The Role of Copper-Induced M2 Macrophage Polarization in Protecting Cartilage Matrix in Osteoarthritis.

BACKGROUND The pathological mechanism of osteoarthritis is still unclear. The regulation of the immune microenvironment has been of growing interest in the progression and treatment of osteoarthritis. Macrophages with different phenotypes, producing different cytokines, have been linked to the mechanism of cartilage injury in osteoarthritis. Copper ions play a role in the immune response and are involved in the pathological mechanisms of osteoarthritis by affecting the metabolism of the cartilage matrix. Bioactive glass (BG) is an osteogenic material with superior biocompatibility. Here, we report on the regulatory behavior of macrophages using a copper-based composite BG material. MATERIAL AND METHODS Cu-BGC powder was prepared by sol-gel method, and scaffolds were fabricated and characterized using 3D printing. Macrophage cultures grown with Cu-BGC were examined for cell culture and proliferation. The effect of Cu-BGC on the degradation metabolism of chondrocytes, cultured in the environment of inflammatory cytokine IL-1ß, was determined. In addition, the morphology of macrophages, secretion of inflammatory cytokines, and expression of surface markers were examined. RESULTS The results show that Cu-BGC promotes macrophage proliferation at a range of concentrations and increases the secretion of anti-inflammatory cytokines while inhibiting proinflammatory cytokines. At the same time, M2-type cell surface markers are definitely expressed and the morphology of macrophages is altered. In addition, Cu-BGC inhibited the degradation metabolism of chondrocytes in the inflammatory environment induced by IL-1ß. CONCLUSIONS These results suggest that Cu-BGC induced macrophage polarization into an M2 type anti-inflammatory phenotype, and inhibition of immune injury response may play a role in delaying cartilage matrix damage in osteoarthritis.

[Immunoadjuvant Effect of Chitosan Oligosaccharide and Its Feasibility of Being Used as an Adjuvant for Attenuated Live Bacteria Vector Vaccines]. / å£³å¯¡ç³–çš„å ç–«ä½å‰‚æ•ˆæžœåŠä½œä¸ºå‡æ¯’æ´»èŒè½½ä½“ç–«è‹—ä½å‰‚çš„å¯è¡Œæ€§æŽ¢ç´¢.

Objective: To study the immunoadjuvant effects of chitosan oligosaccharide (COS), including the immune activation and the triggering of lysosomal escape, and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines. Methods: 1) Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL (the control group) and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay. Mouse macrophages RAW264.7 were treated with COS at 0 (the control group), 1, 2, and 4 mg/mL for 24 h. Then, the mRNA expression levels of the cytokines, including IFN-γ, IL-10, TGF-ß, and TLR4, were determined by RT-qPCR assay. 2) RAW264.7 cells were treated with 1 mL of PBS containing different components, including calcein at 50 µg/mL, COS at 2 mg/mL, and bafilomycin A1, an inhibitor, at 1 µmol/mL, for culturing. The cells were divided into the Calcein group, Calcein+COS group, and Calcein+COS+Bafilomycin A1 group accordingly. Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein, a fluorescent dye, in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape. 3) LM∆E6E7 and LI∆E6E7, the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer, were encapsulated with COS at the mass concentrations of 0.5 mg/mL, 1 mg/mL, 2 mg/mL , 4 mg/mL, and 8 mg/mL. Then, the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria. Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM∆E6E7 and LI∆E6E7 were coated with COS at 2 mg/mL. Results: 1) CCK8 assays showed that, compared with the findings for the control group, the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation, with the difference being statistically significant (P<0.05). According to the RT-qPCR results, compared with those of the control group, the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells, while it inhibited the mRNA expression levels of TGF-ß and IL-10, with the most prominent effect being observed in the 4 mg/mL COS group (P<0.05). 2) Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group. In other words, a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention. Furthermore, this process could be blocked by bafilomycin A1. 3) The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL. Before and after the vaccine strain was encapsulated by COS, the phagocytosis of LM∆E6E7 by RAW264.7 cells was 5.70% and 22.00%, respectively, showing statistically significant differences (P<0.05); the phagocytosis of LI∆E6E7 by RAW264.7 cells was 1.55% and 6.12%, respectively, showing statistically significant differences (P<0.05). Conclusion: COS has the effect of activating the immune response of macrophages and triggering lysosomal escape. The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages. Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14.

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

Ethanol Extract of Limonium bicolor Improves Dextran Sulfate Sodium-Induced Ulcerative Colitis by Alleviating Inflammation and Restoring Gut Microbiota Dysbiosis in Mice.

Ulcerative colitis (UC) is a kind of inflammatory bowel condition characterized by inflammation within the mucous membrane, rectal bleeding, diarrhea, and pain experienced in the abdominal region. Existing medications for UC have limited treatment efficacy and primarily focus on symptom relief. Limonium bicolor (LB), an aquatic traditional Chinese medicine (TCM), exerts multi-targeted therapeutic effects with few side effects and is used to treat anemia and hemostasis. Nevertheless, the impact of LB on UC and its mechanism of action remain unclear. Therefore, the objective of this study was to investigate the anti-inflammatory effects and mechanism of action of ethanol extract of LB (LBE) in lipopolysaccharide-induced RAW 264.7 macrophages and dextran sulfate sodium (DSS)-induced UC. The results showed that LBE suppressed the secretion of cytokines in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. LBE had protective effects against DSS-induced colitis in mice, decreased the disease activity index (DAI) score, alleviated symptoms, increased colon length, and improved histological characteristics, thus having protective effects against DSS-induced colitis in mice. In addition, it reversed disturbances in the abundance of proteobacteria and probiotics such as Lactobacillus and Blautia in mice with DSS-induced UC. Based on the results of network pharmacology analysis, we identified four main compounds in LBE that are associated with five inflammatory genes (Ptgs2, Plg, Ppar-γ, F2, and Gpr35). These results improve comprehension of the biological activity and functionality of LB and may facilitate the development of LB-based compounds for the treatment of UC.

Microfluidic-assisted formulation of cell membrane-camouflaged anisotropic nanostructures.

Anisotropic gold (Au) nanostructures have been widely explored for various nanomedicine applications. While these nanomaterials have shown great promise for disease theranostics, particularly for cancer diagnosis and treatment, the utilization and clinical translation of anisotropic Au nanostructures have been limited by their high phagocytic uptake and clearance and low cancer targeting specificity. Numerous efforts have thus been made toward mitigating these challenges. Many conventional strategies, however, rely on all-synthetic materials, involve complex chemical processes, or have low product throughput and reproducibility. Herein, by integrating cell membrane coating and microfluidic technologies, a high-throughput bioinspired approach for synthesizing biomimetic anisotropic Au nanostructures with minimized phagocytic uptake and improved cancer cell targeting is reported. Through continuous hydrodynamic flow focusing, mixing, and sonication, Au nanostructures are encapsulated within the macrophage and cancer cell membrane vesicles effectively. The fabricated nanostructures are uniform and highly stable in serum. Importantly, the macrophage membrane vesicle-encapsulated Au nanostructures can be preferentially internalized by breast cancer cells, but not by macrophages. Overall, this study has demonstrated the feasibility of employing an integrated microfluidic-sonication technique to formulate uniform and highly stable biomimetic anisotropic nanostructures for enhanced cancer theranostic applications.

Lipoic acid-mediated oral drug delivery system utilizing changes on cell surface thiol expression for the treatment of diabetes and inflammatory diseases.

Lipoic acid (LA), which has good safety and oral absorption, is obtained from various plant-based food sources and needs to be supplemented through human diet. Moreover, substances with a disulfide structure can enter cells through dynamic covalent disulfide exchange with thiol groups on the cell membrane surface. Based on these factors, we constructed LA-modified nanoparticles (LA NPs). Our results showed that LA NPs can be internalized into intestinal epithelial cells through surface thiols, followed by intracellular transcytosis via the endoplasmic reticulum-Golgi pathway. Further mechanistic studies indicated that disulfide bonds within the structure of LA play a critical role in this transport process. In a type I diabetes rat model, the oral administration of insulin-loaded LA NPs exhibited a more potent hypoglycemic effect, with a pharmacokinetic bioavailability of 5.42 ± 0.53%, representing a 1.6 fold enhancement compared to unmodified PEG NPs. Furthermore, a significant upregulation of surface thiols in inflammatory macrophages was reported. Thus, we turned our direction to investigate the uptake behavior of inflammatory macrophages with increased surface thiols towards LA NPs. Inflammatory macrophages showed a 2.6 fold increased uptake of LA NPs compared to non-inflammatory macrophages. Surprisingly, we also discovered that the antioxidant resveratrol facilitates the uptake of LA NPs in a concentration-dependent manner. This is mainly attributed to an increase in glutathione, which is involved in thiol uptake. Consequently, we employed LA NPs loaded with resveratrol for the treatment of colitis and observed a significant alleviation of colitis symptoms. These results suggest that leveraging the variations of thiol expression levels on cell surfaces under both healthy and diseased states through an oral drug delivery system mediated by the small-molecule nutrient LA can be employed for the treatment of diabetes and certain inflammatory diseases.

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Activin A signaling stimulates neutrophil activation and macrophage migration in pancreatitis.

Acute Pancreatitis (AP) is associated with high mortality and current treatment options are limited to supportive care. We found that blockade of activin A (activin) in mice improves outcomes in two murine models of AP. To test the hypothesis that activin is produced early in response to pancreatitis and is maintained throughout disease progression to stimulate immune cells, we first performed digital spatial profiling (DSP) of human chronic pancreatitis (CP) patient tissue. Then, transwell migration assays using RAW264.7 mouse macrophages and qPCR analysis of "neutrophil-like" HL-60 cells were used for functional correlation. Immunofluorescence and western blots on cerulein-induced pancreatitis samples from pancreatic acinar cell-specific Kras knock-in (Ptf1aCreER™; LSL-KrasG12D) and functional WT Ptf1aCreER™ mouse lines mimicking AP and CP to allow for in vivo confirmation. Our data suggest activin promotes neutrophil and macrophage activation both in situ and in vitro, while pancreatic activin production is increased as early as 1 h in response to pancreatitis and is maintained throughout CP in vivo. Taken together, activin is produced early in response to pancreatitis and is maintained throughout disease progression to promote neutrophil and macrophage activation.

Intelligent berberine-loaded erythrocytes attenuated inflammatory cytokine productions in macrophages.

Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl2 and bis-sulfosuccinimidyl-suberate, and targeting was evaluated using flow cytometry and Wright-Giemsa staining. Differentiated macrophages were stimulated with lipopolysaccharide, and the inflammatory profiles of macrophages were evaluated using ELISA, western blotting, and real-time PCR. Findings indicated that the endocytosis method is preferred due to its low impact on the erythrocyte's structural integrity. Maximum loading achieved (1386.68 ± 22.43 µg/ml) at 1500 µg/ml berberine treatment at 37 °C for 2 h. Berberine successfully inhibited NF-κB translation in macrophages, and inflammatory response markers such as IL-1ß, IL-8, IL-23, and TNF-α were decreased by approximately ninefold, sixfold, twofold, eightfold, and twofold, respectively, compared to the LPS-treated macrophages. It was concluded that berberine-loaded erythrocytes can effectively target macrophages and modulate the inflammatory response.

Fucoidan from the cell wall of Silvetia siliquosa with immunomodulatory effect on RAW 264.7 cells.

Silvetia siliquosa, the only species of the family Fucaceae in China, is used as a medicine food homology. Fucoidan from S. siliquosa was extracted by hot water twice thoroughly (13 % of total yield), and a purified fucoidan SSF with a molecular weight of 93 kD was obtained. Chemical composition analysis demonstrated that SSF was primarily composed of sulfate (21.68 wt%) and fucose (84 % of all neutral monosaccharides). IR, methylation analysis, NMR and ESI-MS results indicated SSF had the backbone of mainly (1 â†’ 3)-α-L-fucopyranose and minor (1 â†’ 4)-α-L-fucopyranose, with little 1,3 and 1,4 branched ß-D-Xylp and ß-D-Galp. The in vitro immunomodulatory test on RAW 264.7 cells showed that SSF could up-regulate the expression of immune related factors and proteins in a concentration-dependent manner, but the immunomodulatory effect disappeared from desulfated SSF. This research indicated that highly sulfated fucan possessed immunomodulatory effect and the importance of sulfate groups in the activity of SSF.

Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds.

A chemical study of Aesculus wilsonii Rehd. (also called Suo Luo Zi) and the in vitro anti-inflammatory effects of the obtained compounds was conducted. Retrieving results through SciFinder showed that there were four unreported compounds, aeswilosides I-IV (1-4), along with fourteen known isolates (5-18). Their structures were elucidated by extensive spectroscopic methods such as UV, IR, NMR, [α]D, and MS spectra, as well as acid hydrolysis. Among the known ones, compounds 5, 6, 8-10, and 12-16 were obtained from the Aesculus genus for the first time; compounds 7, 11, 17, and 18 were first identified from this plant. The NMR data of 5 and 18 were reported first. The effects of 1-18 on the release of nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW264.7 cells were determined. The results showed that at concentrations of 10, 25, and 50 µM, the novel compounds, aeswilosides I (1) and IV (4), along with the known ones, 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10) and pisuminic acid (15), displayed significant inhibitory effects on NO production in a concentration-dependent manner. It is worth mentioning that compound 10 showed the best NO inhibitory effect with a relative NO production of 88.1%, which was close to that of the positive drug dexamethasone. The Elisa experiment suggested that compounds 1, 4, 10, and 15 suppressed the release of TNF-α and IL-1ß as well. In conclusion, this study enriches the spectra of compounds with potential anti-inflammatory effects in A. wilsonii and provides new references for the discovery of anti-inflammatory lead compounds, but further mechanistic research is still needed.

pH-responsive size-adjustable liposomes induce apoptosis of fibroblasts and macrophages for rheumatoid arthritis treatment.

In rheumatoid arthritis (RA), macrophages infiltrate joints, while fibroblast-like synovial cells proliferate abnormally, forming a barrier against drug delivery, which hinders effective drug delivery to joint focus. Here we firstly designed a pH-responsive size-adjustable nanoparticle, composed by methotrexate (MTX)-human serum albumin (HSA) complex coating with pH-responsive liposome (Lipo/MTX-HSA) for delivering drugs specifically to inflamed joints in acidic environments. We showed in vitro that the nanoparticles can induce mitochondrial dysfunction, promote apoptosis of fibroblast-like synoviocytes and macrophages, further reduce the secretion of inflammatory factors (TNF-α, IL-1ß, MMP-9), and regulate the inflammatory microenvironment. We also demonstrated similar effects in a rat model of arthritis, in which Lipo/MTX-HSA accumulated in arthritic joints, and at low pH, liposome phospholipid bilayer cleavage released small-sized MTX-HSA, which effectively reduced the number of fibroblast-synoviocytes and macrophages in joints, alleviated joint inflammation, and repaired bone erosion. These findings suggest that microenvironment-responsive size-adjustable nanoparticles show promise as a treatment against rheumatoid arthritis. STATEMENT OF SIGNIFICANCE: Abnormal proliferation of fibroblast synoviocytes poses a physical barrier to effective nanoparticle delivery. We designed size-adjustable nano-delivery systems by preparing liposomes with cholesterol hemisuccinate (CHEM), which were subsequently loaded with small-sized albumin nanoparticles encapsulating the cytotoxic drug MTX (MTX-HSA), termed Lipo/MTX-HSA. Upon tail vein injection, Lipo/MTX-HSA could be aggregated at the site of inflammation via the ELVIS effect in the inflamed joint microenvironment. Specifically, intracellular acidic pH-triggered dissociation of liposomes promoted the release of MTX-HSA, which was further targeted to fibroblasts or across fibroblasts to macrophages to exert anti-inflammatory effects. The results showed that liposomes with adjustable particle size achieved efficient drug delivery, penetration and retention in joint sites; the strategy exerted significant anti-inflammatory effects in the treatment of rheumatoid arthritis by inducing mitochondrial dysfunction to promote apoptosis in fibrosynoviocytes and macrophages.

MAFB in macrophages regulates cold-induced neuronal density in brown adipose tissue.

Transcription factor MAFB regulates various homeostatic functions of macrophages. This study explores the role of MAFB in brown adipose tissue (BAT) thermogenesis using macrophage-specific Mafb-deficient (Mafbf/f::LysM-Cre) mice. We find that Mafb deficiency in macrophages reduces thermogenesis, energy expenditure, and sympathetic neuron (SN) density in BAT under cold conditions. This phenotype features a proinflammatory environment that is characterized by macrophage/granulocyte accumulation, increases in interleukin-6 (IL-6) production, and IL-6 trans-signaling, which lead to decreases in nerve growth factor (NGF) expression and reduction in SN density in BAT. We confirm MAFB regulation of IL-6 expression using luciferase readout driven by IL-6 promoter in RAW-264.7 macrophage cell lines. Immunohistochemistry shows clustered organization of NGF-producing cells in BAT, which are primarily TRPV1+ vascular smooth muscle cells, as additionally shown using single-cell RNA sequencing and RT-qPCR of the stromal vascular fraction. Treating Mafbf/f::LysM-Cre mice with anti-IL-6 receptor antibody rescues SN density, body temperature, and energy expenditure.

The Extracts Derived from Artemisia japonica Thunb. Leaves Mitigate Oxidative Stress and Inflammatory Response Induced by LPS in RAW264.7 Cells through Modulation of the Nrf2/HO-1 Signaling Pathway.

Artemisia japonica Thunb. has been used as a traditional Chinese medicine and a vegetable for thousands of years in China. However, there are few reports on the chemical composition and biological activity of its leaves. Thus, this study aimed to evaluate the chemical composition, antioxidant and anti-inflammatory effects of water extracts of A. japonica leaves and their underlying mechanisms. A total of 48 compounds were identified in the water extract using UPLC-QTOF-MS2 analysis, with phenolic acids, particularly chlorogenic acid compounds, being the predominant components. The ethyl acetate fraction (EAF) contained most of the total phenolic content (385.4217 mg GAE/g) and displayed superior antioxidant capacity with the IC50DPPH•, IC50ABTS•+, and OD0.5reducing power at 10.987 µg/mL, 43.630 µg/mL and 26.883 µg/mL, respectively. Furthermore, EAF demonstrated potent antioxidant and anti-inflammatory effects in LPS-induced RAW264.7 cells by upregulating the Nrf2/HO-1 signal pathway. These findings highlight that A. japonica leaves possess remarkable abilities to mitigate inflammation and oxidative stress, suggesting their potential utilization as medicinal agents and food additives for promoting human health.

Improved synthesis, molecular modeling and anti-inflammatory activity of new fluorinated dihydrofurano-naphthoquinone compounds.

A series of new fluorinated dihydrofurano-napthoquinone compounds were sucessfully synthesized in good yields using microwave-assisted multi-component reactions of 2-hydroxy-1,4-naphthoquinone, fluorinated aromatic aldehydes, and pyridinium bromide. The products were fully characterized using spectroscopic techniques and evaluated for their anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Among 12 new compounds, compounds 8b, 8d, and 8e showed high potent NO inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values ranging from 1.54 to 3.92 µM. The levels of pro-inflammatory cytokines IL-1ß and IL-6 in LPS-stimulated RAW264.7 macrophages were remarkably decreased after the application of 8b, 8d, 8e and 8k. Molecular docking simulations revealed structure-activity relationships of 8b, 8d, and 8e toward NO synthase, cyclooxygenase (COX-2 over COX-1), and prostaglandin E synthase-1 (mPGES-1). Further physicochemical and pharmacokinetic computations also demonstrated the drug-like characteristics of synthesized compounds. These findings demonstrated the importance of fluorinated dihydrofurano-napthoquinone moieties in the development of potential anti-inflammatory agents.

Ameliorative Effect of Areca Nut Polyphenols on Adverse Effects Induced by Lipopolysaccharides in RAW264.7 Cells.

In Asian regions, areca nuts are tropical fruits that are extensively consumed. The areca nut contains a lot of polyphenols and its safety is unknown. In this research, we investigated the effects of lipopolysaccharides (LPS) and areca nut polyphenols (ANP) on normal RAW264.7 cells. The results showed that LPS stimulated adverse effects in normal cells by affecting cytokine production. The GO analysis results mainly affected DNA repair, cell division, and enzyme activities. In the KEGG analysis results, the NOD-like receptor signaling pathway, which is related to NF-κB, MAPK, and the pro-inflammatory cytokines, is the most significant. In the protein-protein interaction network (PPI) results, significant sub-networks in all three groups were shown to be related to cytokine-cytokine receptor interaction. Collectively, our findings showed a comprehensive understanding of LPS-induced toxicity and the protective effects of ANP by RNA sequencing.

Construction of a dual-excitation ratiometric fluorescent probe for determining peroxynitrite levels in living cells and zebrafish.

Peroxynitrite (ONOO-) is a highly reactive oxygen species that plays a critical role in many physiological and pathological processes of cell function. This study aimed to propose a ratiometric fluorescent probe BDHCA derived from coumarin for determining the ONOO- level. ONOO- could specifically induce oxidative cleavage of the conjugated C = C double bond in probe BDHCA, providing a fluorescent ratiometric output. The response of probe BDHCA to ONOO- was selective, fast, and highly sensitive, with a detection limit of 50.3 nM. Biological imaging experiments suggested that probe BDHCA could be used to image ONOO- in living RAW264.7 cells and zebrafish.

The role of JAK2/STAT3 pathway in non-cytotoxic concentrations of DON-induced aggravation of inflammatory response in IL-10 deficient RAW264.7 cells.

Deoxynivalenol (DON) as a mycotoxin was commonly found in food and cereals which can affect immune function and inflammatory response. The majority of foods contain DON at levels below the official limit. This study aimed to evaluate the effects of non-cytotoxic concentration of DON on inflammation and its mechanisms using the IL-10 gene-silenced RAW264.7 cell model. The results showed that a non-cytotoxic concentration of DON at 25 ng/ml aggravated IL-10 knockdown-induced inflammation, which was manifested by increasing IL-1ß and TNF-α mRNA expression, migration and phagocytosis, decreasing IL-10 mRNA expression, and enhancing JAK2/STAT3 phosphorylation. Adding JAK2 inhibitor AG490 attenuated the aggravating effect of DON on IL-10 knockdown-induced inflammation. In conclusion, a non-cytotoxic concentration of DON enhances the inflammatory response through the JAK2/STAT3 signaling pathway when inflammation occurs in the body. These results indicated that non-cytotoxic concentrations of DON could aggravate inflammation when inflammation was induced by IL-10 knockdown, which increases vigilance against DON contamination at low concentration especially when an animal's body has inflammation.

Multifunctional fluorescence probe for simultaneous detection of viscosity, polarity, and ONOO- and its bioimaging in vitro and vivo.

Intracellular microenvironment (viscosity and polarity) and peroxynitrite ions (ONOO-) are involved in maintaining cell morphology, cell function, and signaling so that it is crucial to explore their level changes in vitro and vivo. In this work, we designed and synthesized a mitochondria-targeted fluorescence probe XBL for monitoring the dynamic changes of viscosity, polarity, and ONOO- based on TICT and ICT mechanism. The fluorescence spectra showed obvious changes for polarity at 500 nm as well as ONOO- and viscosity at 660 nm, respectively. The XBL can image simultaneously viscosity, polarity, and ONOO- in cells, and the results showed excess ONOO- leaded to the increase of viscosity in mitochondrial. The ferroptosis process was accompanied by increase of intracellular viscosity and ONOO- levels (or decrease of polarity), which allowed us to better understand the relevant physiological and pathological processes. The XBL can distinguish normal cells and cancerous cells by the fluorescence intensity changes in green and red channels, and image viscosity in inflamed mice. Thus, XBL can provided the chemical tool to understand the physiological and pathological mechanisms of disease by simultaneous detection of viscosity, polarity and ONOO-.