Search Results (381)

Background: Inflammation and oxidative stress are key mechanisms in underlying skin conditions like psoriasis and eczema. While many plants, including Australian native plants, are proposed to target these pathways due to their phytochemical content, studies on whole extracts and their synergistic effects remain limited. Objectives: This study aimed to investigate individual and combined effects of whole plant extracts on skin protection and healing, focusing on their anti-inflammatory and antioxidant properties. Methods: The antioxidant potential of the individual and combined plant extracts were investigated on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) assay followed by luciferase assay in MCF-7 AREc32 cells for nuclear factor erythroid 2-related factor 2 (Nrf2) activation. The anti-inflammatory activities were investigated on lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages for the inhibition of nitric oxide (NO), tumour necrosis factor (TNF)-α, and interleukin (IL)-6. Synergistic interaction was determined by the combination index model (CI < 1). Combination(s) showing synergistic and optimal activity were further investigated on LPS-induced human dermal fibroblasts (HDF) cells for IL-6 inhibition and wound healing activity. Results: Three of the tested Australian native plant extracts demonstrated prominent antioxidant and anti-inflammatory activities including bitter orange, mountain pepper berry and native river mint. In particular, their three-way combination (1:1:1, w/w) showed prominent synergistic (CI < 1) in reducing NO and IL-6, along with enhanced Nrf2 activation. In LPS-inflamed HDF cells, the combination maintained synergistic inhibition of IL-6 levels and promoted wound healing response. Conclusions: These findings highlight the therapeutic potential of Australian native plant as a whole extract for skin protection and repair attributed to antioxidant and anti-inflammatory activities. The observed synergistic anti-inflammatory and antioxidant effects support their use in the development of new cosmetic formulations for skin. Full article

Tuberculosis (TB) is a major global health threat, with the current Bacillus Calmette–Guérin (BCG) vaccine having limited efficacy against adult pulmonary disease. Trained immunity (TI) is a form of innate immune memory that enhances antimicrobial defense. It is characterized by the epigenetic and metabolic reprogramming of innate immune cells and holds promise as a promising approach to prevent TB. In this study, we investigated the capacity of heparin-binding hemagglutinin (HBHA), a methylated antigen of Mycobacterium tuberculosis, to induce TI in murine RAW264.7 macrophages, human-derived THP-1 macrophages, and human peripheral blood mononuclear cells (hPBMCs). HBHA-trained macrophages exhibited the enhanced expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) following secondary lipopolysaccharide stimulation. The epigenetic profiling indicated elevated levels of H3K4me1 and H3K4me3 histone marks at cytokine gene loci. Further, metabolic analysis revealed heightened lactate production and the increased expression of glycolytic enzymes. Functionally, HBHA-trained macrophages exhibited improved control of intracellular mycobacteria, as evidenced by a significant reduction in colony-forming units following BCG infection. These findings elucidate that HBHA induces a functional TI phenotype via coordinated epigenetic and metabolic changes, and suggest HBHA may serve as a valuable tool for studying TI and its relevance to host defense against mycobacterial infections, pending further in vivo and clinical validation. Full article

Background and Objectives: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with limited therapeutic options. Current therapies (pirfenidone, nintedanib) exhibit modest efficacy and significant side effects, underscoring the need for novel strategies targeting early pathogenic drivers. Saroglitazar (SGZ), a dual PPARα/γ agonist with anti-inflammatory properties approved for diabetic dyslipidemia, has not been explored for IPF. We aimed to investigate SGZ’s therapeutic potential in pulmonary fibrosis and elucidate its mechanisms of action. Materials and Methods: Using a bleomycin (BLM)-induced murine pulmonary fibrosis model, we administered SGZ therapeutically. A histopathological assessment (H&E, Masson’s trichrome, collagen I immunofluorescence), Western blotting, and qRT-PCR analyzed the fibrosis progression and inflammatory markers. Flow cytometry evaluated the macrophage polarization. In vitro studies used RAW264.7 macrophages stimulated with BLM/LPS and MRC-5 fibroblast co-cultures. The NF-κB/NLRP3 pathway activation was assessed through protein and gene expression. Results: SGZ significantly attenuated BLM-induced histopathological hallmarks, including alveolar wall thickening, collagen deposition, and inflammatory infiltration. Fibrotic markers (OPN, α-SMA) and pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) were downregulated in the SGZ-treated mice. Mechanistically, SGZ suppressed the M1 macrophage polarization (reduced CD86⁺ populations) and inhibited the NF-κB/NLRP3 pathway activation in the alveolar macrophages. In the RAW264.7 cells, SGZ decreased the NLRP3 inflammasome components (ASC, cleaved IL-1β) and cytokine secretion. Co-cultures demonstrated that the SGZ-treated macrophage supernatants suppressed the fibroblast activation (α-SMA, collagen I) in MRC-5 cells. Conclusions: SGZ attenuates pulmonary fibrosis by suppressing macrophage-driven inflammation via NF-κB/NLRP3 inhibition and disrupting the macrophage–fibroblast crosstalk. These findings nominate SGZ as a promising candidate for preclinical optimization and future clinical evaluation in IPF. Full article

Clinacanthus nutans (Burm.f.) Lindau is a Southeast Asian medicinal plant traditionally used for treating skin inflammation and infections. This study evaluated its wound-healing potential through anti-inflammatory, cytoprotective, and antiviral mechanisms. HPLC-DAD analysis identified schaftoside as the major flavonoid in the 95% ethanolic leaf extract. In the lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW 264.7), both C. nutans extract (5 and 50 μg/mL) and its flavonoid schaftoside (5 and 20 μg/mL) significantly downregulated the expression of pro-inflammatory genes, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and prostaglandin E₂ (PGE₂), under both pre-treatment and post-treatment conditions. ELISA confirmed dose-dependent inhibition of human COX-2 enzymatic activity, reaching up to 99.3% with the extract and 86.9% with schaftoside. In the endothelial cell models (CCL-209), the extract exhibited low cytotoxicity and effectively protected cells from LPS-induced apoptosis, preserving vascular integrity critical to tissue regeneration. Antiviral assays demonstrated suppression of HSV-2 replication, particularly during early infection, which may help prevent infection-related delays in wound healing. Collectively, these findings suggest that C. nutans and schaftoside promote wound repair by attenuating inflammatory responses, supporting endothelial survival, and controlling viral reactivation. These multifunctional properties highlight their potential as natural therapeutic agents for enhancing wound-healing outcomes. Full article

Background and aim. Gold nanoparticles (AuNPs) offer promising potential as nanocarriers in vaccine development due to their biocompatibility, tunable surface properties and capacity to enhance antigen presentation. This study aimed to evaluate the in vitro cytocompatibility, cellular uptake and antigen processing of carcinoembryonic antigen (CEA)-functionalized AuNPs as a nanovaccine candidate. Materials and Methods. AuNPs were synthesized by citrate reduction and subsequently functionalized with CEA through physical adsorption. Nanoparticle size, morphology, and surface charge were characterized using UV–Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Cytocompatibility was assessed via MTT assay on RAW 264.7 murine macrophages. Cellular uptake and antigen processing were evaluated using hyperspectral dark-field microscopy and fluorescence microscopy with proteasomal pathway markers. Results. The synthesized AuNPs displayed a uniform spherical morphology with a mean hydrodynamic diameter of ~50 nm and a stable zeta potential. CEA conjugation slightly altered the surface charge and spectral profile. MTT assays confirmed good cytocompatibility across tested concentrations. Hyperspectral and confocal microscopy revealed the efficient uptake of CEA-AuNPs by RAW 264.7 cells and colocalization with lysosomal compartments, suggesting successful antigen processing. Conclusions. The in vitro data support the safety and biological interaction of CEA-functionalized AuNPs with macrophages. These findings highlight their potential as a nanovaccine delivery platform and warrant further in vivo evaluation to assess immunogenicity and protective efficacy. Full article

Background/Objectives: Pestalotic acid A (PAA), a polyketide derived from Pestalotiopsis vismiae, an endophyte of the Japanese holly (Ilex crenata), is known to exhibit known antimicrobial activity, but its anti-inflammatory properties remain uncharacterized. This study aimed to investigate the anti-inflammatory effects of PAA in lipopolysaccharide (LPS)-stimulated murine macrophages, RAW264.7 cells. Methods: PAA was isolated from P. vismiae endophytes of Ilex crenata, and its structure was confirmed. RAW264.7 macrophages were treated with 0–50 μM of PAA in the presence of 100 ng/mL LPS. Cell viability was assessed by MTS assay; nitric oxide (NO) production was measured via Griess reagent; interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF) were quantified by enzyme-linked immunosorbent assay. Protein expression of inducible NO synthase (iNOS), nuclear factor (NF)-κB p65 phosphorylation, and related signaling proteins was evaluated by Western blot analysis and immunofluorescence staining. Results: PAA significantly increased macrophage viability and dose-dependently inhibited the release of NO by alleviating the protein expression of iNOS in LPS-treated RAW264.7 cells. Furthermore, PAA suppressed the release of IL-6, IL-1β, and TNF induced by LPS. Western blot and immunofluorescence results also indicated that PAA blocked the p65 subunit phosphorylation of NF-κB, which is one of the underlying mechanisms of the anti-inflammatory action of pestalotic acid A. Conclusions: PAA exerts potent anti-inflammatory effects in LPS-stimulated macrophages via inhibition of the NF-κB pathway, highlighting its potential as a natural therapeutic agent for inflammatory diseases. Full article

Polygalacturonic acid (PGA), derived from the natural plant polysaccharide, pectin, has been suggested as a biomaterial for implantable medical devices and tissue engineering; particularly in the field of bone implant materials. As a negatively charged polysaccharide, PGA can be considered similar to hyaluronic acid, a component of the extracellular matrix (ECM). PGA-based biomaterials may therefore exhibit favorable biocompatibility with surface chemistry mimicking the natural ECM. In this study, we synthesized semi-interpenetrating polymer networks (SIPNs) incorporating PGA, and conducted physical characterization and in vitro biocompatibility studies. Biocompatibility testing revealed the SIPNs to be cytocompatible, with the PGA component conferring some resistance to the adherence of the macrophage cell line RAW264.7. In addition, SIPNs did not support the fusion of primary murine macrophages into foreign body giant cells (FBGCs). Macrophage adherence and FBGC formation on implanted biomaterial surfaces are important events in the progression of a foreign body response. Our in vitro studies suggest that PGA-based materials may offer desirable biocompatibility profiles, holding promise for future clinical applications. Full article

Tumor-associated macrophages (TAMs) are pivotal for tumor development and progression. Reprogramming the M2-like pro-tumoral behavior of TAMs towards the M1-like anti-tumor phenotype to unleash their potential against tumors has become one of the most promising anti-tumor immunotherapy strategies. In this work, the natural product pseudolaric acid B (PAB, 1) was found to markedly decrease ARG1 mRNA expression and significantly increase NOS2 expression in the IL-4/IL-13-pre-stimulated RAW 264.7 cells through cellular phenotype screening of a series of pseudolaric acid-related natural products, suggesting its potential to reprogram the pro-tumoral TAMs towards the M1-like phenotype against tumors. Further chemical modification of the carboxylic acid moiety of 1 led to a series of amide or pyranoside derivatives with ARG1- and NOS2-modulating activity. Among them, hydrazineyl amide 12 stands out as the most potent, without significant diminution in cell viability. It inhibited the M2-like polarized tumor-promoting phenotype of macrophages, as evidenced by a decrease in CD206 expression and an increase in CD86 expression in flow cytometry, as well as a decrease in ARG1 protein level in Western blot assays. In addition, 12 could reverse the suppression of Ki67+, IFN γ+, and granzyme B⁺ CD8⁺ T cell proliferation and activation induced by pro-tumoral macrophages. More importantly, it could reshape the tumor immune microenvironment and inhibit tumor growth in immunocompetent murine tumor models. Hsp90 was predicted to be a potential target of 12 by a target fishing software, which was further demonstrated by molecular docking. Collectively, the amide derivative 12 of PAB demonstrated promising anti-tumor TAM-reprogramming activity, which is worthy of further investigation. Full article

The TME is a critical niche for determining the fate of cancer therapy. Tumor cells often polarize nontumor cells, including immune cells, in the TME to favor cancer growth. In pancreatic cancer, macrophages are associated with poor therapy outcomes and unfavorable survival, especially when rendered into M2 macrophages. The latter show features also found in so-called tumor-associated macrophages (TAM), which are described as protecting and propelling tumor growth. In this context, it has been understudied which pancreatic cancer chemokines contribute to macrophage polarization. To this end, we analyzed murine RAW264.7 macrophages and Panc02 and PDA6606 pancreatic cancer cells in mono- and coculture to identify release patterns of 13 chemokines. Artificial macrophage polarization confirmed prominent changes in surface receptor and chemokine secretion profiles. Strikingly, RAW264.7 cocultures with Panc02 or PDA6606 were congruent in showing elevated levels of CCL2, CCL5, CCL17, CCL20, CCL22, CXCL5, and CXCL10. Further underlining the suitability of our in vitro model, both pancreatic cancer cell lines showed similar modulation of the critical macrophage polarization markers arginase, CD206, and iNOS, as well as chemokine receptors CCR2 and CCR4. Collectively, we demonstrated that our model is suitable for testing the roles and functions of chemokines in macrophage polarization by pancreatic cancer cells. Full article

Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506 increase the resistance of mice to Gram-negative pathogens infections. In this work, we advanced the characterization of the CRL1505 and CRL1506 immunomodulatory properties by evaluating their effect on the Toll-like receptor 4 (TLR4)-triggered immune response in macrophages. We performed experiments in murine RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) to evaluate the transcriptomic changes induced by lactobacilli. These in vitro experiments were complemented with in vivo studies in mice to determine the effect of CRL1505 and CRL1506 strains on Peyer’s patches and peritoneal macrophages. Microarray transcriptomic studies and qPCR confirmation showed that the CRL1505 and CRL1506 strains modulated the expression of inflammatory cytokines and chemokines as well as adhesion molecules in LPS-challenged RAW macrophages, making the effect of L. rhamnosus CRL1505 more remarkable. Lactobacilli also modulate regulatory factors in macrophages. L. plantarum CRL1506 increased il10 and socs2 while L. rhamnosus CRL1505 upregulated il27, socs1, and socs3 in RAW cells, indicating a strain-specific effect. However, in vivo, both strains induced similar effects. Peyer’s patches and peritoneal macrophages from mice treated with lactobacilli produced higher levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6, and colony stimulating factor (CSF)-3 after LPS stimulation. This effect would allow improved protection against pathogens. In addition, both lactobacilli equally modulated socs1 and socs2 expressions and IL-10 and IL-27 production in Peyer’s patches macrophages and socs3 and IL-10 in peritoneal cells. Furthermore, lactobacilli reduced the production of IL-1β, IL-12, CSF2, C-C motif chemokine ligand (CCL)-2, and CCL8 in LPS-challenged macrophages. This differential modulation of regulatory and inflammatory factors would allow minimal inflammatory-mediated tissue damage during the generation of the innate immune response. This work provides evidence that L. rhamnosus CRL1505 and L. plantarum CRL1506 modulate macrophages’ TLR4-mediated immunotranscriptomic response, helping to improve protection against Gram-negative bacterial infections. Full article

Prostate cancer remains a significant global health challenge, necessitating the development of novel therapeutic approaches. This study investigated the therapeutic potential of the Antrodia cinnamomea formula (XIANZHIFANG formula, XZF), comprising Antrodia cinnamomea, Sanghuangporus sanghuang, Ganoderma lucidum, Ganoderma sinense, and Inonotus obliquus, in prostate cancer treatment. HPLC analysis confirmed the presence of key triterpenoids, including Antcin A, B, C, K, and Zhankuic acid B, C, and 4,7-dimethoxy-5-methyl-1,3-benzodioxole. Cytotoxicity assays demonstrated that XZF (50–200 μg/mL) exhibited selective activity, maintaining viability in non-cancerous 293T-cells while enhancing the viability of activated CD8+ and CD4+ T-cells in a dose-dependent manner. XZF significantly reduced PD-1 expression in CD8+ T-cells but not in CD4+ T-cells and inhibited the PD-L1/PD-1 interaction, achieving 93% inhibition at 200 μg/mL. Furthermore, when combined with atezolizumab (1 μg/mL), XZF demonstrated complete blockade of PD-L1/PD-1 interaction. In prostate cancer cells, XZF exhibited differential antiproliferative effects. In PC-3 cells, XZF significantly reduced viability across a concentration range of 25–200 μg/mL, whereas DU145 cells showed only partial inhibition at higher concentrations (100–200 μg/mL). LNCaP cells exhibited a dose-dependent reduction in viability, mirroring the response pattern of PC-3 cells. Conditioned medium from XZF-treated macrophages, particularly human THP-1 cells, significantly suppressed the viability and migration of prostate cancer cells in a dose-dependent manner. Notably, the conditioned medium from XZF-treated THP-1 cells exhibited a stronger inhibitory effect on prostate cancer cell viability and migration compared to murine RAW 264.7 macrophages. These findings indicate that XZF exerts its therapeutic potential through multiple mechanisms, including direct antiproliferative effects on cancer cells, enhancement of T-cell responses, modulation of immune checkpoint pathways, and macrophage-mediated suppression of prostate cancer cell survival and migration. The pronounced effects observed in human macrophage models suggest a promising avenue for further investigation in clinical settings, particularly in combination with existing immunotherapies. Full article

Background/Objectives: Compounds derived from pyrimido-diazepine have shown selective inhibition of the susceptible Mycobacterium tuberculosis strain H37Rv. However, there is a need for studies that evaluate the activity of these compounds against multidrug-resistant strains and clinical isolates. This study aims to evaluate the antitubercular potential of FTSD2 against drug-resistant strains of M. tuberculosis. Methods: The compound 4-(2,4-diamino-8-(4-methoxyphenyl)-8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepin-6-yl)-N-(2-(4-(dimethylamino)-6-(4-fluorophenyl)amino-1,3,5-triazin-2-yl)amino)ethyl)benzenesulfonamide (FTSD2) was tested against drug-resistant M. tuberculosis strains at minimal inhibitory and bactericidal concentrations (MIC and MBC). Kill curve assays were performed to assess bactericidal activity, and cytotoxicity was evaluated in human monocyte-derived macrophages and the RAW 264.7 murine macrophage cell line. Intracellular death assays, specifically macrophage infection assays, were also conducted to evaluate the effect of FTSD2 on intracellular M. tuberculosis growth. Results: FTSD2 inhibited the growth of drug-resistant M. tuberculosis at MIC and MBC values between 0.5 and 1 mg/L. Kill curve assays demonstrated concentration-dependent bactericidal activity. No cytotoxicity was observed in macrophages at concentrations below 64 mg/L. Additionally, FTSD2 significantly suppressed intracellular M. tuberculosis growth after 192 h. FTSD2 did not inhibit the growth of nontuberculous mycobacteria, including M. avium, M. abscessus, M. fortuitum, M. chelonae, and M. smegmatis at 50 mg/L. Conclusions: FTSD2 exhibits strong potential as a leading compound for the development of new antitubercular drugs, with selective activity against M. tuberculosis and minimal cytotoxic effects on macrophages. Further studies are needed to explore its mechanisms of action and therapeutic potential. Full article

Background: Diabetes mellitus exacerbates immune dysfunction, leading to higher susceptibility to infections. This study investigated the effects of antibiotics on macrophage functions under high glucose conditions to mimic a diabetic context. Methods: Using murine macrophage cell line RAW 264.7, the present study evaluated the cytotoxicity, phagocytosis, bactericidal activity, and pro-inflammatory cytokine production after treatment with four antibiotics: oxytetracycline, ciprofloxacin, sulfamethoxazole–trimethoprim, and cefotaxime. Results: All antibiotics demonstrated no cytotoxicity across 1×–8× MIC concentrations. Hyperglycemia significantly impaired macrophage phagocytosis and bactericidal activity while inducing pro-inflammatory mediator markers, IL-1, IL-6, TNF-α, and iNOS. Only ciprofloxacin significantly improved phagocytic achieving levels comparable to the low glucose control. Treatments with ciprofloxacin, sulfamethoxazole–trimethoprim, and cefotaxime significantly enhanced bactericidal activity without altering the pro-inflammatory cytokine profile. Conclusions: These findings underscore the negative effect of high glucose on macrophage functions and suggest that ciprofloxacin may be a potential therapeutic option for diabetes-associated infections. Full article

Although interferon-gamma (IFN-γ) is known as a critical factor in polarizing macrophages into the pro-inflammatory state for immune response, how dietary flavonoids regulate IFN-γ response for anti-inflammation is incompletely elucidated. This study aims to investigate the effect of fisetin, a typical flavonol, on the inhibition of IFN-γ-induced inflammation by RNA sequencing (RNA-Seq) and cellular metabolism analysis. RAW264 macrophages pretreated with fisetin following IFN-γ stimulation were subjected to RNA-Seq to analyze alterations in gene expression. Cellular signaling and transcription were investigated using enrichment analysis, motif analysis, and transcription factor prediction. Cellular metabolic state was assessed by measuring the oxygen consumption rate (OCR) and lactate level to reflect mitochondrial respiration and glycolysis. Alterations in signaling proteins were confirmed by Western blot. The results revealed that fisetin downregulated the IFN-γ-induced expression of pro-inflammatory genes and M1 marker genes such as Cxcl9, Il6, Cd80, Cd86, and Nos2. In cellular metabolism, fisetin upregulated the oxidative phosphorylation (OXPHOS) pathway, restored impaired OCR, and reduced lactate production induced by IFN-γ. Motif analysis suggested that fisetin suppressed the activation of IFN-regulatory factor 1 (IRF1). Western blot data further confirmed that fisetin inhibited the phosphorylation of Jak1, Jak2, and STAT1, and decreased the nuclear accumulation of phosphorylated STAT1 and IRF1 induced by IFN-γ. Taken together, our data revealed that fisetin is a potent flavonoid that attenuates IFN-γ-stimulated murine macrophage inflammation and ameliorates disrupted cellular metabolism with a possible Jak1/2-STAT1-IRF1 pathway. Full article

Background: Salmonella Typhimurium poses a substantial health risk to both humans and animals. This study evaluated the potential of using the Salmonella Typhimurium ΔsptP mutant as a live-attenuated vaccine candidate by constructing it through homologous recombination and assessing its key biological properties, including growth characteristics, immunogenicity, and protective efficacy. Methods: We generated the ΔsptP mutant through targeted gene deletion, ensuring the preservation of the bacterial strain’s growth and stability. In vitro and in vivo assays were performed to compare the invasive capabilities between the mutant and the wild-type strains. Specifically, we examined the invasion into RAW264.7 murine macrophages and mice. Furthermore, the virulence of the mutant was evaluated by determining the median lethal dose (LD₅₀). To evaluate immunogenicity and protection, mice were immunized with 2 × 10⁴ CFUs of the ΔsptP mutant, followed by a booster immunization, and then challenged with a virulent strain. Results: The ΔsptP mutant exhibited no significant changes in growth characteristics or genetic stability compared to the wild-type strain. However, it demonstrated a significantly diminished capacity for invasion in both murine macrophages and mice. The LD₅₀ for the mutant was 39.92-fold higher than that of the wild-type, indicating a marked reduction in virulence. Mice immunized with the ΔsptP mutant and administered a booster immunization exhibited 87.5% protection against challenge with a virulent strain, as compared to the PBS control group. Moreover, the mutant induced IgG antibody levels comparable to those induced by the wild-type strain. Conclusions: The ΔsptP mutant of Salmonella Typhimurium exhibits markedly reduced virulence while retaining robust immunogenicity and protective efficacy. These findings suggest that the ΔsptP mutant is a promising candidate for a live-attenuated vaccine, potentially providing an effective strategy to prevent Salmonella Typhimurium infections. Full article