of M1 macrophages than the combination of FMT and β-glucan (FMT+β-glucan) in vivo. In vitro, supernatant from FMT-β-glucan-treated RAW 264.7 cells led to lower cell viability and induced more apoptosis of B16F10 cells than that from the FMT+β-glucan group. Moreover, FMT-β-glucan boosted the expression of M1 type markers, and increased phagocytic activity and ROS in RAW 264.7 cells. Further research indicated that FMT-β-glucan treatment promoted the level of Dectin-1 on the surface of RAW 264.7 cells and that knockdown of Dectin-1 abrogated the phosphorylation levels of several components in MAPK and NF-κB signaling.Conclusion: The nanocomposite FMT-β-glucan suppressed melanoma growth by inducing the M1 macrophage-activated tumor microenvironment.

Natural phenylethanoid glycosides isolated from Callicarpa kwangtungensis suppressed lipopolysaccharide-mediated inflammatory response via activating Keap1/Nrf2/HO-1 pathway in RAW 264.7 macrophages cell

Ethnopharmacological relevanceCallicarpa kwangtungensis, as a characteristic traditional herb in China, has been widely used as indigenous medicine for thousands of years in the treatment of upper respiratory tract infection, tonsillitis, pneumonia and traumatic bleeding in China. Phenylethanoid glycosides (PhGs), as natural polyphenols, are especially abundant in this herb and can be regarded as the representative active ingredients in C. kwangtungensis. Aim of this studyThis study was performed to investigate the anti-inflammatory pharmacodynamic basis of six PhGs (acteoside, forsythoside B, poliumoside, alyssonoside, parvifloroside A, and syringalide A 3′-α-L-rhanmnopyranoside) isolated from C. kwangtungensis from the perspective of antioxidation. Materials and methodsSix PhGs were isolated from the anti-inflammatory extracts of C. kwangtungensis by various chromatographic techniques and their anti-inflammatory activity on RAW 264.7 murine macrophages induced by LPS was investigated by measuring the release of tumor necrosis factor (TNF-α), the colonic interleukin-6 (IL-6), nitric oxide (NO) and reactive oxygen species (ROS). Further, the underlying anti-inflammatory mechanism of two PhGs (forsythoside B and alyssonoside) was explored by determining the expression of Kelch-like ECH-association protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (OH-1) and quinone oxidoreductase 1 (NQO1). Besides, molecular simulation was also employed to evaluate the binding capacity of two PhGs with Keap1. ResultsCompared with the model group, six PhGs revealed obviously inhibitory effects on TNF-α, IL-6, NO and the generation of ROS in RAW 264.7 macrophages. Moreover, forsythoside B and alyssonoside could act as the inhibitors of Keap1-Nrf2 interaction, then activated the nuclear translocation of Nrf2 and promoted the upregulated protein expression of HO-1 and NQO1, finally suppressed LPS-induced inflammatory response in RAW 264.7 macrophages. Molecular modeling exhibited hydrogen bonds played a crucial role for the binding of PhGs with the Nrf2 binding site in Keap1 protein. ConclusionsNatural PhGs-induced protection against LPS-induced inflammatory response via activating Keap1/Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages were confirmed, which provided experimental and theoretical basis for the deeper use of C. Kwangtungensis in the treatment and prevention of diseases related to inflammation and oxidative stress.

Radical scavenging activities and composition identification of phenolic compounds from crowndaisy seeds by offline HPLC combined with LC–ESI–MS/MS

How to screen and evaluate the radical scavenging activity of each compound in the mixture remains a challenging project. In the present study, a sensitive and selective method of offline HPLC coupled with chemical methodology was developed for the evaluation of the radical scavenging activity of each compound in a common vegetable, crowndaisy (Chrysanthemum coronarium L.) seeds alcohol extract (CDAE) without the isolation of monomers. The results of LC–ESI–MS/MS showed that the main components of CDAE were chlorogenic acid (3-CQA) (1693.0 μg/g, dry weight), 1,3-dicaffeoylquinic acid (1,3-DCQA, 1378.5 μg/g), 3,5-DCQA (1027.0 μg/g), and 1,4-DCQA (1031.5 μg/g). The results of scavenging free radical activity analysis showed that, among all components, 1,3-DCQA, 3,5-DCQA, 1,4-DCQA, and 3-CQA were mainly responsible for the radical scavenging activity of the extract. Their scavenging rates for 2,2-diphenyl-1-picrylhydrazyl (DPPH) were 86.72%, 83.50%, 83.50%, and 83.43%, respectively. In the assay of scavenging the reactive oxygen species (ROS), CDAE could effectively decrease ROS in RAW264.7 cells without cell toxicity.

Characterization and Application of Lignin–Carbohydrate Complexes from Lignocellulosic Materials as Antioxidants for Scavenging In Vitro and In Vivo Reactive Oxygen Species

Lignin–carbohydrate complexes (LCCs) have shown antioxidant ability to scavenge the individual free radicals in vitro, while little work has been carried out to show if the LCCs can efficiently scavenge the intracellular and endogenous reactive oxygen species (ROS), which are the multiple radicals derived from the reduction of molecular oxygen during the metabolism process. In this work, carbohydrate-rich LCCs from bamboo (LCCs–B-B) and poplar (LCCs–B-P) were isolated according to the classical method, and their antioxidant activities were evaluated by scavenging intracellular ROS in RAW 264.7 cells in vitro and endogenous ROS in zebrafish in vivo. Results from composition analysis show that both LCC preparations possess similar contents of carbohydrate (52.2% and 51.2%) and lignin (44.1% and 47.8%). However, NMR analysis revealed that the LCCs–B-B contain 16.1/100C9 LCCs linkages, higher than that in LCCs–B-P (12.3/100C9). Antioxidant assays indicated that LCCs–B-B exhibited better antioxidant activities...

Polyphenolic Compound from Brown Macroalga Padina tetrastromatica Imparts Oxidative Stress Tolerance in SH‐SY5Y, RAW 264.7, HeLa Cell Lines and in Caenorhabditis elegans

AbstractMarine brown macroalgae are known for producing potent bioactive secondary metabolites such as phlorotannins. Phlorotannins are the phenolic compounds which can inhibit the oxidative stress via scavenging the free radicals. In this study, phlorotannin from Padina tetrastromatica brown macroalga was collected from Mandapam, Rameswaram, Tamil Nadu and identified by microscopic observation. The compound was extracted and purified by using column chromatography, thin layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). The purified compound showed prominent inhibition of free radicals via reactive oxygen species (ROS) activity in vitro. Further, the compound showed the inhibitory effect of ROS in RAW 264.7 murine macrophage cell line, HeLa human cervical cancer cell line, and SH‐SY5Y human neuroblastoma cell line. Further, the effect of the compound on neurotoxicity was carried out in Caenorhabditis elegans as an animal model. The stress‐responsive genes daf‐16 and sod‐1 gene expression studies were performed and results revealed that the aforementioned genes had shown up‐regulation in compound treated with acrylamide induced worms. Thus, this compound may prospect for therapeutic applications against the oxidative stress‐related complications.

Schisandra chinensis extract ameliorates age-related muscle wasting and bone loss in ovariectomized rats.

Exercise and healthy diet consumption support healthy aging. Schisandra chinensis (Turcz.) also known as "Baill." has anti-inflammatory and antioxidant properties. However, the role of S.chinensis as an antiaging compound has yet to be demonstrated. This study elucidated the antiaging effect of S.chinensis ethanol-hexane extract (C1) and the effect of C1 treatment on muscle and bone following physical exercise in ovariectomized (OVX) rats. RAW 264.7, human diploid fibroblasts (HDFs), C2C12 myoblasts, bone marrow macrophages, and MC3T3-E1 cells were used for in vitro, and muscle and bone of OVX rats were used for in vivo study to demonstrate the effect of C1. The C1 significantly inhibited the expression of inflammatory molecules, β-galactosidase activity, and improved antioxidant activity via down-regulation of reactive oxygen species in RAW 264.7 and aged HDF cells. The C1 with exercise improved muscle regeneration in skeletal muscle of OVX rats by promoting mitochondrial biogenesis and autophagy. C1 induced osteoblast differentiation, and C1+exercise modulated the bone formation and bone resorption in OVX rats. C1 exhibited anti-inflammatory, antioxidant, myogenic, and osteogenic effects. C1 with exercise improved age-related muscle wasting and bone loss. Therefore, S.chinensis may be a potential prevent agent for age-related diseases such as sarcopenia and osteoporosis.

Acinetobacter baumannii outer membrane protein 34 elicits NLRP3 inflammasome activation via mitochondria-derived reactive oxygen species in RAW264.7 macrophages

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium, which acts as an opportunistic pathogen and causes hospital-acquired pneumonia and bacteremia by infecting the alveoli of epithelial cells and macrophages. Evidence reveals that A. baumannii outer membrane protein 34 (Omp34) elicits cellular immune responses and inflammation. The innate immunity NOD-like receptor 3 (NLRP3) inflammasome exerts critical function against pneumonia caused by A. baumannii infection, however, the role of Omp34 in the activation of the NLRP3 inflammasome and its corresponding regulatory mechanism are not clearly elucidated. The present study aimed to investigate whether Omp34 elicited NLRP3 inflammasome activation through the mitochondria-derived reactive oxygen species (ROS). Our results showed that Omp34 triggered cell pyroptosis by up-regulating the expression of NLRP3 inflammasome-associated proteins and IL-1β release in a time- and dose-dependent manner. Omp34 induced the expression of caspase-1-p10 and IL-1β, which was significantly attenuated by NLRP3 gene silencing in RAW264.7 mouse macrophage cells. Additionally, Omp34 stimulated RAW264.7 mitochondria to generate ROS, while the ROS scavenger Mito-TEMPO inhibited the Omp34-triggered expression of NLRP3 inflammasome-associated proteins and IL-1β synthesis. The above findings indicate that mitochondria-derived ROS play an important role in the process of NLRP3 inflammasome activation. In summary, our study demonstrates that the A. baumannii pathogen pattern recognition receptor Omp34 activates NLRP3 inflammasome via mitochondria-derived ROS in RAW264.7 cells. Accordingly, down-regulating the mitochondria-derived ROS prevents the severe infection consequences caused by A. baumannii-induced NLRP3 inflammasome hyper-activation.

Involvement of MAPK/NF-κB signal transduction pathways: Camellia japonica mitigates inflammation and gastric ulcer

Gastric ulcer is an important risk factor for human health globally. Camellia japonica (CJ) is a plant of which the fruits are used as traditional phytomedicine for inflammatory and immunomodulatory diseases; however, the underlying molecular mechanism has not been clarified. The present study aimed to investigate the immunopharmacological activities of Camellia japonica and validate its pharmacological targets. To evaluate the protective roles of Camellia japonica on LPS-induced inflammation in RAW 264.7 cells and HCl/EtOH-induced gastric ulcer in mice; we applied 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitric oxide (NO), reactive oxygen species (ROS), histopathology, malondialdehyde (MDA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and western blot analyses. We also determined the total phenolic and flavonoid content of Camellia japonica which might possess antioxidant and anti-inflammatory properties. We found the production of NO and ROS in RAW 246.7 cells were both suppressed by Camellia japonica. Moreover, Camellia japonica mitigated the HCl/EtOH-induced oxidative stress in gastric mucosa via the reduction of lipid peroxidation and elevation of NO production. Gastric mucosal damages were prominently improved by Camellia japonica, as confirmed by the histopathological evaluation. The gene expression of inflammatory cytokines and enzymes TNF-α, IL-6, IL-1β, iNOS, and COX-2 was notably downregulated by Camellia japonica. In addition, Camellia japonica markedly attenuated the MAPKs (ERK1/2, JNK, and p38) phosphorylation, COX-2 expression, and activation of transcription factor NF-κB and as well as phosphorylation and degradation of IκBα in gastric mucosa. Taken together, the intimated anti-inflammatory and gastroprotective mechanism of Camellia japonica is mediated by modulation of oxidative stress, inflammatory cytokines, and enzymes via suppression of MAPK/NF-κB signaling pathways.

An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages.

Aster yomena (Kitam.) Honda has been widely used as a traditional herbal medicine for centuries to treat cough, asthma, insect bites, etc. Recent reports indicate that A. yomena possesses a wide spectrum of pharmacological activities; however, few experiments have described its anti-inflammatory properties. The present study examined the anti-inflammatory effects of an ethanol extract of A. yomena leaves (EEAY) on lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Treatment with EEAY significantly reduced the secretion of pro-inflammatory molecules, such as nitric oxide and interleukin-1β, in LPS-stimulated RAW 264.7 cells, without incurring any significant cytotoxicity. These protective effects were accompanied by a marked reduction in the expression of regulatory genes at the transcription level. Treatment with EEAY also inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) by suppression of nuclear translocation of NF-κB and by degradation of the inhibitor of NF-κB; these effects were associated with suppression of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. The EEAY treatment also potently suppressed LPS-induced toll like receptor (TLR) 4 expression and attenuated the binding of LPS to the macrophage cell surface. In addition, EEAY treatment markedly inhibited LPS-induced accumulation of intracellular reactive oxygen species in RAW 264.7 macrophages. Therefore, the inhibitory effects of EEAY on LPS-stimulated inflammatory responses in RAW 264.7 macrophages were apparently associated with suppression of the TLR-mediated NF-κB signaling pathway. More work is needed to fully understand the critical role and clinical usefulness of EEAY treatment, but the findings of the present study provide some insights into the potential of EEAY as a therapeutic agent for treatment of inflammatory disorders.