Murine Macrophage RAW Research Articles

Acenocoumarol Exerts Anti-Inflammatory Activity via the Suppression of NF-κB and MAPK Pathways in RAW 264.7 Cells

The repurposing of already-approved drugs has emerged as an alternative strategy to rapidly identify effective, safe, and conveniently available new therapeutic indications against human diseases. The current study aimed to assess the repurposing of the anticoagulant drug acenocoumarol for the treatment of chronic inflammatory diseases (e.g., atopic dermatitis and psoriasis) and investigate the potential underlying mechanisms. For this purpose, we used murine macrophage RAW 264.7 as a model in experiments aimed at investigating the anti-inflammatory effects of acenocoumarol in inhibiting the production of pro-inflammatory mediators and cytokines. We demonstrate that acenocoumarol significantly decreases nitric oxide (NO), prostaglandin (PG)E2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Acenocoumarol also inhibits the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2, potentially explaining the acenocoumarol-induced decrease in NO and PGE2 production. In addition, acenocoumarol inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs), c-Jun N terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK), in addition to decreasing the subsequent nuclear translocation of nuclear factor κB (NF-κB). This indicates that acenocoumarol attenuates the macrophage secretion of TNF-α, IL-6, IL-1β, and NO, inducing iNOS and COX-2 expression via the inhibition of the NF-κB and MAPK signaling pathways. In conclusion, our results demonstrate that acenocoumarol can effectively attenuate the activation of macrophages, suggesting that acenocoumarol is a potential candidate for drug repurposing as an anti-inflammatory agent.

Low Weight Polysaccharide of Hericium erinaceus Ameliorates Colitis via Inhibiting the NLRP3 Inflammasome Activation in Association with Gut Microbiota Modulation.

Ulcerative colitis (UC), one of the typical inflammatory bowel diseases caused by dysregulated immunity, still requires novel therapeutic medicine with high efficacy and low toxicity. Hericium erinaceus has been widely used to treat different health problems especially gastrointestinal sickness in China for thousands of years. Here, we isolated, purified, and characterized a novel low weight polysaccharide (HEP10, Mw: 9.9 kDa) from the mycelia of H. erinaceus in submerged culture. We explored the therapeutic effect of HEP10 on UC and explored its underlying mechanisms. On one hand, HEP10 suppressed the production of TNF-α, IL-1β, IL-6, inducible iNOS, and COX-2 in LPS challenged murine macrophage RAW264.7 cells, as well as in colons from DSS-induced colitis mice. On the other hand, HEP10 treatment markedly suppressed the activation of NLRP3 inflammasome, NF-κB, AKT, and MAPK pathways. Moreover, HEP10 reversed DSS-induced alternation of the gut community composition and structure by significantly increasing Akkermansia muciniphila and also promoting functional shifts in gut microbiota. Structural equation modeling also highlighted that HEP10 can change widely through gut microbiota. In conclusion, HEP10 has a better prebiotic effect than the crude polysaccharides of H. erinaceus, which can be used as a novel dietary supplement and prebiotic to ameliorate colitis.

Magnolia kobus Extract Inhibits Periodontitis-Inducing Mediators in Porphyromonas gingivalis Lipopolysaccharide-Activated RAW 264.7 Cells.

Periodontitis, a disease caused by inflammation of oral bacteria, contributes to the loss of alveolar bone and destruction of connective tissues. Porphyromonas gingivalis, a Gram-negative bacterium, is known to possess important pathogenic factors for periodontal disease. In this study, we investigated the anti-periodontitis effects of Magnolia kobus extract (MKE) and magnolin as a component of Magnolia kobus (MK) in murine macrophage RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Effects of MKE and magnolin on the mechanism of RAW 264.7 cellular inflammation were determined by analyzing nitric oxide (NO) production and Western blot protein expression (n = 3). MKE/magnolin inhibited NO production without affecting cell survival. MKE/magnolin treatment inhibited LPS-induced pro-inflammatory cytokines, expression levels of matrix metalloproteinases (MMPs such as MMP-1, 3, 8, 9, and 13), and protein levels of inflammatory mediators (such as TNF-α, IL-1β, and mPGES-1). MKE/magnolin also suppressed NF-κB activation by inhibiting the TLR4 signaling pathway. These findings suggest that MKE has a therapeutic effect on inflammatory periodontal disease caused by oral bacterium P. gingivalis and that magnolin is a major functional component in the anti-inflammatory effect of MKE.

Microwave‐Assisted Multicomponent Synthesis of New 6‐Arylated 5‐Hydroxy‐benzo[a]phenazine Derivatives and Their Potential Anti‐inflammatory Activity

AbstractA convenient two step “one‐pot” four‐component synthetic approach was developed for the synthesis of new 6‐arylated 5‐hydroxy‐benzo[a]phenazine derivatives using 2‐hydroxy‐1,4‐naphthoquinone, a variety of aromatic aldehydes, benzene‐1,2‐diamine, and tetronic acid. In all cases, the synthesis proceededviathree key steps including condensation, Michael addition, and tautomerization. The structure of synthesized compounds was elucidated by spectral data. The inhibitory effect of newly synthesized compounds on nitric oxide (NO) production in lipopolysaccharide (LPS)‐ stimulated murine macrophage RAW 264.7 cells was assessed. All products exhibited the highly potential NO production inhibition activity with the IC50values in the range of 3.68–12.14 μM, indicating the critical role of these compounds in the development of potential anti‐inflammation agents.

STING antagonists, synthesized via Povarov-Doebner type multicomponent reaction.

The cGAS-STING axis plays an important role in protecting higher organisms against invading pathogens or cancer by promoting the production of cytokines and interferons. However, persistent or uncontrolled activation of this pathway could lead to inflamed environments, which is detrimental to the host in the long run. Persistent activation of STING is known to be the cause of STING-associated vasculopathy with onset in infancy (SAVI) and activated STING is believed to play important roles in worsening various diseased states, such as traumatic brain injury, diabetic kidney disease and colitis. Thus, antagonists of STING could play important roles in managing various inflammatory diseases. Herein, we report the discovery of small molecule STING inhibitors, HSD1077 and analogs, which are facilely synthesized via a Povarov-Doebner type three-component reaction involving an amine, ketone, and aldehyde. Structure-activity relationship, SAR, studies indicate that both the 3H-pyrazolo[4,3-f]quinoline and pyrazole moieties in HSD1077 are critical for STING binding. At concentrations as low as 20 nM, HSD1077 suppressed type-1 interferon expression in both murine RAW macrophages and human THP-1 monocytes upon treatment with 100 μM 2'-3' cGAMP. Compounds containing the 3H-pyrazolo[4,3-f]quinoline moiety have the potential to be translated into anti-inflammatory compounds via STING inhibition.

Cissus quadrangularis (Hexane Fraction) Inhibits RANK-L Induced Osteoclast Differentiation of Murine Macrophage RAW264.7 Cell Line

Cissus quadrangularis (Hexane Fraction) Inhibits RANK-L Induced Osteoclast Differentiation of Murine Macrophage RAW264.7 Cell Line

Activating SIRT1 deacetylates NF-κB p65 to alleviate liver inflammation and fibrosis via inhibiting NLRP3 pathway in macrophages.

Background and aims: Macrophages play a critical role in the development of liver diseases. As an NAD+-dependent histone deacetylase, SIRT1 inhibits liver inflammation and fibrosis, but the mechanisms are not fully understood. Our aim was to investigate the molecular mechanism of SIRT1 in macrophages in liver inflammation and fibrosis. Methods: We employed the CCl4-induced hepatic fibrosis rat models and cultured murine macrophages RAW 264.7 in vitro to explore the anti-fibrosis effect of SIRT1. The content of cytokines was measured with ELISA. The expression of proteins associated with the NF-κB /NLPR3 signaling pathway was detected by Western blot, co-immunoprecipitation, and immunofluorescence. SIRT1, NF-κB, and NLRP3 genes were knocked down in RAW 264.7 cells by small interfering RNA (siRNA) transfection. Results: The expression of NF-κB p65, NLRP3, α-SMA, and iNOS increased in liver tissue, with high plasma LPS level and low expression of SIRT1 in CCl4-induced rat models. Overexpressing SIRT1 could inhibit these protein levels, decrease plasma LPS level, and attenuate liver injury and fibrosis. In vitro, LPS induced cytomorphology changes and up-regulated NF-κB/NLRP3 pathway, with the low expression of SIRT1 in RAW 264.7; meanwhile, the secretion of inflammatory factors increased. Nevertheless, knockdown of NF-κB or NLRP3 and activation of SIRT1 inhibited inflammation of macrophages; inhibition or knockdown of SIRT1 enhanced macrophage inflammation. Furthermore, activation of SIRT1 could inhibit LPS-treated macrophages from activating hepatic stellate cells (HSCs). Conclusions: Activating SIRT1 inhibits the inflammation in macrophages by down-regulating NLRP3 pathway through deacetylating NF-κB p65, which in turn inhibits the activation of HSCs to alleviate hepatic inflammation and fibrosis.

Synthesis and anti-inflammatory activity evaluation of novel AZT and adenosine derivatives

A five-step procedure was used to synthesize four novel conjugates of AZT and adenosine with quinazolinone scaffold. In the last step, alkynes-1 of quinazolinone were coupled to adenosine azide and AZT by Click chemistry to yield the designed conjugates. Their structures were characterized by full-length data of spectra including 1H-, 13C-NMR and MS. Screening for their in vitro anti-inflammatory activity was performed using Murine macrophage RAW 264.7 cells. The relationship between structure and biological activity was also discussed.

Suppressive Effects of Flavonoids on Macrophage-Associated Adipocyte Inflammation in a Differentiated Murine Preadipocyte 3T3-L1 Cells Co-Cultured with a Murine Macrophage RAW264.7 Cells.

The suppressive effects of flavonoids on macrophage-associated adipocyte inflammation in a differentiated murine preadipocyte cell line (3T3-L1) co-cultured with a murine macrophage cell line (RAW264.7) were evaluated. Extracellular lipid accumulation was investigated via Oil Red O staining. The expression levels of adipogenesis- and inflammation-associated proteins, including CCAAT/enhancer-binding protein (C/EBP)-α, inducible nitric oxide synthase (iNOS), C/EBPβ, peroxisome proliferator-activated receptor γ (PPARγ), and cyclooxygenase-2 (COX-2), were determined via Western blotting. Proinflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1) and interleukin-6 (IL-6), were assessed using enzyme-linked immunosorbent assay kits. We found that silybin, formononetin, and diosmetin inhibited lipid accumulation and production of proinflammatory cytokines in the co-cultures of 3T3-L1 and RAW264.7 cells. Moreover, they inhibited the protein expression of PPARγ, C/EBPα, COX-2, C/EBPβ, and iNOS in the co-cultures of 3T3-L1 and RAW264.7 cells. These data support that silybin, formononetin, and diosmetin inhibit macrophage-associated adipocyte inflammation and lipid accumulation.

Immunotoxicity and Anti-Inflammatory Characterizations of Prenylated Flavonoids-The Lipophilic 7-O-Terpenylated Wogonin.

Wogonin, one of the exceptional bioactive flavonoids found abundant in the roots of Huang-Qin (Scutellaria baicalensis Georgi), is a popular health-preserving Chinese medicine. The therapeutic applications can be expanded by improving its bioavailability. The 7-O-terpenylated wogonin consisting one to three prenyl units are chemically synthesized for increasing lipophilic nature for efficient uptake, and also an attempt in mimicry of naturally scarce terpenylated flavonoids found in limited plant families and bee propolis. Wogonin (W) and its lipophilic nature prenyl wogonin (W5), geranyl wogonin (W10), and farnesyl wogonin (W15) were comparatively studied with structure-relationship in immunotoxicity of cell livability on lymphoid, myeloid, and somatic origins cell lines. Anti-inflammatory functions characterized with nitric oxide inhibition and intracellular ROS level of LPS-activated murine macrophage RAW264.7 were assessed. Wogonin and its terpenylated derivatives have selectively influenced livability of lymphoid origin cells but not myeloid and somatic origin cells. The mitotic protein survivin gene expressions analysis further supported the selective suppressions on lymphoid origin YAC-1 cells by wogonin and geranyl wogonin, while oppositely boosted survivin expressions in LPS-activated macrophages. Moreover, wogonin exhibits dose-dependent inhibition on the nitric oxide (NO) production and iNOS gene expressions of LPS-activated RAW264.7 cells. Terpenylated wogonin exhibits profoundly superior control in intracellular ROS level and a sustained action with sound cell integrity than the wogonin. The enhanced cellular uptake with higher lipophilicity to membrane of 7-O-terpenylated wogonin may pose an important biological nature in facilitating better bioavailability and specific immunomodulatory actions of the category of terpenylated flavonoids. The 7-O-terpenylated wogonin having biological merit of fast membrane lipid bilayer integration, lower effective concentration, and better preserving immune cells functions and livability deserved further in-depth investigations and their broadly therapeutic applications.

Innate and Adaptive Immune Responses Induced by Aspergillus fumigatus Conidia and Hyphae.

Previous research indicated that hyphae of Aspergillus fumigatus (A. fumigatus) rather than conidia could successfully build a pulmonary aspergillosis model in immunocompetent mice. In this study, we compared the immune responses induced by hyphae and conidia to explore the possible mechanism of this striking phenomenon. Herein, a novel method was designed and adopted to quantify hyphal fragments. Murine macrophages RAW264.7 and human peripheral blood mononuclear cells were stimulated by A. fumigatus hyphae and conidia in vitro, respectively, and then immunological reactions were measured. Male C57BL/6 mice were challenged with conidia and hyphae through intratracheal inoculation. Dynamic conditions of mice were recorded, and RNA-seq measured corresponding immune responses. The results of the study confirmed that hyphae could induce more intensive inflammation than conidia in vitro and in vivo. However, macrophages revealed a higher production of ROS and M1 polarisation in response to conidia stimuli. Additionally, conidia could promote Th1 cell differentiation, while hyphae could increase the CD4/CD8 ratio. RNA-seq validated the fact that those multiple immunologically relevant pathways were more strongly activated by hyphae than conidia, which also promoted Th2 cell differentiation and suppressed Th1 signalling. Both hyphae and conidia could activate Th17 signalling. In general, conidia and hyphae induced distinctly different host immune responses, and the immune responses induced by conidia played a better protective effect. Therefore, the unique function of hyphae in the spread and infection of Aspergillus should be emphasised, and more research is required to clarify the underlying mechanisms for better understanding and management of aspergillosis.

Radiopaque Iodosilane-Coated Lipid Hybrid Nanoparticle Contrast Agent for Dual-Modality Ultrasound and X-ray Bioimaging.

Here, we report the synthesis of robust hybrid iodinated silica-lipid nanoemulsions (HSLNEs) for use as a contrast agent for ultrasound and X-ray applications. We engineered iodinated silica nanoparticles (SNPs), lipid nanoemulsions, and a series of HSLNEs by a low-energy spontaneous nanoemulsification process. The formation of a silica shell requires sonication to hydrolyze and polymerize/condensate the iodomethyltrimethoxysilane at the oil/water interface of the nanoemulsion droplets. The resulting nanoemulsions (NEs) exhibited a homogeneous spherical morphology under transmission electron microscopy. The particles had diameters ranging from 20 to 120 nm with both negative and positive surface charges in the absence and presence of cetyltrimethylammonium bromide (CTAB), respectively. Unlike CTAB-coated nanoformulations, the CTAB-free NEs showed excellent biocompatibility in murine RAW macrophages and human U87-MG cell lines in vitro. The maximum tolerated dose assessment was evaluated to verify their safety profiles in vivo. In vitro X-ray and ultrasound imaging and in vivo computed tomography were used to monitor both iodinated SNPs and HSLNEs, validating their significant contrast-enhancing properties and suggesting their potential as dual-modality clinical agents in the future.

Preparation of High-Solid Microfibrillated Cellulose from Gelidium amansii and Characterization of Its Physiochemical and Biological Properties.

Microfibrillated cellulose (MFC) is a valuable material with wide industrial applications, particularly for the food and cosmetics industries, owing to its excellent physiochemical properties. Here, we prepared high-solid microfibrillated cellulose (HMFC) from the centrifugation of Gelidium amansii-derived MFC right after fibrillation. Dispersion properties, morphology, and structural changes were monitored during processing. HMFC has a five-fold higher solid concentration than MFC without significant changes to dispersion properties. SEM images and FTIR spectra of HMFC revealed a stable surface and structure against centrifugal forces. HMFC exhibited 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, although it could not scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH). Moreover, HMFC inhibited the generation of LPS-induced excessive nitrite and radial oxygen species in murine macrophage RAW264.7 cells. Additionally, HMFC suppressed LPS-induced Keap-1 expression in the cytosol but did not alter iNOS expression. HMFC also attenuated the UVB-induced phosphorylation of p38, c-Jun N-terminal kinase (JNK) 1/2, and extracellular-signal-regulated kinase (ERK) 1/2, as well as the phosphorylation of c-Jun in the immortalized human skin keratinocyte HaCaT cells. Therefore, the application of centrifugation is suitable for producing high-solid MFC as a candidate material for anti-inflammatory and anti-oxidative marine cosmeceuticals.

Anti-Inflammatory and Antioxidant Properties of Physalis alkekengi L. Extracts In Vitro and In Vivo: Potential Application for Skin Care.

Objective Inflammatory skin disorders are becoming major issues threatening public health with increasing prevalence. This study was to evaluate the anti-inflammatory, antioxidant, and antisenescent activities of traditional folk medicinal plant, Physalis alkekengi L. extracts to alleviate skin inflammation and its possible mechanisms. Methods Lipopolysaccharides (LPS)-treated murine macrophages RAW264.7 and human skin keratinocytes HaCaT were incubated with the plant extracts, respectively. The production of nitric oxide (NO) was tested by using Griess reagents. The activity of nitric oxide synthase (NOS) was detected through a fluorescence microplate reader. Reactive oxygen species (ROS) production and cell apoptosis were quantified by flow cytometry. The proinflammatory cytokines were measured using ELISA and qRT-PCR. Human skin fibroblasts (HFF-1) were coincubated with D-galactose (D-gal) and the plant extracts. The senescence associated-galactosidase (SA-β-gal) was stained to evaluate cellular senescence. The senescence-associated secretory phenotype (SASP), IL-1β, was measured through ELISA. The mRNA of IL-1α in SLS-stimulated and PGE2 in UV-radiated 3D skin models were detected by qRT-PCR. In vivo ROS production and neutrophil recruitment in CuSO4-treated zebrafish models were observed by fluorescence microscopy. Inflammation-related factors were measured by qRT-PCR. Results. In vitro, Physalis alkekengi L. significantly reduced NO production, NOS activity, cell apoptosis, transcription of TNF-α, IL-6, IL-1β and ROS production. These plant extracts markedly attenuated SA-β-gal and IL-1β and downregulated the production of IL-1α and PGE2. In vivo, the plant extracts dramatically dampened ROS production, the number of neutrophils, and proinflammatory cytokines. Conclusions Cumulatively, this work systematically demonstrated the anti-inflammatory, antioxidant, and antisenescent properties of Physalis alkekengi L. and proposed the possible roles of Physalis alkekengi L. in inflammatory signaling pathways, providing an effective natural product for the treatment of inflammatory skin disorders.

First Occurrence of Megastigmane Glucosides in a Plant of Retama Genus.

Chemical investigation of Retama sphaerocarpa collected in Algeria resulted in the isolation of two megastigmane glucosides, compounds 1 and 2, along with a series of isoflavones and phenol derivatives. Compound 1, named retamoside, was new and its structure was determined by extensive application of spectroscopic methods, including HRMS, 1D and 2D NMR and CD. The anti-inflammatory properties of co-occurring main megastigmane, saurobaccioside B (2) and structurally related vomifoliol (3) on LPS-stimulated murine macrophages RAW 274.7 have been evaluated.

Enzyme-assisted extraction of anti-inflammatory compounds from habanero chili pepper (Capsicum chinense) seeds.

Capsaicinoids are the main bioactive compounds extracted from chili pepper seeds (CPSs) but other bioactive compounds such as phenolic compounds may be found. Enzyme-assisted extraction (EAE) improves the extraction of bioactive compounds from fruits and seeds. The aim of this study was to establish the cellulase-assisted extraction conditions of capsaicinoids and phenolic compounds from Habanero CPSs (Capsicum chinense) and to evaluate the anti-inflammatory activity of the obtained extracts on murine macrophages. EAE was performed using different temperatures (T1 = 30°C, T2 = 45°C and T3 = 60°C), enzyme concentrations (E1 = 2,500 UI/L and E2 = 250 UI/L), and extraction time periods (0-150 min). Total phenolic compounds were quantified using the Folin-Ciocalteu assay, capsaicin (CAP) and dihydrocapsaicin (DHC) contents were evaluated by HPLC, and anti-inflammatory activity was performed with Griess assay on murine macrophage RAW 264.7 cell culture. The highest phenolic compound content (337.96 mg GAE/L) was achieved at 30°C, 2,500 UI/L, and 150 min of extraction. The highest CAP content (310.23 μg/ml) was obtained at 45°C with 250 UI/L for 150 min, while for DHC (167.72 μg/ml), the conditions were 60°C, 2,500 UI/L, and 120 min. The highest anti-inflammatory response was obtained when 60°C, E2, and 150 min were used for the extraction, and nitric oxide (NO) production was reduced to 22.56%. Based on the results obtained in this research, EAE allowed the recovery of compounds with anti-inflammatory activity from CPS using water as a solvent. There was a correlation between the extraction of CAP and DHC. But although a moderate direct correlation between the concentration of capsaicinoids and total phenolic compounds (TPCs) and an inverse correlation of the presence of the bioactive compounds (TPC, CAP, and DHC) with the NO synthesis, these were not statistically significant. We demonstrated that Habanero seeds are an important raw material to recover anti-inflammatory compounds beyond capsaicinoids using water in EAE.

A Comparative Study of Hesperetin, Hesperidin and Hesperidin Glucoside: Antioxidant, Anti-Inflammatory, and Antibacterial Activities In Vitro.

The antioxidant, anti-inflammatory and antibacterial activities of hesperetin, hesperidin and hesperidin glucoside with different solubility were compared in vitro. Hesperetin was prepared by enzymatic hydrolysis from hesperidin, and hesperidin glucoside composed of hesperidin mono-glucoside was prepared from hesperidin through enzymatic transglycosylation. Solubility of the compounds was different: the partition coefficient (log P) was 2.85 ± 0.02 for hesperetin, 2.01 ± 0.02 for hesperidin, and −3.04 ± 0.03 for hesperidin glucoside. Hesperetin showed a higher effect than hesperidin and hesperidin glucoside on radical scavenging activity in antioxidant assays, while hesperidin and hesperidin glucoside showed similar activity. Cytotoxicity was low in the order of hesperidin glucoside, hesperidin, and hesperetin in murine macrophage RAW264.7 cells. Treatment of the cells with each compound reduced the levels of inflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hesperetin was most effective at relatively low concentrations, however, hesperidin glucoside was also effective at higher concentration. Hesperetin showed higher antibacterial activity than hesperidin in both Gram-positive and -negative bacteria, and hesperidin glucoside showed similarly higher activity with hesperetin depending on the bacterial strain. In conclusion, hesperetin in the form of aglycone showed more potent biological activity than hesperidin and hesperidin glucoside. However, hesperidin glucoside, the highly soluble form, has been shown to increase the activity compared to poorly soluble hesperidin.

Human Umbilical Cord Mesenchymal Stem Cells Promote Macrophage PD-L1 Expression and Attenuate Acute Lung Injury in Mice.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains a serious clinical problem but has no approved pharmacotherapy. Mesenchymal stem cells (MSCs) represent an attractive therapeutic tool for tissue damage and inflammation owing to their unique immunomodulatory properties. The present study aims to explore the therapeutic effect and underlying mechanisms of human umbilical cord MSCs (UC-MSCs) in ALI mice. In this study, we identify a novel mechanism for human umbilical cord-derived MSCs (UC-MSCs)-mediated immunomodulation through PGE2-dependent reprogramming of host macrophages to promote their PD-L1 expression. Our study suggests that UC-MSCs or primed- UC-MSCs offer new therapeutic approaches for lung inflammatory diseases. Lipopolysaccharide (LPS)-induced ALI mice were injected with 5×105 UC-MSCs via the tail vein after 4 hours of LPS exposure. After 24 hours of UC-MSC administration, the total protein concentration and cell number in the bronchoalveolar lavage fluid (BALF) and cytokine levels in the lung tissue were measured. Lung pathological changes and macrophage infiltration after UCMSC treatment were analyzed. Moreover, in vitro co-culture experiments were performed to analyze cytokine levels of RAW264.7 cells and Jurkat T cells. UC-MSC treatment significantly improved LPS-induced ALI, as indicated by decreased total protein exudation concentration and cell number in BALF and reduced pathological damage in ALI mice. UC-MSCs could inhibit pro-inflammatory cytokine levels (IL-1β, TNF-α, MCP-1, IL-2, and IFN-γ), while enhancing anti-inflammatory cytokine IL-10 expression, as well as reducing macrophage infiltration into the injured lung tissue. Importantly, UC-MSC administration increased programmed cell death protein ligand 1 (PD-L1) expression in the lung macrophages. Mechanistically, UC-MSCs upregulated cyclooxygenase-2 (COX2) expression and prostaglandin E2 (PGE2) secretion in response to LPS stimulation. UC-MSCs reduced the inflammatory cytokine levels in murine macrophage Raw264.7 through the COX2/PGE2 axis. Furthermore, UC-MSC- derived PGE2 enhanced PD-L1 expression in RAW264.7 cells, which in turn promoted programmed cell death protein 1 (PD-1) expression and reduced IL-2 and IFN-γ production in Jurkat T cells. Our results suggest that UC-MSCs attenuate ALI via PGE2-dependent reprogramming of macrophages to promote their PD-L1 expression.

Cholesterolinhibits autophagy in RANKL-induced osteoclast differentiation through activating the PI3K/AKT/mTOR signaling pathway.

A dysregulated balance between bone formation and bone resorption controlled by osteoblast and osteoclast will lead to osteoporosis. Cholesterol (CHO) is a crucial factor leading to osteoporosis, and autophagy appears to involve it. Therefore, we aimed to study the molecular mechanism of autophagy in CHO-induced osteoclasts differentiation. Nuclear factor-κ B ligand as a receptor activator was used to induce osteoclasts differentiation of murine macrophage RAW264.7 treated with CHO, PI3-kinase inhibitor (LY294002), and Rapamycin (RAPA), respectively. Western blot assay was used to detect the expression of TRAP/ACP5 and the proteins involved in autophagy and the PI3K/AKT/mTOR signaling pathway. In addition, TRAP staining, bone resorption assay, and F-actin immunofluorescence were performed to evaluate the ability of osteoclast formation. Transmission electron microscopy and immunofluorescence were also executed to observed the expression of LC3B, and autophagosome. When RAW264.7 was treated with 20μg/mL CHO for 5 consecutive days, It exhibited the optimal osteoclast activity. In addition, CHO could inhibit autophagy and activate the PI3K/AKT/mTOR signaling pathway. Moreover, the effects of CHO on osteoclast differentiation and autophagy could partially be reversed by LY294002 and RAPA. Therefore, our results demonstrated that CHO could inhibit autophagy during osteoclast differentiation by activating the PI3K/AKT/mTOR signaling pathway. These findings provided important theoretical basis for CHO in bone resorption and formation.

Using immune cell-based bioactivity assays to compare the inflammatory activities of oil sands process-affected waters from a pilot scale demonstration pit lake

In this study, we provide evidence that oil sands process-affected waters (OSPW) contain factors that activate the antimicrobial and proinflammatory responses of immune cells. Specifically, using the murine macrophage RAW 264.7 cell line, we establish the bioactivity of two different OSPW samples and their isolated fractions. Here, we directly compared the bioactivity of two pilot scale demonstration pit lake (DPL) water samples, which included expressed water from treated tailings (termed the before water capping sample; BWC) as well as an after water capping (AWC) sample consisting of a mixture of expressed water, precipitation, upland runoff, coagulated OSPW and added freshwater. Significant inflammatory (i.e. macrophage activating) bioactivity was associated with the AWC sample and its organic fraction (OF), whereas the BWC sample had reduced bioactivity that was primarily associated with its inorganic fraction (IF). Overall, these results indicate that at non-toxic exposure doses, the RAW 264.7 cell line serves as an acute, sensitive and reliable biosensor for the screening of inflammatory constituents within and among discrete OSPW samples.