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Fucoidans possess potent anti-inflammatory effects. In the present study, the anti-inflammatory effect of the fucoidan (SFF-PS-F5) isolated from fermented Sargassum fusiforme was evaluated in vitro in RAW 264.7 macrophages and in vivo in zebrafish. The in vitro test results demonstrate that SFF-PS-F5 effectively inhibited nitric oxide (NO) production induced by lipopolysaccharides (LPS) in RAW 264.7 cells. SFF-PS-F5 effectively and concentration-dependently improved the viability of LPS-stimulated RAW 264.7 cells, and reduced the level of prostaglandin E

The major quercetin metabolite, quercetin-3-glucuronide, exerts various biological activities, including anti-inflammatory effects. This study aimed to evaluate the metabolic profiles and biological properties of the positional isomers of quercetin monoglucuronides (Q3G, Q7G, Q3’G, and Q4’G) in activated macrophages. In addition to quercetin aglycone, Q7G was more cytotoxic than the other quercetin monoglucuronides (QGs), which corresponded to its lower stability under neutral pH conditions. Q3G was most effective in inhibiting both LPS-dependent induction of IL-6 and RANKL-dependent activation of tartrate-resistant acid phosphatase; however, Q3’G and Q4’G may also help exert biological activities without potential cytotoxicity. The deconjugation efficacy to generate quercetin aglycone differed among QGs, with the highest efficacy in Q3G. These results suggest that the chemical or biological properties and metabolic profiles may depend on the stability of QGs to generate quercetin aglycone using β-glucuronidase.

A soluble soybean polysaccharide SSPS1 with a molecular weight of 2737 kDa was extracted and purified from soy whey. SSPS1 was composed of glucose (97.3 %) and a small amount of mannose (2.7 %). Structural analysis results suggested that SSPS1 had a → 6)-α-d-Glcp-(1 → glucan structure, with a trace amount of α-d-Glcp-(1 → connected to the main chain via O-3. In vitro immunological experiments suggested that SSPS1 enhanced the growth rate and phagocytic activity of RAW 264.7 macrophages. In addition, SSPS1 stimulated the secretion of cytokines (TNF-α, INF-β, IL-6 and IL-1β) as well as nitric oxide (NO) production through upregulating the expression of the related genes and proteins in RAW 264.7 cells. This study provided a new method for efficient utilization of soy whey, and the results indicate that SSPS1 extracted from soy whey could be used as a novel immunomodulator.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis.

Recently, the potential of β-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.

Sevoflurane is identified as an effective candidate drug for acute lung injury (ALI) treatment, but its curing effects and detailed mechanisms have not been fully disclosed. The present study was designed to resolve this academic issue.The ALI mice models were established, and Hematoxylin-eosin staining assay was performed to examine tissue morphologies. Cell viability was determined by CCK-8 assay, and Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The expression levels of proteins were determined by performing Western Blot analysis and immunofluorescence staining assay. ROS levels were examined by using DCFH-DA staining assay.In this study, we investigated this issue and the ALI models were respectively established by treating the BALB/c mice and the murine macrophage cell line RAW264.7 with different concentrations of lipopolysaccharide (LPS) in vivo and in vitro, which were subsequently subjected to sevoflurane co-treatment. The results showed that sevoflurane reduced LPS-induced ALI in mice and suppressed LPS-triggered oxidative stress and apoptotic cell death in the RAW264.7 cells. Interestingly, we evidenced that the elimination of reactive oxygen species (ROS) by N-acetyl-L-cysteine (NAC) reversed LPS-induced cell apoptosis in RAW264.7 cells. Then, we verified that sevoflurane suppressed oxidative damages to restrain LPS-induced apoptotic cell death in the RAW264.7 cells through activating the anti-oxidant Keap1/Nrf2 pathway. Mechanistically, sevoflurane down-regulated Keap1 and upregulated Nrf2 in nucleus to activate the downstream anti-oxidant signaling cascades, which further ameliorated LPS-induced cell apoptosis and lung injury by eliminating oxidative damages.Taken together, our study illustrated that the sevoflurane attenuates LPS-induced ALI by inhibiting oxidative stress-mediated apoptotic cell death and inflammation, and the Keap1/Nrf2 pathway played an important role in this process.

A novel polysaccharide (MSP-1) was isolated from the fruiting body of Morchella sextelata and purified using DEAE-52 and Sephadex G-75. The molecular weight of MSP-1 was 1.17 × 10

A new polysaccharide fraction HLP-1 (2.55 × 10

Polysaccharides from Pimpinella anisum were isolated using water at elevated temperature and DEAE Sepharose FF chromatography to examine their chemical structure and activation capacity on immune cells. P. anisum fractions (PAF1, PAF2 and PAF3) were mainly composed of neutral sugars (84.0-98.2%) and uronic acids (2.1-11.8%) with weight average molecular weight (M

The pathogenesis of ulcerative colitis (UC) is associated with severe inflammation, damaged colonic barriers, increased oxidative stress, and intestinal dysbiosis. The majority of current medications strive to alleviate inflammation but fail to target additional disease pathologies. Addressing multiple symptoms using a single 'magic bullet' remains a challenge. To overcome this, a smart epigallocatechin-3-gallate (EGCG)-loaded silk fibroin-based nanoparticle (NP) with the surface functionalization of antimicrobial peptides (Cathelicidin-BF, CBF) was constructed, which could be internalized by Colon-26 cells and RAW 264.7 macrophages with high efficiencies. The resulting CBF-EGCG-NPs efficiently restored colonic epithelial barriers by relieving oxidative stress and promoting epithelium migration. They also alleviated immune responses through downregulation of pro-inflammatory factors, upregulation of anti-inflammatory factors, M2 macrophage polarization, and lipopolysaccharide (LPS) elimination. Interestingly, oral administration of hydrogel (chitosan/alginate)-embedding CBF-EGCG-NPs could not only retard progression and treat UC, but also modulate intestinal microbiota by increasing their overall diversity and richness and augmenting the abundance of beneficial bacteria (e.g., Firmicutes and Lactobacillaceae). Our work provides a "many birds with one stone" strategy for addressing UC symptoms using a single NP-based oral platform that targets immune microenvironment modulation, LPS clearance, and microbial remodeling.

This study aimed to reveal the mechanism of transcription factor Kruppel-like factor 4 (KLF4) in regulating M1 polarization of macrophages in rheumatoid arthritis (RA) in order to induce inflammatory response. The results suggested that KLF4 overexpression promoted the M1 polarization of RAW 264.7 cells, increased STAT1 expression and up-regulated the phosphorylation level. After KLF4 silencing, the M1 polarization level was down-regulated. Besides, the induced M1 macrophages were co-cultured with articular chondrocytes. KLF4 overexpression further aggravated chondrocyte injury, increased the cell apoptosis rate and activated the inflammatory injury. However, pretreatment with STAT1 inhibitor Cerulomycin resisted the effect of KLF4, and significantly suppressed STAT1 expression and M1 polarization of cells. KLF4 overexpression aggravated synovial tissue injury in mouse joints, up-regulated the expression of inflammatory factors, and increased the levels of CD86 and STAT1. It was discovered that transcription factor KLF4 promoted the transcription of STAT1 to regulate the M1 polarization of macrophages, thus aggravating the progression of RA and inflammatory response.

The inflammatory response is a common feature of many pathological conditions, and there is urgent necessity for new substances that minimize the harmful effects of inflammation. Chromenes represent a class of compounds with multiple pharmacological actions that have already been described and may be potential candidates for studies of therapeutic action. This study aimed to test novel 4-aryl-4H-chromene-derived molecules in an in vitro model of inflammation using lipopolysaccharide (LPS)-induced Raw 264.7 cells. Seven compounds derived from 4-aryl-4H-chromene were tested on Raw 264.7 cells to evaluate their cytotoxic effects. Next, the effect of the selected compounds on the pro-inflammatory mediators (tumor necrosis factor-alpha [TNF-α], monocyte chemoattractant protein-1 [MCP-1], interleukin [IL]-6) and on the anti-inflammatory mediators (IL-10 and IL-13) was analyzed, and finally, the effect of the compounds on macrophage apoptosis and expression of surface receptors (toll-like receptor 4 [TLR-4] and mannose) was evaluated. The results of this study demonstrated that changes in the molecular structure of 4-aryl-4H-chromene altered its cytotoxic profile. Therefore, derivatives that showed safe results were selected for further analyses (named compounds: 4-6). In these experiments, the compounds were able to decrease nitric oxide (NO) levels and production of MCP-1, IL-6, IL-10, and IL-13. Furthermore, these derivatives were effective in reducing macrophage apoptosis and the expression of surface receptors, as TLR-4/CD284. Moreover, compounds 5 and 6 also were effective in increasing mannose receptor (CD206) expression. The results indicate, for the first time to our knowledge, that the anti-inflammatory effect produced by chromenes is linked to macrophage repolarization (M1 to M2).

Growing evidence suggests that excessive inflammation hampers the regenerative capacity of periodontal ligament cells (PDLCs) and that activation of the Wnt/β-catenin pathway is crucial in suppressing immune dysregulation.This study aimed to establish the role of the Wnt/β-catenin in regulating the immune microenvironment and its subsequent impact on periodontal regeneration.Lithium chloride (LiCl, Wnt activator) was administered daily into the standard periodontal defects created in 12-week-old Lewis rats. Harvested at 1-week and 2-week post-surgery, samples were then subjected to histological and immunohistochemical evaluation of macrophage distribution and phenotype (pro-inflammatory M1 and anti-inflammatory M2). A murine macrophage cell line, RAW 264.7, was stimulated with LiCl to activate Wnt/β-catenin. Following treatment with the conditioned medium derived from the LiCl-activated macrophages, the expression of bone- and cementum-related markers of the PDLCs was determined. The involvement of Wnt/β-catenin in the immunoregulation and autophagic activity was further investigated with the addition of cardamonin, a commercially available Wnt inhibitor.A significantly increased number of macrophages were detected around the defects during early healing upon receiving the Wnt/β-catenin signaling cue. The defect sites in week 2 exhibited fewer M1 and more M2 macrophages along with an enhanced regeneration of alveolar bone and cementum in the Wnt/β-catenin activation group. LiCl-induced immunomodulatory effect was accompanied with the activation Wnt/β-catenin signaling, which was suppressed in the presence of Wnt inhibitor. Exposure to LiCl could induce autophagy in a dose-dependent manner, thus maintaining macrophages in a regulatory state. The expression level of bone- and cementum-related markers was significantly elevated in PDLCs stimulated with LiCl-activated macrophages.The application of Wnt activator LiCl facilitates the recruitment of macrophages to defect sites and regulates their phenotypic switching in favor of periodontal regeneration. Suppression of Wnt/β-catenin pathway could attenuate the LiCl-induced immunomodulatory effect. Taken together, the Wnt/β-catenin pathway may be targeted for therapeutic interventions in periodontal diseases.

A novel acidic polysaccharide named SSPA50-1 was isolated from Scapharca subcrenata using a simulated gastric fluid extraction method. SSPA50-1 is a heteropolysaccharide with an average molecular weight of 44.7 kDa that is composed of galacturonic acid, glucose, galactose, mannose, ribose, rhamnose, fucose, xylose and arabinose at a molar ratio of 1.00:5.40:9.04:3.10:1.59:4.01:2.10:2.21:2.28. The structural characterization based on the methylation and 1D/2D NMR analyses indicated that SSPA50-1 is composed of →3)-β-L-Rhap-(1→, →3)-β-L-2-O-Me-Fucp-(1→, →2)-α-D-Xylp-(1→, →5)-α-L-Araf-(1→, →3)-β-D-Galp-(1→, →6)-α-D-Glcp-(1→, →3,4)-β-D-Manp-(1→, →3,4)-β-D-Galp-(1→, β-D-Ribf-(1→, α-D-Glcp-(1→, and α-D-GalAp6Me-(1→. Furthermore, SSPA50-1 possessed potent immunoregulatory activity by enhancing the phagocytosis and NO, iNOS, TNF-α and IL-6 secretion capacity of RAW264.7 cells. Otherwise, SSPA50-1 significantly promoted the proliferation of splenic lymphocytes and RAW264.7 macrophages. These results indicated that SSPA50-1 could be developed as a potential ingredient for immunostimulatory agents.

Several experimental models have been designed to promote the development of new anti-inflammatory drugs. The in vitro model using RAW 264.7 cells has been widely used. However, there is still no consensus on which inflammatory mediators should initially be measured to screen for possible anti-inflammatory effects. To determine the rationality of measuring inflammatory mediators together with NO, such as the levels of tumor necrosis factor (TNF)-α, and interleukins (IL) 1β and 6, we carried out this systematic review (SR) and meta-analysis (MA).We conducted this SR and MA in accordance with the Preferred Reporting of Systematic Reviews and Meta-Analysis and the Cochrane Handbook for Systematic Reviews of Intervention. This review was registered in the Open Science Framework ( https://doi.org/10.17605/OSF.IO/8C3HT ).LPS-induced cells produced high NO levels compared to non-LPS induced, and this production was not related to cell density. TNF-α, IL-1β, and IL-6, also showed high levels after cells had been stimulated with LPS. Though with some restrictions, all studies were reliable, as the risk of bias was detected in the test compounds and systems.Measurement of NO levels may be sufficient to screen for possible anti-inflammatory action in the context of LPS-induced RAW 264.7 cells.

Inflammation is a host defense response to harmful agents, such as pathogenic invasion, and is necessary for health. Excessive inflammation may result in the development of inflammatory disorders. Levilactobacillus brevis KU15151 has been reported to exhibit probiotic characteristics and antioxidant activities, but the effect of this strain on inflammatory responses has not been determined. The present study aimed to investigate the anti-inflammatory potential of L. brevis KU15151 in Staphylococcus aureus lipoteichoic acid (aLTA)-induced RAW264.7 macrophages. Treatment with L. brevis KU15151 reduced the production of nitric oxide and prostaglandin E

A bioactive polysaccharide (TS2-2A) with a molecular weight of 15 kDa was isolated from Trametes sanguinea Lloyd, a medicinal food homologous fungus, by water extraction-alcohol precipitation and chromatographic separation. NMR analysis of polysaccharide and MS/MS analysis of its oligosaccharide indicated that TS2-2A featured a novel straight chain with a backbone of 1,3-α-d-glucopyranose and 1,4-β-d-glucopyranose at a molar ratio of 1:4. Moreover, TS2-2A, recognized by Toll-like receptor 4 (TLR4), stimulated RAW 264.7 macrophages to release related cytokines and contributed to immune-enhancing effects. Briefly, with remarkable immune-enhancing activity and noncytotoxicity, TS2-2A was proposed to be a potential immune enhancer for supplementing drugs or functional foods.

Inflammation is a contributing factor to the initiation and progression of many diseases, and some food-derived biofunctional peptides show high anti-inflammatory activity. In our previous study, we demonstrated that peptides derived from trypsin hydrolysis of rice protein show good immunological activity. In the present study, proteins of broken rice were extracted and identified by macroporous resin fractionation and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Subsequently, a bioinformatics prediction and in silico simulation approach was used to screen for peptides showing anti-inflammatory activity, including inhibition of the production of nitric oxide and proinflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) by lipopolysaccharide-stimulated RAW264.7 mice macrophages. Three peptides (DNIQGITKPAIR, IAFKTNPNSMVSHIAGK, and IGVAMDYSASSKR) that demonstrated the highest binding affinity were synthesized, and their in vitro anti-inflammatory activity was investigated. This is the first study that integrates LC-MS/MS identification and bioinformatics prediction for reporting the anti-inflammatory activity of anti-inflammatory peptides derived from broken rice protein. The study findings revealed that the peptides derived from the byproduct of rice milling could be potentially used as natural anti-inflammatory alternativities.

In this study, gradient ethanol precipitation method was applied to obtain the extracellular polysaccharides of Lactobacillus paracasei VL8 (VL8-EPS). The yields, physicochemical properties, and immunomodulatory effects of VL8-EPS obtained by precipitation at different ethanol concentrations (30%, 50%, and 70%, v/v) were compared. The results showed that VL8-EPSs were high molecular weight sulfated heteropolysaccharides, composed mainly of glucose and galactose, and the alteration of ethanol concentration had an effect on their chemical compositions, molecular weight distributions, monosaccharide composition, and surface structure, while the primary structure remained the same. Among the three polysaccharide fractions, VL8-EPS50 displayed better immunomodulatory activities compared with VL8-EPS30 and VL8-EPS70. VL8-EPS50 was found to exert immunomodulatory effects by enhancing the phagocytic activity of RAW264.7 cells and to promote their secretion of more nitric oxide; it also showed stronger thermal and solution stability. In summary, there was a correlation between the structural characteristics of polysaccharides and their immunomodulatory activity, and VL8-EPS50 was preferentially used for in vivo immunomodulatory activity. Practical Application This study opens up the source of raw materials for functional foods, which can provide some theoretical basis for the research and development of extracellular polysaccharides of lactic acid bacteria and promote their application in the future development of food industry.

Icariin (ICA), isolated from Herba Epimedii, is a natural flavonoid glycoside that possesses antioxidant properties and inhibits osteoclastogenesis. However, the mechanism underlying osteoclastogenesis inhibition by ICA remains unclear. Here, we investigated the effects of ICA on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. ICA inhibited the expression of osteoclastogenesis-related genes in RAW264.7 cells induced by RANKL. ICA could inhibit osteoclastogenesis without inhibiting the viability of RAW264.7 cells. In addition, ICA inhibited reactive oxygen species production in RANKL-induced RAW264.7 cells. ICA reduced the expression of nuclear factor in activated T cells, cytoplasmic 1, and tartrate-resistant acid phosphatase, which are osteoclast-related molecules. Moreover, ICA decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), specifically NOX1 and NOX4, in RANKL-induced RAW264.7 cells. Our findings suggest that ICA can be used as a potential therapeutic agent for osteolytic diseases such as osteoporosis.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1β, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8–15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti-inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme-linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL-6. The expression levels of NF-κB, IL-6, TNF-α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF-κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB-α and suppressing IKK-β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB-α, while siIKK-β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF-κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.

In this study, polysaccharides of Erythronium sibiricum bulb were extracted using enzyme-assisted extraction technology and then optimised by response surface methodology. The characteristics and immunomodulatory activities of the polysaccharide (E1P) were investigated. Setting the yield of polysaccharides as the index, the effects of amylase content, zymolytic time, extraction pH and zymolytic temperature were investigated. The optimal extraction conditions for polysaccharides were as follows: amylase content, 1% weight of pre-treated powder; zymolytic time, 2 h; extraction pH, 7.5; and zymolytic temperature, 55 °C. The yield was predicted to be 61.10%, which agreed with the value obtained in confirmatory experiments (59.71% ± 2.72%). Further research indicated that the primary component of E1P is glucose; however, it also contains a small quantity of galactose and arabinose. In vitro assays showed that E1P and ESBP (another kind of E. sibiricum bulb polysaccharide extracted by water decoction in our previous study) could significantly promote the cellular viability and phagocytosis of RAW264.7 cells without cytotoxicity. Moreover, they could enhance the ability to secrete nitric oxide and cytokines such as TNF-α and IL-1β. However, the immunomodulatory activities of E1P were better than those of ESBP. According to the results of this study, enzyme-assisted extraction represents a new strategy for extracting E. sibiricum bulb polysaccharides with higher yield and better immune activity.

Objectives Hydrolysable tannin fraction (HTF) derived from Terminalia chebula fruit pericarps was assessed for its anti-inflammatory potential in LPS-induced RAW 264.7 cells. Its molecular mechanism was also established and compared with individual tannins – chebulagic acid (CH) and corilagin (CO). Methods The effect of HTF on LPS-stimulated RAW 264.7 cells was studied by estimating the release of NO, ROS, cytokines and changes in nuclear morphology by DAPI staining. Furthermore, the effect of HTF, CO and CH was compared with the expression of p65, p38 and pERK proteins by immunoblotting and the mRNA transcript level of COX-2, iNOS and TNF-α by quantitative PCR. The in-silico interactions of various hydrolysable tannins present in HTF with molecular targets of inflammation were studied using Maestro software. Key findings HTF at the dose levels of 25, 50 and 100 µg/ml was able to decrease the release of NO, ROS and cytokines from LPS-induced RAW 264.7 cells without disturbing the cell nuclear morphology. Investigation of molecular mechanism revealed that inhibition of NF-κB and MAPK signalling pathways was responsible for its anti-inflammatory action. The effect of HTF was higher than the individual tannins CH and CO. Conclusion HTF can be developed as an effective anti-inflammatory agent.

Rheumatoid arthritis (RA) is an ordinarily occurring autoimmune disease with systemic inflammatory. Targeted drug delivery systems have many successful applications in the treatment of rheumatoid arthritis. In order to develop nanoparticles for targeted delivery of Celastrol (Cel) to rheumatoid arthritis and specific drug release, the dextran sulfate (DS) was modified as the targeting molecular by binding to the scavenger receptor of macrophage. The dextran-sulfate-PVGLIG-celastrol (DS-PVGLIG-Cel), named DPC, amphiphilic polymeric prodrug was synthesized and characterized. The resulting DPC@Cel micelles had the average size of 189.9 nm. Moreover, the micelles had ultrahigh entrapment efficiency (about 44.04%) and zeta potential of −11.91 mV. In the in vitro release study, due to the excessive production of matrix metalloproteinase-2 (MMP-2) at the inflammatory joint, the MMP-2 reactive peptide was used to crack in the inflammatory microenvironment to accelerate the release of Cel. The results have shown that the nanoparticles can effectively deliver Cel to activated macrophages and significantly improve the bioavailability. In vivo experiments showed that DPC@Cel have better anti-rheumatoid arthritis effects and lower systemic toxicity than free Cel. This study provided a new therapeutic strategy for the treatment of RA.

Osteoclasts (OCs), the main cause of bone resorption irregularities, may ultimately cause various bone diseases, including osteoarthritis. The objective of this study was to investigate the effect of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) on OC formation induced by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and to further explore its underlying mechanism. IFIT1 expression in Raw264.7 cells treated with macrophage colony-stimulating factor (M-CSF) and RANKL was determined by qRT-PCR. OC formation was detected using tartrate-resistant acid phosphatase (TRAP) staining. The effect of IFIT1 on STAT3 activation was detected using Western blotting. Additionally, Western blotting was used to measure the change in the expression of OC-specific proteins. IFIT1 was highly expressed in Raw264.7 cells after stimulation with M-CSF and RANKL. IFIT1 overexpression accelerated the formation of OCs, as evidenced by the increased number and size of multinuclear cells, and the upregulation of OC-specific proteins, and activated the STAT3 pathway, by inducing phosphorylation of JAK1 and STAT3. However, silencing of IFIT1 inhibited the formation of OCs and a STAT3 inhibitor Stattic weakened the effects of IFIT1. In conclusion, IFIT1 accelerates the formation of OCs, which is caused by RANKL by STAT3 pathway regulation. This study provides a potential basis for further research and for development of drugs for treating bone resorption-related diseases.

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.

Perillaldehyde (PAH), one of the active ingredients of the traditional Chinese medicine (TCM) plant Perilla frutescens, is widely used and exerts crucial anti-cancer activities. The aim of current study is to illustrate the potential mechanisms of PAH-mediated regulation of bone metastasis and osteoclastogenesis in prostate cancer (PCa) cell lines. Effects of PAH on proliferation, invasion and migration of PC-3 cells were assessed with the Cell Counting Kit-8 (CCK-8) assay and Transwell assays, respectively. Effects of PAH on stem cell characteristics of PC-3 cells were evaluated by cell-matrix adhesion assay, colony formation assay, spheroid formation assay, as well as western blot . The anti-metastasis and anti-osteoclastogenesis activity of PAH in RAW264.7 cells was examined by osteoclast differentiation assay and western blot. The protein levels of CD133 and CD44 in PC-3 cells and the activity of nuclear factor kappa B (NF-κB) signaling pathway in RAW264.7 cells were measured by western blot. PAH suppressed proliferation, invasion and migration of PC-3 cells, prevented stem cell characteristics including cell-matrix adhesion, colony formation, spheroid formation as well as CD133 and CD44 expression. PAH inhibited bone metastasis and osteoclastogenesis via repressing the activation of NF-κB pathway as well as (RANKL)- and cancer cell- induced osteoclastogenesis in PCa cells. These findings suggested the potential therapeutic effects of PAH on the metastasis of patients with PCa.

Transcription factor forkhead box protein 1 (FOXP1) has been shown cardiovascular protection. We aimed to analyze the role of FOXP1 in oxidized low-density lipoprotein (ox-LDL)-induced macrophages and its possible regulatory effect on sestrin1 (SESN1) expression. After stimulation with ox-LDL, FOXP1 expression in RAW264.7 cells was evaluated with RT-qPCR and Western blotting. Then, FOXP1 was overexpressed, followed by detection of inflammatory mediator levels using ELISA kits and RT-qPCR. Lipid accumulation was detected with oil red O staining. Additionally, the JASPAR database was used to predict the potential genes that could be transcriptionally regulated by FOXP1. ChIP and luciferase reporter assays were used to verify this combination. To further clarify the regulatory effects of FOXP1 on SESN1 in damage of macrophages triggered by ox-LDL, SESN1 was silenced to determine the inflammation and lipid accumulation under the condition of FOXP1 overexpression. Results indicated that ox-LDL stimulation led to a significant decrease in FOXP1 expression. FOXP1 overexpression notably reduced the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, accompanied by a decreased in phosphorylated NF-κB p65 expression. Besides, FOXP1-upregulation inhibited lipid accumulation and reduced CD36 expression level in RAW264.7 cells upon ox-LDL stimulation. Moreover, results of ChIP and luciferase reporter assays suggested that FOXP1 could transcriptionally regulate SESN1 expression. Further experiments supported that SESN1 silencing restored the inhibitory effects of FOXP1 overexpression on the inflammation and lipid accumulation in RAW264.7 cells exposed to ox-LDL. Collectively, FOXP1 transcriptionally activates SESN1 for the alleviation of ox-LDL-induced inflammation and lipid accumulation in macrophages.

Ulcerative colitis is a common inflammatory bowel disease with a complex genetic and immune etiology. Immune infiltration plays a vital role in the development of ulcerative colitis. To explore potential biomarkers for ulcerative colitis and analyze characteristics of immune cell infiltration, we used bioinformatic analyses, including machine learning algorithms, cell type deconvolution methods, and pathway enrichment methods. In this study, we identified 216 differentially expressed mRNAs (DEMs), of which 153 were upregulated, and 63 were downregulated genes. DEMs were mainly enriched in infiltrating neutrophils and regulation of leukocyte migration. Moreover, eight candidate biomarkers, DPP10, MST1L, DPP10‐AS1, CEP55, ACSL1, MGP, OLFM4, and SGK1, were identified. Of these candidate biomarkers, MST1L, OLFM4, and DPP10 were then validated in the GSE48958 dataset and were predicted to be strongly correlated with infiltrating immune cells of ulcerative colitis. The underlying mechanism of these key genes in the development of colitis was also predicted by gene set variation analysis. To further validate these biomarkers' expression in ulcerative colitis, we determined mRNA levels of SGK1, CEP55, ACSL1, OLFM4, and DPP10 in lipopolysaccharides (LPS)‐stimulated Raw264.7 cells by quantitative reverse transcription‐polymerase chain reaction. We also examined SGK1, CEP55, ACSL1, OLFM4, DPP10, and MGP expression in the colon tissues of dextran sodium sulfate‐induced colitis mice. Consistent with the predicted computational results, the mRNA levels of these candidate genes were markedly changed in LPS‐stimulated Raw264.7 cells and inflamed colon tissues. Hence, our findings indicated that these critical genes may act as diagnostic biomarkers for ulcerative colitis and that differential immune infiltration cells may help illustrate the progression of ulcerative colitis., In this study, we identified eight critical genes closely related to ulcerative colitis by machine learning algorithms and validated them in GSE48958 and quantitative reverse transcription‐polymerase chain reaction in in vitro bioassays. For these genes, we also analyzed their characteristics of immune cell infiltration and their downstream signaling pathways. Hence, our findings indicated that these genes may act as diagnostic biomarkers for ulcerative colitis.

Eight new polyketides, including three dimeric benzophenones, named dipleosporones A-C (

When infected by Helicobacter pylori, it often causes gastritis, gastric ulcer, or gastric cancer. Antibiotics are used to treat H. pylori infection, as they inhibit or kill H. pylori often ex-tending to reduce the incidences of gastric adenoma and cancer. However, H. pylori has developed drug resistance to many clinically used antibiotics over the years, thereby providing no warranty of successful treatment whenever H. pylori infection befalls. We report here that fucoidan from Sargassumhemiphyllum can effectively reduce infection of H. pylori without development of drug resistance. Fucoidan demonstrated a strong anti-inflammation activity in RAW264.7 cell model. Using AGS cell model, fucoidan decreased H. pylori adhesion to host cells and thus reduced its infection rate, especially in post-treatment where the infection rate was reduced to 40%. Mechanistically, fucoidan intervenes the proper functions of adhesion molecules BabA and AlpA of H. pylori. Moreover, fucoidan is able to significantly lower the total count of H. pylori and the levels of IL-6 and TNF-α in vivo. Added together, these convergent results suggest that fucoidan is an effective agent in a position to protect stomach from H. pylori infection by reducing its total count and induced inflammation.

As a dietary supplement, enzymatic modification of mannose (phosphatidylmannoside, PtdMan) via phospholipase d catalyzed transphosphatidylation is to be used as a way to enhance the modulatory effect of mannose on colitis.

Sepsis often leads to multiple organ failure or even death and is a significant health problem that contributes to a heavy economic burden. The lung is the first organ to be affected by sepsis. Presently, there is no specific drug or method to treat sepsis and sepsis-induced acute lung injury (ALI). H2S, along with CO and NO, is a physiological gas that acts as a signaling molecule and plays an active role in fighting various lung infections. GYY4137 is a novel H2S donor that is stable in vivo and in vitro. However, particularly in the context of ferroptosis, GYY4137 affects cecal ligation and puncture (CLP)-induced ALI by a mechanism that is not understood. Ferroptosis is a new form of cell necrosis. The primary mechanism is the accumulation of cellular lipid ROS in an iron-dependent manner. The principal objective of this project was to investigate the effects of GYY4137 on ferroptosis and autophagy in a mouse model of sepsis-induced ALI. We divided the experimental mice randomly into 5 groups: (1) sham group; (2) CLP group; (3) CLP + DMSO group: (4) CLP + GYY4137 (25 mg/kg) group; and (5) CLP + GYY4137 (50 mg/kg) group. (6) CLP + Rapamycin (2.0 mg/Kg) group. (7) CLP + Chloroquine (80 mg/Kg) group. (8) the Chloroquine (80 mg/Kg) + GYY (50 mg/Kg) group. The findings showed that GYY4137 significantly protected against CLP-induced ALI by improving sepsis-induced lung histopathological changes, diminishing lung tissue damage, ameliorating oxidative stress, and attenuating the severity of lung injury in mice. In this study, we found that GYY4137 could alleviate septicemia-induced ferroptosis in ALI by increasing the expression of GPx4 and SLC7A11 in lung tissue after CLP. One unexpected finding was the extent to which the levels of ferritin and ferritin light chain increased after CLP, which may be a compensatory mechanism for storing abnormally increased iron. We also found that the expression of p-mTOR, P62, and Beclin1 was significantly increased and that LC3II/LC3I declined after LPS stimulation, but the effect was inhibited by treatment with GYY4137, indicating that GYY4137 could inhibit the activation of autophagy in sepsis-induced ALI by blocking mTOR signaling.

In the present study, a water-soluble neutral polysaccharide (CAPW-1) with an average molecular weight of 64 kDa was purified from the root of Cynanchum atratum Bunge (Apocynaceae). The monosaccharide residue analysis revealed that CAPW-1 was composed of arabinose and galactose with a relative molar ratio of 7: 3. The backbone of CAPW-1 was consisted of 1,3-Galp and 1,3,6-Galp, the branches were attached to the O-6 of 1,3-Galp, and the side chains contained 1,6-Galp, 1,3,6-Galp, 1,5-linked, 1,3-linked, 1,3,5-linked, and terminal-Araf, which was attached to the O-3 of side 1,6-Galp. The bioactivity study indicated CAPW-1 could stimulate the proliferation of RAW264.7 cells and promote the secretion of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with no cytotoxicity. The results suggested a potential application of CAPW-1 as an immunostimulant for the treatment of diseases such as infection and tumor.

Fruit juice is one of the most popular drinks, which requires strict processing conditions to ensure its quality, especially to prevent enzymatic browning and turbidity loss. In this work, a new strategy for the preparation of composite nanofibers for juice clarification and anti-browning control was proposed. The strategy used microfluidic spinning to combine Fucoxanthin (Fx), hydroxypropyl-γ-cyclodextrin (HP-γ-CD) and polyvinyl pyrrolidone (PVP) to prepare Fx/HP-γ-CD-PVP (PCF) nanofibers, which not only reflected the excellent antioxidant properties of cyclodextrin-wrapped Fx, but also achieved a more optimized juice clarification agent dosage. Molecular docking technique was used to prove that the stable inclusion complex of Fx and HP-γ-CD could be formed by hydrogen bonding when the molar ratio of Fx to HP-γ-CD was 1 : 2, and the binding energy was as low as -10.23 kcal mol

Increasing knowledge of probiotics has shown that co-cultures of probiotics can achieve better fermentation and beneficial effects, and adding LAB to fermented milk fat products can increase the production of polyunsaturated fatty acids. In this study, the fatty acid profiles of milk fermented by

Atherosclerosis is a global disease with an extremely high morbidity and fatality rate, so it is necessary to develop effective treatments to reduce its impact. In this work, we successfully prepared a multifunctional drug-loaded nano-delivery system with pH-responsive, CD44-targeted, and chemical-photothermal synergistic treatment. Dendritic mesoporous silica nanoparticles capped with copper sulfide (CuS) were synthesized

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

Androgenic alopecia is a common type of hair loss, usually caused by testosterone metabolism generating dihydrotestosterone and hair follicular micro-inflammation. These processes induce dermal papilla cells to undergo apoptosis. Currently approved effective medications for alopecia are Finasteride, an oral 5α-reductase inhibitor, Minoxidil, a topical hair growth promoter, and Diclofenac, an anti-inflammatory agent, all of which, however, have several adverse side effects. In our study, we showed the bioactivity of Acanthus ebracteatus Vahl. (AE) extract performed by 95% ethanol, and verbascoside (VB), a biomarker of AE extract. Both AE extract and VB were studied for their effects on dermal papilla cell viability and the cell cycle by using MTT assay and flow cytometry. The effect of an anti-inflammatory activity of AE extract and VB on IL-1β, NO, and TNF-α, released from LPS induced RAW 264.7 cells, and IL-1α and IL-6 released from irradiated dermal papilla cells were detected using ELISA technique. The preventive effect on dermal papilla cell apoptosis induced by testosterone was determined by MTT assay. In controlled in vitro assays it was found that AE extract and VB at various concentrations induced dermal papilla cell proliferation which was indicated by an increase in the number of cells in the S and G2/M phases of the cell cycle. AE extract at 250 µg/mL concentration or VB at 62.50 µg/mL concentration prevented cell apoptosis induced by testosterone at a statistically significant level. In addition, both AE extract and VB greatly inhibited the release of pro-inflammatory cytokines from RAW 264.7 and dermal papilla cells. The release of IL-1β, TNF-α, and NO from RAW 264.7 cells, as well as IL-1α and IL-6 from dermal papilla cells, was also diminished by AE extract 250 µg/mL and VB 125 µg/mL. Our results indicate that AE extract and VB are promising ingredients for anti-hair loss applications. However, further clinical study is necessary to evaluate the effectiveness of AE extract and VB as treatment for actual hair loss.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation., Oxylipins are lipid mediators generated during infection for regulating inflammatory responses, but how they are removed is not completely clear. Here the authors show that cellular oxylipin removal is linked to mitochondria β-oxidation by CPT1, a mitochondria lipid importer protein, to serve as a metabolic checkpoint for oxylipin homeostasis and inflammation.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder that inflicts damage to the joints of the hands and wrist. The aim of this study was to investigate the protective effect of β-Arrestin-2 (βArr2) on RA in vivo and in vitro. The βArr2 adenovirus (βArr2-Ad) or the control (Con-Ad) was injected into the ankle joint cavity of collagen-induced arthritis (CIA) mice. According to the results, an improvement was shown in the symptoms and pathological injury of RA after an upregulation of βArr2. Correspondingly, the inflammatory response was attenuated, as evidenced by the decreased serum pro-inflammatory cytokines levels and NF-κB pathway-related proteins. Nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome activation was inhibited in CIA mice treated with βArr2-Ad injection, as reflected by the diminished IL-18 level and declined protein levels of ankle inflammasome components in the ankle joint. Likewise, the anti-inflammatory effect of macrophages was also validated by in vitro experiments. In summary, βArr2 effectively ameliorates ankle inflammation in CIA mice via NF-κB/NLRP3 inflammasome, providing theoretical and clinical basis for RA therapy.Key wordsRheumatoid arthritis; β-arrestin-2; NF-κB; NLRP3.

Inflammation causes a protective immune response, which can be observed by examining the inflammatory responses of macrophages. Macrophages release various immunostimulatory factors when destroying external pathogens. We induced lipopolysaccharides (LPS) in RAW 264.7 cells, a macrophage cell line, to determine whether Helixor-M can cause immuno-suppression. Helixor-M is known to have anticancer and immune effects. However, an indicator that regulates immunity has not been clearly confirmed. To this end, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to confirm Helixor-M was not cytotoxic. Western blotting and real-time polymerase chain reaction (RT-PCR) confirmed the anti-inflammatory effects. Additionally, immunofluorescence assay confirmed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, a representative inflammatory pathway. Helixor-M was found to be non-cytotoxic, induce the NF-κB pathway, and reduce the levels of pro-inflammatory cytokine and mitogen-activated protein kinase (MAPK). We found Helixor-M affected the PI3K/AKT/JNK pathway. Therefore, we confirmed Helixor-M acts as an anti-inflammatory agent through NF-κB, TLR4 and PI3K inhibition and that it could be an effective immunosuppressive drug.

LPS causes lysosome tubulation in macrophages and dendritic cells. The PI3K-Akt-mTOR pathway is necessary for LPS-induced lysosome tubulation, and mTOR is required for MHC-II presentation in dendritic cells. Evidence shows that mTOR may control lysosome tubulation by modulating microtubule motor activity through Arl8b., Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.

Specific changes in phospholipid content are a hallmark of the membranes of maturing endosomes and phagosomes, but is it unclear how this is controlled. Naufer et al. now show that acidification of the lumen of endosomes and phagosomes triggers dissociation of the Vps34 lipid kinase from these organelles, which terminates PtdIns(3)P synthesis and signaling., Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

Phosphoinositide (PtdInsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them

Breast cancer is one of the most common types of cancer in female patients with high morbidity and mortality. Multi-drug chemotherapy has significant advantages in the treatment of malignant tumors, especially in reducing drug toxicity, increasing drug sensitivity and reducing drug resistance. The objective of this research is to fabricate lipid nanoemulsions (LNs) for the co-delivery of PTX and docosahexaenoic acid (DHA) with folic acid (FA) decorating (PTX/DHA-FA-LNs), and investigate the anti-tumor activity of the PTX/DHA-FA-LNs against breast cancer both in vitro and in vivo. PTX/DHA-FA-LNs showed a steady release of PTX and DHA from the drug delivery system (DDS) without any burst effect. Furthermore, the PTX/DHA-FA-LNs exhibited a dose-dependent cytotoxicity and a higher rate of apoptosis as compared with the other groups in MCF-7 cells. The cellular uptake study revealed that this LNs were more readily uptaken by MCF-7 cells and M2 macrophages in vitro. Additionally, the targeted effect of PTX/DHA-FA-LNs was aided by FA receptor-mediated endocytosis, and its cytotoxicity was proportional to the cellular uptake efficiency. The anti-tumor efficiency results showed that PTX/DHA-FA-LNs significant inhibited tumor volume growth, prolonged survival time, and reduced toxicity when compared with the other groups. These results indicated that DHA increases the sensitivity of tumor cells and tumor-associated macrophages (ATM2) to PTX, and synergistic effects of folate modification in breast cancer treatment, thus PTX/DHA-FA-LNs may be a promising nanocarrier for breast cancer treatment.