Macrophage RAW264 Research Articles

Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B

BackgroundDysregulated and excessive inflammatory reactions can lead to tissue damage, which is the underlying cause of most human diseases. Menisoxoisoaporphine A (MA), a novel oxoisoaporphine alkaloid, was obtained from Menispermi Rhizoma, a traditional Chinese medicinal herb used in the treatment of inflammatory conditions in clinical practice. This suggests that MA has very promising potential for the development of anti-inflammatory therapeutics. Hence, this study aims to investigate the anti-inflammatory effects and underlying mechanisms of MA.MethodThe anti-inflammatory effects of MA were evaluated in lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 cells. Its underlying mechanisms were explored through RNA sequencing and Western blotting. The binding modes and interactions sites between MA and phosphodiesterase 4B (PDE4B) were predicted using molecular docking and validated by molecular dynamics simulation.ResultsMA treatment significantly reduced LPS-induced morphological changes, inflammatory cytokine relesae, and proinflammatory genes expression in RAW264.7 cells compared to the LPS-induced controls. Transcriptome sequencing analysis suggested that PDE4B might be a key target for MA to exert its therapeutic effect. Mechanismly, MA directly acted on Tyr405 site of PDE4B, thus leading to a sustained elevation of the cyclic adenosine monophosphate (cAMP) levels, which subsequently inactivated NF-κB signaling pathway by phosphorylating protein kinase A (PKA). MA inhibited the NF-κB-mediated inflammatory response depending on PDE4B.ConclusionMA, a natural and novel compound, exerted anti-inflammatory effects in LPS-induced RAW264.7 macrophage cells. It demonstrated a strong binding ability to the Tyr405 sites of PDE4B, thereby inhibiting NF-κB signaling pathway by regulating the cAMP-PKA axis. Elucidating the interaction between MA and PDE4B holds significant potential for the advancement of innovative therapeutic strategies aimed at inflammatory diseases. By strategically modulating this interaction, it may be feasible to achieve more precise regulation of inflammatory responses, thereby offering promising therapeutic benefits for conditions such as rheumatoid arthritis, asthma, and inflammatory bowel disease.

Immunostimulatory activity of sea buckthorn polysaccharides via TLR2/4-mediated MAPK and NF-κB signaling pathways in vitro and in vivo

SP0.1–1, derived from Sea buckthorn (Hippophae rhamnoides L.), has been discovered to exhibit unique antioxidant activity. In this study, we investigated the immunomodulatory activity and mechanisms of SP0.1–1 on macrophage RAW 264.7 cells in vitro and immunosuppressive mice induced by cyclophosphamide in vivo. The results indicated SP0.1–1 strengthened the immune functions via promoting the proliferation of RAW264.7 cells and phagocytic activity, along with stimulating the release of NO, ROS and cytokines including TNF-α, IL-6, IL-1β and IFN-γ. Western blot and molecular docking analysis demonstrated that SP0.1–1 attached to the prime receptors TLR2 and TLR4 in RAW264.7 cells, and triggered the activation of MyD88-mediated MAPK and NF-κB signaling pathways, thereby exerting the immune response in RAW264.7 cells. However, the intervention of specific inhibitors against TLR2, TLR4, JNK, ERK, p38 and NF-κB blocked the TLR-mediated MAPK and NF-κB signaling pathways and downregulated the levels of NO and the aforementioned cytokines, thus suppressing the activation of macrophages. Therefore, it can be speculated that SP0.1–1 activated the macrophages principally via the TLR2/4-MyD88-mediated MAPK and NF-κB signaling pathways. Additionally, SP0.1–1 could protect against the cyclophosphamide-induced immunosuppression in mice, manifested by the improvement of body weight, immune organ indices, phagocytic index, and the relievement of spleen damage, along with the enhancement of cytokines TNF-α, IL-6, IFN-γ and immunoglobulin IgG and IgM. These findings will shed light on the molecular mechanism of SP0.1–1 on the immunoregulatory effect, and lay the foundation for exploiting a potential immunostimulatory agent of SP0.1–1.

Surface-modified dendrimer nanoconjugate for targeting delivery of rifampicin

ObjectiveThe study aimed to formulate and evaluate the efficiency of surface-modified Polyamidoamine dendrimer (PAMAM) as a targeted nanocarrier for the antimycobacterial agent rifampicin. MethodsThe periphery of dendrimer was modified with mannose using α-D-mannopyranosylphenyl isothiocyanate and characterized using analytical techniques i.e., infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). Then, rifampicin was encapsulated into mannosylated dendrimers and evaluated for size, shape, encapsulation efficiency (EE%), drug-dendrimer interaction, and drug release. The toxicity of the nanoconjugates were assessed towards macrophages using the MTT technique. The study investigated the internalization of dendrimer nanoparticles into macrophages to assess the impact of mannose conjugation. Dendrimers were fluorescently labelled and the internalization was quantified using a fluorescence spectroscopy and flow cytometer methods. ResultsMannosylated dendrimers with different mannose densities were successfully developed. The nanoparticles were within the nanoscopic scale. The dendrimers appeared spheroidal under scanning electron microscopy (SEM), and thermal analysis confirmed the conjugation of rifampicin into the dendrimers. The nanoparticles exhibited a good EE% for rifampicin at 10.34 % w/w. After surface mannosylation, the EE% further increased, ranging from 43.43 % to 57.91 % w/w. Mannose-functionalized dendrimers prolonged rifampicin release in physiological pH, while a rapid release in pH 4.5 medium was noticed. The cytotoxicity of the dendrimers in RAW macrophages was considerably reduced after mannose functionalization. In vitro studies indicated that mannose significantly increased the macrophage uptake of nanoconjugates, likely via lectin receptor-mediated endocytosis. ConclusionOverall results suggested mannosylated dendrimers as an effective targeted pulmonary delivery system for rifampicin with negligible cytotoxicity.

Anti-Inflammatory and Anti-proliferative Role of Essential Oil of Leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry.

Phytochemicals have long remained an essential component of the traditional medicine system worldwide. Advancement of research in phytochemicals has led to the identification of novel constituents and metabolites from phytochemicals, performing various vital functions ranging from antimicrobial properties to anticarcinogenic roles. This plant is traditionally used by local people to manage inflammation. In this study, we aim to extract and chemically profile the essential oil from the leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry and study of the anti-inflammatory and anti-proliferative role of essential oil. The hydro distillation method was used for the extraction of essential oil, and the GC-MS was applied for the chemical profiling. The percentage of cell viability was calculated using a crystal violet assay, colony formation assay was performed using Semiquantitative PCR, Propodium iodite staining was used for cell death assay, and Western blotting was used to determine antibodies and proteins. Schrodinger 2015 software was used for molecular docking. Myrcene, a monoterpene, constitutes 56% of the oil and could be attributed to its anti-inflammatory potential. Treatment of LPS-challenged mouse macrophages RAW264.7 cells with essential oil resulted in a decline in the inflammatory markers, such as IL-1β, TNFα, iNOS, COX-2, and NFκB. Further, essential oil inhibited cancer PC-3, A431, A549, and MCF-7 cell lines at concentrations lower than normal PNT2 and HEK-293 cell lines. This decline in proliferative potential can be attributed to a decline in anti-apoptotic proteins, such as procaspase 3 and PARP, an increase in CKIs, such as p21, and a decline in the Akt signaling responsible for survival. The essential oil of the plant Cleistocalyx operculatus may be a potential lead for anti-inflammatory and anti-proliferative function.

Uricase-Expressing Engineered Macrophages Alleviate Murine Hyperuricemia.

Background: Uricase, or urate oxidase (Uox) is a key enzyme in uric acid (UA) metabolism and has been applied in clinical treatment of human hyperuricemia (HUA). However, the current clinically applied uricases, despite their potent urate-lowering capacity, tend to form anti-drug antibodies because of their immunogenicity, leading to increased risk of anaphylaxis, faster drug clearance and reduced or even complete loss of therapeutic effect, limiting their clinical application. In this study, we constructed engineered macrophages that stably expressed uricase, which might serve as a promising alternative to the direct injection of uricases. Materials and Methods: Engineered macrophages RAW264.7 cells were injected intravenously to treat hyperuricemic KM mice. Serum uric acid and bio-indicators for renal and hepatic functions were detected by an automatic biochemical analyzer; inflammatory cytokines were determined by ELISA; the livers and kidneys of the mice were sectioned for histological examination. Results: The uricase-expressing macrophages reduced UA levels from 300 ± 1.5 μmol/L to 101 ± 8.3 μmol/L in vitro. And in an HUA mouse model established by gavage with yeast extract, intravenous injection of the engineered macrophages could reduce the serum uric acid (sUA) of mice to normal level on the 14th day of modeling, with a decrease of 48.6%, and the urate-lowering effect was comparable to that of the first-line clinical drug allopurinol. In terms of safety, engineered macrophages did not cause liver or kidney dysfunction in mice, nor did they induce systemic immune response. Conclusions: Using macrophages as a chassis to deliver uricase might be a new, safe and effective strategy for the treatment and control of hyperuricemia.

The role of CISD1 reduction in macrophages in promoting COPD development through M1 polarization and mitochondrial dysfunction

BackgroundThe mitochondrial dysfunction and oxidative stress imbalance caused by macrophage polarization play a role in the progression of COPD, with CDGSH iron–sulfur domain-containing protein 1 (CISD1) playing a key role. This study revealed the role and mechanism of CISD1 in smoke-induced macrophages.MethodsUsing a pure cigarette smoke exposure-induced COPD mouse model, stimulation of Raw264.7 macrophages with cigarette smoke extract mimics the COPD environment. Knocking down CISD1 expression in macrophages and combining it with high-throughput sequencing to obtain subsequent differentially expressed genes and pathways. Macrophage polarization tendency under different treatments was determined using flow cytometry. Meanwhile, Mitosox, JC-1, DCFH-DA fluorescence intensity was measured to detect mitochondrial function and cellular oxidative stress levels. Western Blot technique was employed to validate autophagy (mitochondrial autophagy) pathway-related proteins. In addition, Elisa technique was used to measure inflammatory factors (IL-6, TNF-a) in the cell supernatant after co-culturing macrophages (Raw264.7) with epithelial cells (MLE12).ResultsCISD1 is underexpressed in peripheral blood monocytes of COPD patients. Under in vitro conditions, we verified that cigarette smoke (smoke extract) indeed inhibits CISD1 expression in macrophages. Subsequently, we found that macrophages with knocked-down CISD1 tend to polarize towards M1 phenotype, and exhibit signs of mitochondrial dysfunction and oxidative stress imbalance. In addition, we observed significant activation of the autophagy pathway in CISD1-inhibited macrophages, with upregulation of LC3A/B and downregulation of p62 protein, as well as increased expression of mitochondrial autophagy-related proteins (PINK1, PARKN). Furthermore, co-culturing CISD1-knockdown macrophages (Raw264.7) with epithelial cells (MLE12) resulted in upregulation of inflammatory factors in the supernatant.ConclusionsSmoke-induced reduction of CISD1 in macrophages promotes M1 polarization and mitochondrial dysfunction by activating the autophagy pathway, thereby promoting the occurrence and development of COPD.

Anti-Inflammatory Secoiridoids from the Medicinal Herb Gentianopsis barbata.

Five new secoiridoids, gentianopsins A-E (1-5), along with two known analogues (6 and 7) were isolated from the whole plants of the medicinal herb Gentianopsis barbata. Their structures were elucidated by a comparison of extensive spectroscopic analysis (1D and 2D NMR, and HRMS) and quantum chemical calculations. Gentianopsins A (1) and B (2) represented two unusual skeletons of trihomo-secoiridoids. Anti-inflammatory activity of these isolates was evaluated via suppressing the secretion of cytokines TNF-α and IL-6 in LPS-induced macrophages RAW264.7. Significant inhibitory activity was observed for compounds 3 and 7 on IL-6 secretion with IC50 values of 10.22 and 13.30 μM, respectively.

Dynamic reorganization of multivesicular bodies and exosome production impacted by sonoporation

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production.

Curculigosides J–K and curcorchidihydrobenzofuran A, a dihydrobenzofuran with anti-proliferative properties from Curculigo orchioides

Phytochemical investigation of the rhizomes and the leaves of Curculigo orchioides led to the isolation of fourteen compounds. They consist of one undescribed dihydrobenzofuran, curcorchidihydrobenzofuran A (1), two undescribed benzyl benzoate glycosides, curculigoside J (2) and K (3), and eleven known compounds (4–14). The structures of the isolated compounds were elucidated by thorough analysis of spectroscopic (IR, NMR and ECD) and spectrometric (MS) data. Compound 1 displayed significant anti-proliferative activities against cervical cancer cells (HelaS3, IC50 = 3.6 µM) and breast cancer cells (MCF-7, IC50 = 13.9 µM) while showing moderate activities against lung cancer cells (A549, IC50 = 29.8 µM). Importantly, it was non-toxic to Vero cells. Compound 1 also inhibited the tyrosinase enzyme in a moderate manner (IC50 = 120.8 µM). Eventually, its permethylated synthetic analog 1a showed a significant suppression of lipopolysaccharide (LPS)-induced NO production in murine macrophage RAW 264.7 (IC50 = 23.4 µM).

The Effects of the oxLDL/β2GPI/anti-β2GPI Complex on Macrophage Autophagy and its Mechanism.

Previous research has established that the oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex can stimulate macrophages to secrete molecules associated with atherosclerosis (AS), such as monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), and tumor necrosis factor-α (TNF-α). This complex also enhances the uptake of oxLDL, thereby accelerating foam cell formation through the Toll-like receptor-4/nuclear factor kappa B (TLR4/NF-κB) pathway. Given the critical role of macrophage autophagy in the instability of vulnerable atherosclerotic plaques, it is imperative to investigate whether the oxLDL/β2GPI/anti-β2GPI complex influences macrophage autophagy in AS. This study aims to elucidate the effects and underlying mechanisms of the oxLDL/β2GPI/anti-β2GPI complex on macrophage autophagy in AS. Experiments were conducted using murine macrophage RAW264.7 cells and the human monocytic cell line THP-1. Western blot analysis was employed to determine the expressions of autophagy-associated markers and signaling pathway proteins. Autophagosomes were detected through mRFP-GFP-LC3 adenoviral transfection and transmission electron microscopy (TEM). Treatment of macrophages with the oxLDL/β2GPI/anti-β2GPI complex resulted in decreased expressions of Beclin1 and LC3 proteins, alongside an upregulation of SQSTM1/P62 protein expression. Additionally, there was a reduction in the number of autophagosomes and autolysosomes. An increase in the phosphorylation levels of phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) was also observed. Notably, the expressions of autophagy-associated markers were partially restored when the TLR4/NF-κB and PI3K/AKT/mTOR pathways were inhibited by their respective inhibitors. Our findings indicate that the oxLDL/β2GPI/anti-β2GPI complex inhibits macrophage autophagy in AS via the TLR4/NF-κB and PI3K/AKT/mTOR signaling pathways.

Cefiderocol has immunoregulative effects in LPS-induced vitro experimental model via inhibiting inflammation and ferroptosis

BackgroundCefiderocol is a new catecholamine-containing siderophore cephalosporin. It, however, remains unclear how cefiderocol modulates the immune response of the host. ObjectivesThis study elucidated whether cefiderocol exerts immunoprotective effects in an in vitro experimental model induced with lipopolysaccharide (LPS). MethodsMouse macrophage RAW 264.7 cells were exposed to LPS (100 ng/mL) or LPS + cefiderocol (40 mg/L) to assess the immunomodulatory effect of cefiderocol in vitro. ELISA was performed on cell culture supernatants to estimate cytokine levels. Ferroptosis level was also quantified by detecting intracellular reactive oxygen species (ROS) and iron levels through flow cytometry analysis. Malondialdehyde (MDA) and glutathione (GSH) levels were estimated by ELISA. We conducted western blotting assay for evaluating key ferroptosis pathway proteins. ResultsCefiderocol alleviated LPS-induced inflammation by reducing IL-6, TNF-α, and IL-1β production levels and enhancing the IL-10 production level. Further analysis to determine the underlying mechanism revealed that cefiderocol inhibited ferroptosis; this was confirmed by reduced ROS, MDA, and Fe2+ ion levels; increased GSH levels; upregulated expression of solute carrier family 7 member 11, glutathione peroxidase 4, nuclear factor erythroid 2-related factor 2, and ferroptosis suppressor protein 1; and downregulated expression of acyl-CoA synthetase long-chain family member 4. ConclusionsCefiderocol may play a key role in reducing inflammation by decreasing inflammatory cytokine release and suppressing ferroptosis.

Anti-Inflammatory and Antinociceptive Properties of the Quercetin-3-Oleate AV2, a Novel FFAR1 Partial Agonist.

Free fatty acid receptor 1 (FFAR1) has emerged as the most targeted isoform of the free fatty acid receptors because of its involvement in the modulation of energy balance and its potential role in the control of inflammatory and pain conditions. Quercetin-3-oleate (AV2), recognized as a new FFAR1 partial agonist, was investigated for its ability to modulate inflammation and nociception. Human immortal neuroblastoma SH and the murine macrophagic RAW 264.7 cells were used to evaluate cell viability, the potential cytoprotective activity, and the anti-inflammatory properties of AV2 in vitro. Paw edema, caused by zymosan-A, and the formalin test were used to assess the in vivo anti-inflammatory and antinociceptive effects in CD-1 mice. In vitro, AV2 was devoid of cytotoxicity, significantly reduced ROS in both cell types, and protected RAW 264.7 cells from lipopolysaccharide damage by reducing tumor necrosis factor-α production. Interestingly, AV2 induced a transient elevation of intracellular calcium that was reduced in cells, pre-incubated with the FFAR1 antagonist DC260126. In vivo, AV2 reduced formalin-induced nociception and zymosan A-induced paw edema, and both effects were reversed by the FFAR1 antagonist GW1100. In conclusion, these data strongly support the AV2-mediated antioxidant, anti-inflammatory, and antinociceptive activity. AV2 represents a promising molecule for the clinical management of inflammatory-related pain conditions.

Anti-inflammatory polyketides from Santalum album derived endophytic fungus Hypomontagnella sp. TX-09

ABSTRACT Four new lactones, including hypomonacid A (1) and hypomonone A–C (4–6), as well as nine known polyketide analogues (2–3 and 7–13) were obtained from endophytic fungus Hypomontagnella sp. TX-09 derived from Santalum album. Their planar structures were extensively established by analysing HRESIMS and NMR spectroscopic data. Stereochemistry of new compounds was determined by X-ray diffraction analysis and modified Mosher’s method in combination with quantum-chemical ECD calculation. In addition, compounds 1 and 2 showed anti-inflammatory activity by inhibition of lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells at 50 μmol/L without cytotoxicity. Among them, compound 1 inhibited the production of LPS-stimulated inflammation in mouse macrophage RAW264.7 cells by suppressing the expression of iNOS, TNF-α, IL-1β, and IL-6.

A Novel Trichinella spiralis Galectin Strengthens the Macrophage ADCC Killing of Larvae via Driving M1 Polarization.

Galectin recognizes β-galactosides through its carbohydrate recognition domains (CRDs). This study aimed to determine the biological features of a novel Trichinella spiralis galectin (galactoside-binding lectin family protein, TsGLFP) and its role in driving macrophage M1 polarization and enhancing ADCC killing of larvae. TsGLFP belongs to the galectin family and has two CRDs. The complete TsGLFP cDNA sequence was cloned and then expressed in Escherichia coli BL21. The results of qPCR, Western blot, and indirect immunofluorescence tests (IIFTs) revealed that TsGLFP was expressed in various stages of T. spiralis worms and principally localized at the cuticle and around the female embryos of the nematode. rTsGLFP had the function of agglutinating mouse erythrocytes, and this agglutination activity could be inhibited by lactose. After the mouse macrophage RAW264.7 was incubated with rTsGLFP, the expression level of the M1 genes (iNOS, IL-6, and TNF-α) and NO production were obviously increased. After incubating macrophages with rTsGLFP, there was a noticeable rise in the expression levels of p-IκB-α and p-NF-κB p65. Additionally, rTsGLFP enhanced the macrophage's ability to kill newborn larvae by ADCC cytotoxicity. When the macrophages were pretreated with the specific p-NF-κB p65 inhibitor PDTC, and then stimulated with rTsGLFP, the expression levels of iNOS, NO, and p-NF-κB p65 and the macrophages' ADCC cytotoxicity were distinctly decreased. These findings indicated that rTsGLFP enhanced the macrophage ADCC killing of larvae by driving M1 polarization through activating the NF-κB pathway.

Predictive Production of a New Highly Soluble Glucoside, Corylin-7-O-β-Glucoside with Potent Anti-inflammatory and Anti-melanoma Activities.

Computational tools can now facilitate screening precursors and selecting suitable biotransformation enzymes for producing new bioactive compounds. This study applied the data-mining approach to screen for candidate precursors of glycosyltransferases to produce new glucosides from 412 commercial natural compounds. Among five candidates, experimental results showed that only corylin could be glycosylated by the bacterial glycosyltransferase, BsUGT489. Analysis of interaction potential between candidates and glycosyltransferase by molecular docking tools also found that corylin was the only compatible substrate. The new glucoside was purified and confirmed to be corylin-7-O-β-glucoside. The aqueous solubility of corylin-7-O-β-glucoside was 14.2 times more than its precursor aglycone, corylin. Corylin-7-O-β-glucoside retained anti-inflammatory activity in lipopolysaccharide-induced nitric oxide production of murine macrophage RAW 264.7 cells, with an IC50 value of 121.1 ± 9.5µM. Further, corylin-7-O-β-glucoside exhibited more potent anti-melanoma activity against murine B16 and human A2058 melanoma cells than corylin. Together, predictive studies facilitate the production of a new glucoside, corylin-7-O-β-glucoside, which is highly soluble and possesses anti-inflammatory and anti-melanoma activities and therefore has promising future applications in pharmacology.

Cloning, characterization of β-glucosidase from Furfurilactobacillus rossiae in bioconversion and its efficacy.

Minor ginsenosides produced by β-glucosidase are interesting biologically and pharmacologically. In this study, new ginsenoside-hydrolyzing glycosidase from Furfurilactobacillus rossiae DCYL3 was cloned and expressed in Escherichia coli strain BL21. The enzyme converted Rb1 and Gyp XVII into Rd and compound K following the pathways: Rb1→Rd and Gyp XVII→F2→CK, respectively at optimal condition: 40°C, 15min, and pH 6.0. Furthermore, we examined the cytotoxicity, NO production, ROS generation, and gene expression of Gynostemma extract (GE) and bioconverted Gynostemma extract (BGE) in vitro against A549 cell lines for human lung cancer and macrophage RAW 264.7 cells for antiinflammation, respectively. As a result, BGE demonstrated significantly greater toxicity than GE against lung cancer at a dose of 500µg/mL but in normal cells showed lower toxicity. Then, we indicated an enhanced generation of ROS, which may be boosting cancer cell toxicity. By blocking the intrinsic way, BGE increased p53, Bax, Caspase 3, 9, and while Bcl2 is decreased. At 500µg/mL, the BGE sample was less toxic in normal cells and decreased the LPS-treated NO and ROS level to reduce inflammation. In addition, BGE inhibited the expression of pro-inflammatory genes COX-2, iNOS, IL-6, and IL-8 in RAW 264.7 cells than the sample of GE. In conclusion, FrBGL3 has considerable downstream applications for high-yield, low-cost, effective manufacture of minor ginsenosides. Moreover, the study's findings imply that BGE would be potential materials for anti-cancer and anti-inflammatory agent after consideration of future studies.

GRN Activates TNFR2 to Promote Macrophage M2 Polarization Aggravating Mycobacterium Tuberculosis Infection.

The polarization of macrophages plays a critical role in the immune response to infectious diseases, with M2 polarization shown to be particularly important in various pathological processes. However, the specific mechanisms of M2 macrophage polarization in Mycobacterium tuberculosis (Mtb) infection remain unclear. In particular, the roles of Granulin (GRN) and tumor necrosis factor receptor 2 (TNFR2) in the M2 polarization process have not been thoroughly studied. To investigate the effect of macrophage M2 polarization on Mtb infection and the mechanism of GRN and TNFR2 in M2 polarization. Forty patients with pulmonary tuberculosis (PTB) and 40 healthy volunteers were enrolled in this study, and peripheral blood samples were taken to detect the levels of TNFR2 and GRN mRNA by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR); monocytes were isolated and then assessed by Flow Cytometry (FC) for M1 and M2 macrophage levels. To further validate the function of TNFR2 in macrophage polarization, we used interleukin 4 (IL-4) to induce mouse monocyte macrophages RAW264.7 to M2 polarized state. The expression of TNFR2 was detected by Western Blot and RT-qPCR. Next, we constructed a GRN knockdown plasmid and transfected it into IL-4-induced mouse monocyte macrophage RAW264.7, and detected the expression of TNFR2, M1 macrophage-associated factors tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6), and the M2 macrophage-associated factors CD206, IL-10, and Arginase 1 (Arg1); Immunofluorescence staining was used to monitor the expression of CD86+ and CD206+, and FC was used to analyze the macrophage phenotype. Subsequently, immunoprecipitation was used to detect the binding role of GRN and TNFR2. Finally, the effects of GRN and TNFR2 in macrophage polarization were further explored by knocking down GRN and simultaneously overexpressing TNFR2 and observing the macrophage polarization status. The results of the study showed elevated expression of TNFR2 and GRN and predominance of M2 type in macrophages in PTB patients compared to healthy volunteers (p < 0.05). Moreover, TNFR2 was highly expressed in M2 macrophages (p < 0.05). Additionally, GRN knockdown was followed by elevated expression of M1 polarization markers TNF-α, iNOS and IL-6 (p < 0.05), decreased levels of M2 polarization-associated factors CD206, IL-10 and Arg1 (p < 0.05), and macrophage polarization towards M1. Subsequently, we found that GRN binds to TNFR2 and that GRN upregulates TNFR2 expression (p < 0.05). In addition, knockdown of GRN elevated M1 polarization marker expression, decreased M2 polarization marker expression, and increased M1 macrophages and decreased M2 macrophages, whereas concurrent overexpression of TNFR2 decreased M1 polarization marker expression, elevated M2 polarization marker expression, and decreased M1 macrophages and increased M2 macrophages. TNFR2 and GRN are highly expressed in PTB patients and GRN promotes macrophage M2 polarization by upregulating TNFR2 expression.

Sulfated polysaccharide from Apostichopus japonicus viscera exhibits anti-inflammatory properties in vitro and in vivo

Polysaccharides from sea cucumbers are known for their biological activities, but little is known about those from sea cucumber viscera. The present study isolated a sulfated polysaccharide (SCVP-2) from the viscera of Apostichopus japonicas, which had a molecular weight of 209.1 kDa. SCVP-2 comprised 66.3 % total sugars, 2.1 % uronic acid, 4.5 % proteins, and 25.5 % sulfate groups, containing glucosamine, galactosamine, glucose, galactose, and fucose. FT-IR and NMR analyses identified SCVP-2 as a fucoidan sulfate with sulfation patterns of the fucose branches as Fuc2S, Fuc4S, and Fuc0S. SEM and AFM analyses showed irregular clusters and linear conformations. SCVP-2 demonstrated strong anti-inflammatory properties both in vitro and in vivo. In lipopolysaccharide (LPS)-induced inflammation in macrophage RAW264.7 cells, SCVP-2 significantly reduced nitric oxide (NO) and cytokine secretion (IL-1β, IL-6, TNF-α). Additionally, it downregulated the expression of these cytokine genes. Furthermore, the anti-inflammatory mechanism of SCVP-2 was related to the inhibition of the MAPKs and NF-κB pathways. SCVP-2's anti-inflammatory capacity was confirmed in acute inflammation models, including xylene-induced ear swelling and acetic acid-induced peritoneal capillary permeability, and in high-fat diet-induced systemic low-grade chronic inflammation. In conclusion, SCVP-2 exhibits significant anti-inflammatory activity, suggesting its potential for development as a functional food ingredient or therapeutic agent for inflammation-related diseases.

Sulfated polysaccharide from Antrodia cinnamomea mycelium cultured with zinc sulfate stimulates M1 polarization of macrophages through AKT/mTOR pathways

Antrodia cinnamomea-derived sulfated polysaccharides (Ac-SPSs) have health benefits, but their yield is low. This study explores a strategy to increase Ac-SPS yield and elucidates the biofunctions of Ac-SPS. For this, A. cinnamomea mycelia were treated with zinc sulfate (ZnSO4) administered at 1, 10, and 100 μM. Firstly, functional assay indicated that ZnSO4 increases the Ac-SPS yield by 20 %–30 % compared with the control treatment. ZnSO4 engenders a population of middle-molecular-weight (~200 kDa) Ac-SPSs. Ac-SPS (ASZ-10) from A. cinnamomea treated with 10 μM ZnSO4 exhibits the best anti-proliferation ability against lung cancer A549 cells. Co-treatment of ASZ-10 does not inhibit lipopolysaccharide-induced inflammation but does induce M1-related markers of macrophage RAW264.7 cells. Secondly, immunomodulatory properties showed that ASZ-10 increases the expression of CD80+ and CD86+ in M-CSF-stimulated bone-marrow-derived macrophages. ASZ-10 induces M1 polarization through up-regulation of the AKT/mTOR pathway as confirmed by AKT and mTOR inhibitors eliminating ASZ-10-induced M1-like markers of macrophages. Through systemic chemical and functional analysis, this study shows that trace amounts (10 μM) of ZnSO4 increase Ac-SPS yield and it reveals that ASZ-10 exhibits anti-cancer activity and acts as a stimulator for M1 macrophages by stimulation of AKT and mTOR.

The Immunosuppressive Receptor CD32b Regulation of Macrophage Polarization and Its Implications in Tumor Progression.

Macrophages, pivotal components of the immune system, orchestrate host defense mechanisms in humans and mammals. Their polarization into classically activated macrophages (CAMs or M1) and alternatively activated macrophages (AAMs or M2) dictates distinct functional roles in immunity and tissue homeostasis. While the negative regulatory role of CD32b within the FC gamma receptor (FCγR) family is recognized across various immune cell types, its influence on macrophage polarization remains elusive. This study aimed to elucidate the regulatory role of CD32b in macrophage polarization and discern the differential expression markers between the M1 and M2 phenotypes following CD32b siRNA transfection. The results revealed a decrease in the CD32b levels in lipopolysaccharide (LPS)-treated M1 and an increase in interleukin-4 (IL-4)-treated M2 macrophages, as observed in macrophage Raw264.7 cells. Furthermore, CD32b siRNA transfection significantly downregulated the M2 markers (IL-10, VEGF, Arg-1, and STAT6), while upregulating the M1 markers (IL-6, NF-κB, NOS2, and STAT1) in the Raw264.7 cells. Similar findings were recapitulated in macrophage-rich adherent cells isolated from mouse spleens. Additionally, the cytopathological analysis of pleural effusions and ascitic fluids from patients with cancer revealed a positive correlation between advanced tumor stages, metastasis, and elevated CD32b levels. In conclusion, this study highlights the regulatory influence of CD32b in suppressing M1 expression and promoting M2 polarization. Moreover, heightened M2 activation and CD32b levels appear to correlate with tumor progression. A targeted CD32b blockade may serve as a novel therapeutic strategy to inhibit M2 macrophage polarization and is promising for anti-tumor intervention.