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Introduction: Triple-negative breast cancer (TNBC) represents a significant global health crisis due to its resistance to conventional therapies and lack of specific molecular targets. This study explored the potential of Eriocephalus racemosus (E. racemosus) as an alternative treatment for TNBC. The cytotoxic properties and high-resolution respirometry mitochondrial activities of E. racemosus against the MDA-MB 231 TNBC cell line were evaluated. Methods: Hexane solvent and bioactive fraction extractions of E. racemosus were performed, while mass spectrometry-based metabolite profiling was used to identify the phytochemical constituents of the extracts. The extracts were further tested against MDA-MB 231 TNBC cells to determine their cytotoxicity. The mode of cell death was determined using flow cytometry. The activities of caspases 3, 8, and 9 were assessed using a multiplex activity assay kit. Glycolytic activity and High-resolution respirometry measurements of mitochondrial function in the MDA-MB 231 cell line were conducted using the Seahorse XFp and Oroboros O2K. Results: Metabolite profiling of E. racemosus plant crude extracts identified the presence of coumarins, flavonoids, sesquiterpenoids, triterpenoids, and unknown compounds. The extracts demonstrated promising cytotoxic activities, with a half maximal inhibitory concentration (IC50) of 12.84 µg/mL for the crude hexane extract and 15.49 µg/mL for the bioactive fraction. Further, the crude hexane and bioactive fraction extracts induced apoptosis in the MDA-MB-231 TNBC cells, like the reference drug cisplatin (17.44%, 17.26% and 20.25%, respectively) compared to untreated cells. Caspase 3 activities confirmed the induction of apoptosis in both cisplatin and the plant crude extracts, while caspase 8 and 9 activities confirmed the activation of both the intrinsic and extrinsic apoptosis pathways. Increased levels of glycolytic activity were observed in the hexane crude extract. High-resolution respiratory measurements showed elevated mitochondrial activities in all mitochondrial states except for complex-IV activity. Conclusion: These findings support further exploration of E. racemosus as a potential therapeutic agent for TNBC, offering a promising avenue for the development of targeted treatments with minimal adverse effects. [ABSTRACT FROM AUTHOR]

Background: While COVID-19 has been controlled and deaths have decreased, the long-term consequences of COVID-19 remain a challenge we face today. This study was conducted to determine the relationship between the apoptosis of lymphocyte cells with DNA damage and oxidative stress and the therapeutic and clinical outcomes of elderly patients with COVID-19. Methods: This study was conducted from April 2020 to May 2021 (the period of severe attacks of the epidemic peak of COVID-19) and September 2022 (the post-COVID-19 period). The study groups included elderly patients with COVID-19 hospitalized in the ICU and normal wards of the hospital as well as elderly patients with influenza. A polymerase chain reaction was used to check the validity of the studied diseases. The Annexin V/Propidium Iodide method was used to evaluate the level of apoptosis. Genotoxic effects and DNA damage were assessed by the comet assay method. Total antioxidant status (TAS), total oxidant status (TOS), and myeloperoxidase activity (MPO) were measured by photometric methods. Results: The highest level of apoptosis in peripheral blood lymphocytes and the highest level of DNA damage were observed at both times in the intubated-ICU and non-intubated-ICU groups. In all groups, there was a significant increase in peripheral blood lymphocyte apoptosis levels and DNA damage levels compared to the healthy control group (p < 0.01). The level of apoptosis and DNA damage decreased significantly in the post-COVID-19 period (p < 0.01). In the investigation of oxidative stress biomarkers, the oxidative stress index, including TOS and MPO levels, increased in patients (p < 0.01), and the TAS level decreased (p < 0.01). Conclusion: It shows that the apoptosis of lymphocyte cells, DNA damage, and oxidative stress can be effective in prognostic decisions and is a suitable predictor for diagnosing the condition of patients with viral infections such as COVID-19 and influenza. [ABSTRACT FROM AUTHOR]

Background: Open fractures are challenging due to susceptibility to Staphylococcus aureus infections. This study examines the impact of Vancomycin-Loaded Calcium Sulfate (VLCS) and negative pressure wound therapy (NPWT) on macrophage behavior in enhancing healing and infection resistance. Both VLCS and NPWT were evaluated individually and in combination to determine their effects on macrophage polarization and infection resistance in open fractures. Methods: Through single-cell RNA sequencing, genomic expressions in macrophages from open fracture patients treated with VLCS and NPWT were compared to a control group. The analysis focused on MBD2 gene changes related to macrophage polarization. Results: Remarkable modifications in MBD2 expression in the treatment group indicate a shift towards M2 macrophage polarization. Additionally, the combined treatment group exhibited greater improvements in infection resistance and healing compared to the individual treatments. This shift suggests a healing-promoting atmosphere with improved infection resilience. Conclusions: VLCS and NPWT demonstrate the ability to alter macrophage behavior toward M2 polarization, which is crucial for infection prevention in open fractures. The synergistic effect of their combined use shows even greater promise in enhancing outcomes in orthopedic trauma care. [ABSTRACT FROM AUTHOR]

Background: Failure of treatment with gemcitabine in most cholangiocarcinoma (CCA) patients is due to drug resistance. The therapeutic potential of natural plant secondary compounds with minimal toxicity, such as cannabidiol (CBD), is a promising line of investigation in gemcitabine-resistant CCA. We aim to investigate the effects of CBD on gemcitabine-resistant CCA (KKU-213BGemR) cells in vitro and in vivo. Materials: In vitro, cell proliferation, colony formation, apoptosis and cell cycle arrest were assessed using MTT assay, clonogenicity assay and flow cytometry. The effect of CBD on ROS production was evaluated using the DCFH-DA fluorescent probe. The mechanism exerted by CBD on ER stress-associated apoptosis was investigated by western blot analysis. A gemcitabine-resistant CCA xenograft model was also used and the expression of PCNA and CHOP were evaluated by immunohistochemical analysis. Results: The IC50 values of CBD for KKU-213BGemR cells ranged from 19.66 to 21.05 µM. For a non-cancerous immortalized fibroblast cell line, relevant values were 18.29 to 19.21 µM. CBD suppressed colony formation by KKU-213BGemR cells in a dose-dependent manner in the range of 10 to 30 µM. CBD at 30 µM significantly increased apoptosis at early (16.37%) (P = 0.0024) and late (1.8%) stages (P < 0.0001), for a total of 18.17% apoptosis (P = 0.0017), in part by increasing ROS production (P < 0.0001). Multiphase cell cycle arrest significantly increased at G0/G1 with CBD 10 and 20 µM (P = 0.004 and P = 0.017), and at G2/M with CBD 30 µM (P = 0.005). CBD treatment resulted in increased expression of ER stress-associated apoptosis proteins, including p-PERK, BiP, ATF4, CHOP, BAX, and cytochrome c. In xenografted mouse, CBD significantly suppressed tumors at 10 and 40 mg/kg·Bw (P = 0.0007 and P = 0.0278, respectively), which was supported by an increase in CHOP, but a decrease in PCNA expression in tumor tissues (P < 0.0001). Conclusion: The results suggest that CBD exhibits potent anti-cancer activity against gemcitabine-resistant CCA in vitro and in vivo, in part via ER stress-mediated mechanisms. These results indicate that clinical explorative use of CBD on gemcitabine-resistant CCA patients is warranted. [ABSTRACT FROM AUTHOR]

Background: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. Results: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. Conclusions: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer. [ABSTRACT FROM AUTHOR]

Methionine, an indispensable amino acid crucial for dietary balance, intricately governs metabolic pathways. Disruption in its equilibrium has the potential to heighten homocysteine levels in both plasma and tissues, posing a conceivable risk of inducing inflammation and detriment to the integrity of vascular endothelial cells. The intricate interplay between methionine metabolism, with a specific focus on S-adenosyl-L-methionine (SAM), and the onset of thoracic aortic dissection (TAD) remains enigmatic despite acknowledging the pivotal role of inflammation in this vascular condition. In an established murine model induced by β-aminopropionitrile monofumarate (BAPN), we delved into the repercussions of supplementing with S-adenosyl-L-methionine (SAM) on the progression of TAD. Our observations uncovered a noteworthy improvement in aortic dissection and rupture rates, accompanied by a marked reduction in mortality upon SAM supplementation. Notably, SAM supplementation exhibited a considerable protective effect against BAPN-induced degradation of elastin and the extracellular matrix. Furthermore, SAM supplementation demonstrated a robust inhibitory influence on the infiltration of immune cells, particularly neutrophils and macrophages. It also manifested a notable reduction in the inflammatory polarization of macrophages, evident through diminished accumulation of MHC-IIhigh macrophages and reduced expression of inflammatory cytokines such as IL1β and TNFα in macrophages. Simultaneously, SAM supplementation exerted a suppressive effect on the activation of CD4 + and CD8 + T cells within the aorta. This was evidenced by an elevated proportion of CD44- CD62L + naïve T cells and a concurrent decrease in CD44 + CD62L- effector T cells. In summary, our findings strongly suggest that the supplementation of SAM exhibits remarkable efficacy in alleviating BAPN-induced aortic inflammation, consequently impeding the progression of thoracic aortic dissection. [ABSTRACT FROM AUTHOR]

Background: Microplastics, widely present in the environment, are implicated in disease pathogenesis through oxidative stress and immune modulation. Prevailing research, primarily based on animal and cell studies, falls short in elucidating microplastics' impact on human cardiovascular health. This cross-sectional study detected blood microplastic concentrations in patients presenting with chest pain using pyrolysis–gas chromatography/mass spectrometry and evaluating inflammatory and immune markers through flow cytometry, to explore the potential effects of microplastic on acute coronary syndrome. Results: The study included 101 participants, comprising 19 controls and 82 acute coronary syndrome cases. Notably, acute coronary syndrome patients exhibited elevated microplastic concentrations, with those suffering from acute myocardial infarction presenting higher loads compared to those with unstable angina. Furthermore, patients at intermediate to high risk of coronary artery disease displayed significantly higher microplastic accumulations than their low-risk counterparts. A significant relationship was observed between increased microplastic levels and enhanced IL-6 and IL-12p70 contents, alongside elevated B lymphocyte and natural killer cell counts. Conclusion: These results suggest an association between microplastics and both vascular pathology complexity and immunoinflammatory response in acute coronary syndrome, underscoring the critical need for targeted research to delineate the mechanisms of this association. Highlights: Blood microplastic levels escalate from angiographic patency, to angina patients, peaking in myocardial infarction patients. Microplastics in acute coronary syndrome patients are predominantly PE, followed by PVC, PS, and PP. Microplastics may induce immune cell-associated inflammatory responses in acute coronary syndrome patients. [ABSTRACT FROM AUTHOR]

Background: To investigate the effects of Isoorientin on the apoptosis, proliferation, invasion, and migration of human gastric cancer cells (HGC27 cells). Methods: We used network pharmacology to predict the targets of drugs and diseases. The CCK-8 assay was used to determine the effects of Isoorientin on the proliferation of HGC27 cells. Flow cytometry was employed to analyze the effects of Isoorientin on cell apoptosis and cell cycle distribution of HGC27 cells. Scratch test and transwell chamber test were conducted to assess the effects of Isoorientin on invasion and migration, respectively. Additionally, qPCR and western blot were performed to examine the impact of Isoorientin on apoptosis-related genes and protein expression, respectively. Results: The Isoorientin significantly inhibited the proliferation, migration, and invasion of HGC27 cells compared to the control group. Furthermore, Isoorientin induced apoptosis in HGC27 cells by upregulating the relative expression of Bax and caspase-3 while downregulating the relative expression of p-PI3K, p-AKT, and Bcl-2 proteins. Conclusion: The Isoorientin exhibits inhibitory effects on the proliferation, invasion, and migration of HGC27 cells, and induces apoptosis in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence shows the pivotal significance of miRNAs in the pathogenesis of osteoporosis. miR-381-3p has been identified as an inhibitor of osteogenesis. This study explored the role and mechanism of miR-381-3p in postmenopausal osteoporosis (PMOP), the most common type of osteoporosis. Methods: Bilateral ovariectomy (OVX) rat model was established and miR-381-3p antagomir was administrated through the tail vein in vivo. The pathological changes in rats were assessed through the evaluation of serum bone turnover markers (BALP, PINP, and CTX-1), hematoxylin and eosin (H&E) staining, as well as the expression of osteoblast differentiation biomarkers. Moreover, isolated bone marrow mesenchymal stem cells from OVX-induced rats (OVX-BMMSCs) were utilized to explore the impact of miR-381-3p on osteoblast differentiation. In addition, the target gene and downstream pathway of miR-381-3p were further investigated both in vivo and in vitro. Results: miR-381-3p expression was elevated, whereas KLF5 was suppressed in OVX rats. miR-381-3p antagomir decreased serum levels of bone turnover markers, improved trabecular separation, promoted osteoblast differentiation biomarker expression in OVX rats. ALP activity and mineralization were suppressed, and levels of osteoblast differentiation biomarkers were impeded after miR-381-3p overexpression during osteoblast differentiation of OVX-BMMSCs. While contrasting results were found after inhibition of miR-381-3p. miR-381-3p targets KLF5, negatively affecting its expression as well as its downstream Wnt/β-catenin pathway, both in vivo and in vitro. Silencing of KLF5 restored Wnt/β-catenin activation induced by miR-381-3p antagomir. Conclusion: miR-381-3p aggravates PMOP by inhibiting osteogenic differentiation through targeting KLF5/Wnt/β-catenin pathway. miR-381-3p appears to be a promising candidate for therapeutic intervention in PMOP. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is a malignant bone tumor that commonly occurs in children and adolescents under the age of 20. Dysregulation of microRNAs (miRNAs) is an important factor in the occurrence and progression of OS. MicroRNA miR-744-5p is aberrantly expressed in various tumors. However, its roles and molecular targets in OS remain unclear. Methods: Differentially expressed miRNAs in OS were analyzed using the Gene Expression Omnibus dataset GSE65071, and the potential hub miRNA was identified through weighted gene co-expression network analysis. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-744-5p in OS cell lines. In vitro experiments, including CCK-8 assays, colony formation assays, flow cytometry apoptosis assays, and tube formation assays, were performed to explore the effects of miR-744-5p on OS cell biological behaviors. The downstream target genes of miR-744-5p were predicted through bioinformatics, and the binding sites were validated by a dual-luciferase reporter assay. Results: The lowly expressed miRNA, miR-744-5p, was identified as a hub miRNA involved in OS progression through bioinformatic analysis. Nuclear factor I X (NFIX) was confirmed as a direct target for miR-744-5p in OS. In vitro studies revealed that overexpression of miR-744-5p could restrain the growth of OS cells, whereas miR-744-5p inhibition showed the opposite effect. It was also observed that treatment with the conditioned medium from miR-744-5p-overexpressed OS cells led to poorer proliferation and angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, NFIX overexpression restored the suppression effects of miR-744-5p overexpression on OS cell growth and HUVECs angiogenesis. Conclusion: Our results indicated that miR-744-5p is a potential tumor-suppressive miRNA in OS progression by targeting NFIX to restrain the growth of OS cells and angiogenesis in HUVECs. [ABSTRACT FROM AUTHOR]

Background: Several studies have been reported previously on the bioactivities of different extracts of marine molluscs. Therefore, we decided to evaluate the cytotoxic and antimicrobial activities of S. pharaonis ink as a highly populated species in the Red Sea. We extracted the flavonoids from the ink and analyzed their composition. Then we evaluated systematically the cytotoxic and antimicrobial properties of this extract. A pharmacokinetic study was also conducted using SwissADME to assess the potential of the identified flavonoids and phenolic compounds from the ink extract to be orally active drug candidates. Results: Cytotoxic activity was evaluated against 5 cell lines (MCF7, Hep G2, A549, and Caco2) at different concentrations (0.4 µg/mL, 1.6 µg/mL, 6.3 µg/mL, 25 µg/mL, 100 µg/mL). The viability of examined cells was reduced by the extract in a concentration-dependent manner. The highest cytotoxic effect of the extract was recorded against A549 and Hep G2 cancer cell lines cells with IC50 = 2.873 and 7.1 µg/mL respectively. The mechanistic analysis by flow cytometry of this extract on cell cycle progression and apoptosis induction indicated that the extract arrests the cell cycle at the S phase in Hep G2 and MCF7, while in A549 cell arrest was recorded at G1 phase. However, it causes G1 and S phase arrest in Caco2 cancer cell line. Our data showed that the extract has significant antimicrobial activity against all tested human microbial pathogens. However, the best inhibitory effect was observed against Candida albicans ATCC 10,221 with a minimum inhibitory concentration (MIC) of 1.95 µg/mL. Pharmacokinetic analysis using SwissADME showed that most flavonoids and phenolics compounds have high drug similarity as they satisfy Lipinski's criteria and have WLOGP values below 5.88 and TPSA below 131.6 Å2. Conclusion: S. pharaonis ink ethanolic extract showed a promising cytotoxic potency against various cell lines and a remarkable antimicrobial action against different pathogenic microbial strains. S. pharaonis ink is a novel source of important flavonoids that could be used in the future in different applications as a naturally safe and feasible alternative of synthetic drugs. [ABSTRACT FROM AUTHOR]

Background: Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. Method: We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. Results: We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. Conclusion: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages. [ABSTRACT FROM AUTHOR]

Background: Proper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation. Methods: We utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance. Results: In this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p. Conclusions: miR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs. [ABSTRACT FROM AUTHOR]

Background: Oxylipins including lipoxin A4 (LXA4) facilitate the resolution of inflammation and possess analgesic properties by inhibiting macrophage infiltration and transient receptor potential (TRP) protein expression. Yu-Xue-Bi Tablet (YXB) is a traditional Chinese patent medicine used to relieve inflammatory pain. Our previous research has shown that the analgesic effect of YXB is related to inhibiting peripheral inflammation and regulating macrophage infiltration, but the mechanism is not yet clear. The purpose of this study is to explore the mechanisms of YXB on mice models with Complete Freund's Adjuvant (CFA)-induced inflammatory pain from the perspective at the resolution of inflammation. Methods: Mechanical allodynia thresholds and heat hypersensitivity were measured using the Von Frey test and the hot plate test respectively. The open field test and the tail suspension test were employed to measure anxiety and depressive behaviors respectively. The expression of CD68+ and the proportion of F4/80+CD11b+ cells were measured by immunofluorescence staining and flow cytometry. The expression of transient receptor potential ankyrin 1(TRPA1) was measured by immunofluorescence staining and western blotting. Oxylipins omics analysis provided quantitative data on oxylipins in the paws, and enzyme linked immunosorbent assay (ELISA) was used to measure the levels of LXA4 there. Immunofluorescence staining was used to perform the expression of Leukotriene A4 hydroxylase (LTA4H) in the paws of mice. The impact of injecting the formyl peptide receptor 2(FPR2) antagonist WRW4 and the TRPA1 agonist AITC into the left paws was observed, focusing on the expression of mechanical allodynia thresholds, the expression of CD68+, TRPA1 in the paws, and Calcitonin gene-related peptide (CGRP) in the L5 spinal dorsal horn. Results: YXB elevated mechanical allodynia thresholds, alleviated heat hypersensitivity and anxiety and depressive behaviors in CFA mice. It significantly reduced the number of CD68+ and proportion of F4/80+CD11b+ within the paws, thereby decreasing macrophage infiltration. Additionally, it diminished the expression of TRPA1 in the paws and TRPV1 in the DRG, leading to an inhibition of peripheral sensitization. Through quantitative analysis, it was found that YXB could modulate DHA-derived oxylipins and LXA4. ELISA results indicated that YXB elevated the levels of LXA4 and inhibited the expression of LAT4H in the paws. Furthermore, the pro-resolution and analgesic effects of YXB were hindered after administration of the FPR2 antagonist. Compared with the AITC group, YXB showed no significant improvement in anti-inflammatory and analgesic effects. Conclusions: YXB can regulate the oxylipins of paws in CFA mice to promote the resolution of inflammation. The LXA4-FPR2-TRPA1 pathway is a key mechanism for the resolution of inflammation and analgesic effects. [ABSTRACT FROM AUTHOR]

Background: Prolonged interferon-γ signaling activation induces cancer resistance to therapeutics, especially immunotherapy. However, the detailed mechanisms are not well characterized. In present study, we explored cancer intrinsic resistant mechanisms employing for evading immune checkpoint blockade (ICB) and searched for key immune checkpoints contributing to the constitution of suppressive immune microenvironment of glioblastoma (GBM). Methods: We screened key immune checkpoint (IC) associated with IFN signaling activation in GBM according to integrated transcriptomic profiling on the ICs. Expression analysis and functional assays revealed that malignant cells elevated the key IC, TNFRSF14 expression under IFN-γ stimulation, which enhanced their proliferation and in vivo tumorigenicity. Therapeutic efficiency of TNFRSF14 disruption in GBM was evaluated with in vitro and in vivo functional assays, including immunofluorescence, transwell, RT-qPCR, flow cytometry, mass cytometry, and mice preclinical GBM models. Moreover, the improvement of TNFRSF14 blockade on the efficacy of PD-L1 treatment was examined in mice intracranial xenograft bearing models. Results: TNFRSF14, a previously poorly characterized IC, was disclosed as a checkpoint with malignant intrinsic elevation closely associated with type II not type I IFN signaling activation in GBM. Anti-PD-L1 treatment induces compensatory TNFRSF14 elevation, while enhancing IFN-γ production. TNFRSF14 phosphorylates FAK at Y397 and consequently activates NF-κB, which not only strengthens the tumorigenicity of GBM cells, but also enhances TAMs recruitment through elevating CXCL1/CXCL5 secretion from GBM cells. TNFRSF14 ablation reduces the tumorigenicity of GBM cells, reshapes the immunosuppressive microenvironment, and enhances therapeutic efficacy of anti-PD-L1 in mouse orthotopic GBM model. Conclusion: Our findings highlight a malignant TNFRSF14/FAK axis as a potential target to blunt cancer-intrinsic resistance to ICB treatment, which may help improve the therapeutic efficiency of immunotherapy in malignancies. [ABSTRACT FROM AUTHOR]

Purpose: Osteosarcoma is a primary bone tumor lacking optimal clinical treatment options. Tumor-associated macrophages in the tumor microenvironment are closely associated with tumor development and metastasis. Studies have identified the macrophage receptor with collagenous structure (MARCO) as a specific receptor expressed in macrophages. This study aimed to investigate whether anti-MARCO mAb treatment can induce macrophage polarization in the tumor microenvironment and elicit anti-tumor effects. Methods: THP-1 cells were treated with 20 ng/mL phorbol 12-myristate 13-acetate and 80 ng/mL interleukin-4 for 48 h to induce macrophage polarization to alternatively activated macrophages (M2). Enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, flow cytometry, and bioinformatic analyses were performed to evaluate macrophage polarization. The co-culture groups included a blank group, an M2 macrophage and U2OS co-culture group, and an anti-MARCO mAb-treated M2 macrophage group. Cell viability assays, cell scratch tests, apoptosis, and cell cycle analyses were performed to determine the effects of anti-MARCO mAb-treated macrophages on osteosarcoma cells. Results: It was demonstrated that anti-MARCO mAb can drive macrophages toward classically activated macrophage (M1) polarization. Anti-MARCO mAb promoted the secretion of pro-inflammatory factors by macrophages, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta, interleukin-6 and interleukin-23. Studies on in vitro co-culture models have revealed that macrophages treated with anti-MARCO mAb can suppress the growth and migration of osteosarcoma cells, induce cell apoptosis, and inhibit cell cycle progression of osteosarcoma cells through M1 polarization of macrophages in vitro. Conclusion: Anti-MARCO mAb treatment exerts anti-osteosarcoma effects by affecting macrophage polarization toward M1 macrophages, offering a potential new therapeutic approach for treating osteosarcoma. [ABSTRACT FROM AUTHOR]

Background: Molecularly targeted therapies have recently become a hotspot in the treatment of LUAD, with ongoing efforts to identify new effective targets due to individual variability. Among these potential targets, the mitochondrial transcription elongation factor (TEFM) stands out as a crucial molecule involved in mitochondrial synthetic transcriptional processing. Dysregulation of TEFM has been implicated in the development of various diseases; however, its specific role in LUAD remains unclear. Methods: We conducted a comprehensive analysis of TEFM expression in LUAD, leveraging data from the TCGA database. Subsequently, we validated these findings using clinical specimens obtained from the First Affiliated Hospital of Soochow University, employing western blotting and qRT-PCR techniques. Further experimental validation was performed through the transfection of cells with TEFM overexpression, knockdown, and knockout lentiviruses. The effects of TEFM on LUAD were evaluated both in vitro and in vivo using a range of assays, including CCK-8, colony formation, EdU incorporation, Transwell migration, Tunel assay, flow cytometry, JC-1 staining, and xenograft tumour models. Results: Our investigation uncovered that TEFM exhibited elevated expression levels in LUAD and exhibited co-localization with mitochondria. Overexpression of TEFM facilitated malignant processes in LUAD cells, whereas its silencing notably curbed these behaviors and induced mitochondrial depolarization, along with ROS production, culminating in apoptosis. Moreover, the absence of TEFM substantially influenced the expression of mitochondrial transcripts and respiratory chain complexes. Results from nude mouse xenograft tumors further validated that inhibiting TEFM expression markedly hindered tumor growth. Conclusion: TEFM promotes LUAD malignant progression through the EMT pathway and determines apoptosis by affecting the expression of mitochondrial transcripts and respiratory chain complexes, providing a new therapeutic direction for LUAD-targeted therapy. [ABSTRACT FROM AUTHOR]

Background: Ischemia-reperfusion (I/R) injury is a major factor in liver damage following hepatic resection and liver transplantation, with anesthetics demonstrating the ability to shield organs from this type of injury. Methods: Hypoxia-reoxygenation (H/R) was used to create in vitro I/R hepatocyte cell injury models. The CCK-8 assay, flow cytometer, LDH assay, and ELSIA were utilized to assess hepatocyte injury. The in vivo I/R injury rat model was then built. HE and TUNEL staining were used to assess liver tissue damage. Western-blot was applied to assess the activation of the MAPK/ERK pathway. Results: Remimazolam (RMZL) remarkably improved cell viability and decreased apoptosis in H/R-induced hepatocyte injury. RMZL reduced the release of H/R-induced inflammatory mediators (TNF-α and IL-6) as well as LDH levels. We also discovered that RMZL inhibited p38 and ERK1/2 phosphorylation in vivo and in vitro. The stimulation of MAPK/ERK, on the other hand, abolished RMZL's anti-inflammation effects in H/R-induced hepatocyte injury. Furthermore, RMZL reduced liver tissue injury in I/R rats. Conclusion: RMZL prevented hepatic I/R damage by inhibiting MAPK/ERK signaling. [ABSTRACT FROM AUTHOR]

Background: Cellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment. Methods: The expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1β for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1β. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting. Results: cGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1β for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1β. Conclusion: cGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence. [ABSTRACT FROM AUTHOR]

Background: Mucosal-associated invariant T (MAIT) cells assume pivotal roles in numerous autoimmune inflammatory maladies. However, scant knowledge exists regarding their involvement in the pathological progression of oral lichen planus (OLP). The focus of our study was to explore whether MAIT cells were altered across distinct clinical types of OLP. Methods: The frequency, phenotype, and partial functions of MAIT cells were performed by flow cytometry, using peripheral blood from 18 adults with non-erosive OLP and 22 adults with erosive OLP compared with 15 healthy adults. We also studied the changes in MAIT cells in 15 OLP patients receiving and 10 not receiving corticosteroids. Surface proteins including CD4, CD8, CD69, CD103, CD38, HLA-DR, Tim-3, Programmed Death Molecule-1 (PD-1), and related factors released by MAIT cells such as Granzyme B (GzB), interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-17A, and IL-22 were detected. Results: Within non-erosive OLP patients, MAIT cells manifested an activated phenotype, evident in an elevated frequency of CD69+ CD38+ MAIT cells (p < 0.01). Conversely, erosive OLP patients displayed an activation and depletion phenotype in MAIT cells, typified by elevated CD69 (p < 0.01), CD103 (p < 0.05), and PD-1 expression (p < 0.01). Additionally, MAIT cells exhibited heightened cytokine production, encompassing GzB, IFN-γ, and IL-17A in erosive OLP patients. Notably, the proportion of CD103+ MAIT cells (p < 0.05) and GzB secretion (p < 0.01) by MAIT cells diminished, while the proportion of CD8+ MAIT cells (p < 0.05) rose in OLP patients with corticosteroid therapy. Conclusions: MAIT cells exhibit increased pathogenicity and pro-inflammatory capabilities in OLP. Corticosteroid therapy influences the expression of certain phenotypes and functions of MAIT cells in the peripheral blood of OLP patients. [ABSTRACT FROM AUTHOR]

Background: Minimal access breast surgery (MABS) is commonly employed in the management of breast cancer, but there is limited research on the postoperative immune function associated with MABS. Objective: This study aimed to assess the postoperative immune function in breast patients who underwent MABS or conventional open breast surgery (COBS). Methods: We retrospectively analyzed the medical records of 829 breast cancer patients treated with either MABS or COBS at a single hospital between January 2020 and June 2023. Among them, 116 matched pairs were obtained through 1:1 propensity score matching (PSM). Flow cytometry was used to measure the percentages of CD3+, CD4+, and CD8+ cells, as well as the CD4+/CD8+ ratio, on three different time points: preoperative day 1 (PreD1), postoperative day 1 (PostD1), and postoperative day 7 (PostD7). Results: Both the MABS and COBS groups demonstrated a significant reduction in the percentages of CD3+, CD4+, and CD8+ cells, along with the CD4+/CD8+ ratio, from PreD1 to PostD1. Interestingly, the MABS group showed a reversal of these parameters, returning to preoperative levels by PostD7. Conversely, the COBS group showed an increase in these parameters from PostD1 to PostD7, but they still remained significantly lower than preoperative levels at PostD7. Conclusion: MABS treatment may result in reduced postoperative immune suppression and faster recovery of preoperative immune function compared to COBS in patients. [ABSTRACT FROM AUTHOR]

Immune response is known to play an important role in local tumor control especially in renal cell carcinoma (RCC), which is considered highly immunogenic. For localized tumors, operative resection or local ablative procedures such as cryoablation are common therapeutical options. For thermal ablative procedures such as cryoablation, additional immunological anti-tumor effects have been described. The purpose of this prospective study was to determine changes in peripheral blood circulating lymphocytes and various of their subsets in RCC patients treated with cryoablation or surgery in a longitudinal approach using extensive flow cytometry. Additionally, lymphocytes of RCC patients were compared to a healthy control group. We included 25 patients with RCC. Eight underwent cryoablation and 17 underwent surgery. Univariate and multivariable analysis revealed significantly lower values of B cells, CD4 and CD8 T cells, and various of their subsets in the treatment groups versus the healthy control group. Comparing the two different therapeutical approaches, a significant decline of various lymphocyte subsets with a consecutive normalization after three months was seen for the surgery group, whereas cryoablation led to increased values of CD69 + CD4 + and CD69 + CD8 + cell counts as well as memory CD8 + cells after three months. Treatment-naïve RCC patients showed lower peripheral blood lymphocyte counts compared to healthy controls. The post-treatment course revealed different developments of lymphocytes in the surgery versus cryoablation group, and only cryoablation seems to induce a sustained immunological response after three months. [ABSTRACT FROM AUTHOR]

Cryptococcal meningitis (CM) is a severe fungal disease in immunocompromised patients affecting the central nervous system (CNS). Host response and immunological alterations in the cerebrospinal fluid (CSF) after invasion of Cryptococcus neoformans to the central nervous system have been investigated before but rigorous and comprehensive studies examining cellular changes in the CSF of patients with cryptococccal meningitis are still rare. We retrospectively collected CSF analysis and flow cytometry data of CSF and blood in patients with CM (n = 7) and compared them to HIV positive patients without meningitis (n = 13) and HIV negative healthy controls (n = 7). Within the group of patients with CM we compared those with HIV infection (n = 3) or other immunocompromised conditions (n = 4). Flow cytometry analysis revealed an elevation of natural killer cells and natural killer T cells in the CSF and blood of HIV negative patients with CM, pointing to innate immune activation in early stages after fungal invasion. HIV positive patients with CM exhibited stronger blood-CSF-barrier disruption. Follow-up CSF analysis over up to 150 days showed heterogeneous cellular courses in CM patients with slow normalization of CSF after induction of antifungal therapy. [ABSTRACT FROM AUTHOR]

Background: Hypercoagulability emerges as a central pathological feature and clinical complication in nephrotic syndrome. Increased platelet activation and aggregability are closely related to hypercoagulability in nephrotic syndrome. Monocyte-platelet aggregates (MPAs) have been proposed to represent a robust biomarker of platelet activation. The aim of this study was to investigate levels of the circulating MPAs and MPAs with the different monocyte subsets to evaluate the association of MPAs with hypercoagulability in nephrotic syndrome. Methods: Thirty-two patients with nephrotic syndrome were enrolled. In addition, thirty-two healthy age and sex matched adult volunteers served as healthy controls. MPAs were identified by CD14 monocytes positive for CD41a platelets. The classical (CD14 + + CD16-, CM), the intermediate (CD14 + + CD16+, IM) and the non-classical (CD14 + CD16++, NCM) monocytes, as well as subset specific MPAs, were measured by flow cytometry. Results: Patients with nephrotic syndrome showed a higher percentage of circulating MPAs as compared with healthy controls (p < 0.001). The percentages of MPAs with CM, IM, and NCM were higher than those of healthy controls (p = 0.012, p < 0.001 and p < 0.001, respectively). Circulating MPAs showed correlations with hypoalbuminemia (r=-0.85; p < 0.001), hypercholesterolemia (r = 0.54; p < 0.001), fibrinogen (r = 0.70; p < 0.001) and D-dimer (r = 0.37; p = 0.003), but not with hypertriglyceridemia in nephrotic syndrome. The AUC for the prediction of hypercoagulability in nephrotic syndrome using MPAs was 0.79 (95% CI 0.68–0.90, p < 0.001). The sensitivity of MPAs in predicting hypercoagulability was 0.71, and the specificity was 0.78. Conclusion: Increased MPAs were correlated with hypercoagulability in nephrotic syndrome. MPAs may serve as a potential biomarker for thrombophilic or hypercoagulable state and provide novel insight into the mechanisms of anticoagulation in nephrotic syndrome. [ABSTRACT FROM AUTHOR]

Background: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. Methods: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting. Results: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells. Conclusion: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype. [ABSTRACT FROM AUTHOR]

Background: Shotgun metagenomics for microbial community survey recovers enormous amount of information for microbial genomes that include their abundances, taxonomic, and phylogenetic information, as well as their genomic makeup, the latter of which then helps retrieve their function based on annotated gene products, mRNA, protein, and metabolites. Within the context of a specific hypothesis, additional modalities are often included, to give host-microbiome interaction. For example, in human-associated microbiome projects, it has become increasingly common to include host immunology through flow cytometry. Whilst there are plenty of software approaches available, some that utilize marker-based and assembly-based approaches, for downstream statistical analyses, there is still a dearth of statistical tools that help consolidate all such information in a single platform. By virtue of stringent computational requirements, the statistical workflow is often passive with limited visual exploration. Results: In this study, we have developed a Java-based statistical framework (https://github.com/KociOrges/cviewer) to explore shotgun metagenomics data, which integrates seamlessly with conventional pipelines and offers exploratory as well as hypothesis-driven analyses. The end product is a highly interactive toolkit with a multiple document interface, which makes it easier for a person without specialized knowledge to perform analysis of multiomics datasets and unravel biologically relevant patterns. We have designed algorithms based on frequently used numerical ecology and machine learning principles, with value-driven from integrated omics tools which not only find correlations amongst different datasets but also provide discrimination based on case–control relationships. Conclusions: CViewer was used to analyse two distinct metagenomic datasets with varying complexities. These include a dietary intervention study to understand Crohn's disease changes during a dietary treatment to include remission, as well as a gut microbiome profile for an obesity dataset comparing subjects who suffer from obesity of different aetiologies and against controls who were lean. Complete analyses of both studies in CViewer then provide very powerful mechanistic insights that corroborate with the published literature and demonstrate its full potential. EDLr-6-tH-9CdVCxoa6z_8 Video Abstract [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (LncRNAs) are recognized as a pivotal element in the processes of fracture healing and the osteogenic differentiation of stem cells. This study investigated the molecular mechanism and regulatory significance of lncRNA MAGI2-AS3 (MAGI2-AS3) in fracture healing. Methods: Serum levels of MAGI2-AS3 in patients with normal and delayed fracture healing were verified by RT-qPCR assays. The predictive efficacy of MAGI2-AS3 for delayed fracture healing was analyzed by ROC curve. Osteogenic markers were quantified by RT-qPCR assays. MC3T3-E1 cell viability was detected using CCK-8 assay, and flow cytometry was utilized to measure cell apoptosis. The dual-luciferase reporter gene assay was used to determine the targeted binding between MAGI2-AS3 and miR-223-3p. Results: Serum MAGI2-AS3 expression was decreased in patients with delayed fracture healing compared with patients with normal healing. Elevated MAGI2-AS3 resulted in an upregulation of the proliferative capacity of MC3T3-E1 cells and a decrease in mortality, along with increased levels of both osteogenic markers. However, after transfection silencing MAGI2-AS3, the trend was reversed. Additionally, miR-223-3p was the downstream target of MAGI2-AS3 and was controlled by MAGI2-AS3. miR-223-3p mimic reversed the promoting effects of MAGI2-AS3 overexpression on osteogenic marker levels and cell growth, and induced cell apoptosis. Conclusion: The upregulation of MAGI2-AS3 may expedite the healing of fracture patients by targeting miR-223-3p, offering a novel biomarker for diagnosing patients with delayed healing. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is the most common type of cancer diagnosed in women. Finding novel therapeutic agents with significant cytotoxic action and minimal adverse impact on normal cells becomes crucial. Today, natural anticancer agents present an unconventional method of treating cancer, either as a curative or preventative agent, with considerable concern for marine organisms. Methods: The anticancer effect of the alcoholic extract of different Red Sea Seagrasses on MCF-7 human breast cancer cell line has been investigated. Seagrasses were collected from Wadi El Gamal, Red Sea and extracted. Qualitative HPLC analysis was performed on the extracts for the identification of their active biomarkers. This study was aimed to explore the cytotoxic impact of Thalassia hemprichii (Ehren.) and Enhalus acoroides (L.f.) Royle on MCF-7 and their mode of action. Their anti-proliferative effects on cancer cells were performed using Neutral red assay. On the other hand, their apoptotic effect and their capacity to induce cell cycle arrest were investigated by flow cytometry assay. The effect of Seagrasses on the mitochondrial membrane potential (ΔψM) was studied by using JC-1 mitochondrial membrane potential assay kit in Seagrasses treated cancer cells to Δψ Caspases 3/7activity was examined using the colorimetric method. Gene expression analysis and quantitative real time RT-PCR for the sea grasses on MCF-7 was performed. Immune-blotting technique for Bcl-2 and p53 was investigated. Results: HPLC analysis demonstrated that the extracts contained mainly flavonoids and polyphenols such as Caffeic acid, Chlorogenic acids, catechin and kaempferol that might be responsible for these anticancer effects. Seagrasses alcoholic crude extract markedly suppressed the growth and expansion of MCF-7 cells concentration-dependently with no toxicity against normal human skin fibroblast HSF. Thalassia hemprichii and Enhalus acoroides trigger mode of cell death primarily via apoptosis as confirmed by the flow cytometry. Additionally, they have ability to induce G0/S cell cycle arrest in MCF-7. The data showed the depletion in mitochondrial membrane potential (ΔψM) in the treated cells dose-dependently Caspases 3/7activities markedly increased following 24 h treatment. Finally, Gene expression analysis showed a marked reduction in Bcl-2, Survivin and CDC2 gene expression levels and a significant increase in the expression of p53 and CC2D1A as compared to control cells. Conclusion: In summary, the Methanolic extract of seagrass, Thalassia hemperchii and Enhalus ocoroides are able to induce concentration-dependent cytotoxic effects in human MCF-7 cells through intrinsic pathway of apoptosis in MCF-7 cells. This study reveals the beneficial importance of sea grasses as a source of anticancer agents. Further in vivo study is recommended for the active isolated biomolecules. [ABSTRACT FROM AUTHOR]

Background: Extended contact with silica particles can lead to Silicosis, a chronic lung condition lacking established treatment protocols or clear mechanisms of development. The urgency for innovative treatments arises from the unavailability of effective treatment methodologies. The origin of silica-induced pulmonary fibrosis includes essential processes such as macrophage activation and the conversion of fibroblasts into myofibroblasts, with oxidative stress playing a pivotal role. Shionone (SHI), a triterpenoid extracted from the Aster tataricus plant, is recognized for its extensive health benefits. This study explores the capability of SHI to alleviate the effects of silica-induced lung fibrosis in mice. Methods: This investigation explored the impact of SHI on lung inflammation and fibrosis at different stages (early and late) triggered by silica in mice, focusing specifically on the initial and more developed phases. It comprised an analysis of isolated peritoneal macrophages and fibroblasts extracted from mice to elucidate SHI's therapeutic potential and its underlying mechanism. The methodology employed encompassed quantitative PCR, immunofluorescence, flow cytometry, and western blotting to examine macrophage activity and their transition into myofibroblasts. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by SHI was confirmed via immunofluorescence and western blot studies. SHI's antioxidative properties were evidenced by the measurement of reactive oxygen species (ROS) and mitochondrial ROS within both macrophages and fibroblasts, using 2′, 7′-dichlorodihydrofluorescein diacetate and MitoSOX, respectively. The relevance of SHI was further underscored by applying ML385 and Nrf2 siRNA to gauge its effectiveness. Results: Starting SHI treatment early countered the harmful effects of lung inflammation and fibrosis caused by silica, while initiating SHI at a later phase decelerated the advancement of fibrosis. SHI's action was linked to the activation of the Nrf2 signaling pathway, a boost in antioxidant enzyme levels, and a decrease in oxidative stress and inflammation in macrophages affected by silica. Furthermore, SHI prevented the conversion of fibroblasts into myofibroblasts prompted by TGF-β, along with the resultant oxidative stress. The beneficial outcomes of SHI were negated when ML385 and Nrf2 siRNA were applied, highlighting the pivotal role of the Nrf2 pathway in SHI's efficacy. Conclusion: SHI plays a significant role in stimulating the Nrf2 pathway, thereby defending against silica-induced oxidative stress and inflammatory reactions in macrophages, and inhibiting the conversion of fibroblasts to myofibroblasts due to TGF-β. This suggests that SHI is a viable option for treating lung inflammation and fibrosis in mice suffering from silicosis. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence suggests that DXS253E is critical for cancer development and progression, but the function and potential mechanism of DXS253E in colorectal cancer (CRC) remain largely unknown. In this study, we evaluated the clinical significance and explored the underlying mechanism of DXS253E in CRC. Methods: DXS253E expression in cancer tissues was investigated using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The Kaplan-Meier plot was used to assess the prognosis of DXS253E. The cBioPortal, MethSurv, and Tumor Immune Estimation Resource (TIMER) databases were employed to analyze the mutation profile, methylation, and immune infiltration associated with DXS253E. The biological functions of DXS253E in CRC cells were determined by CCK-8 assay, plate cloning assay, Transwell assay, flow cytometry, lactate assay, western blot, and qRT-PCR. Results: DXS253E was upregulated in CRC tissues and high DXS253E expression levels were correlated with poor survival in CRC patients. Our bioinformatics analyses showed that high DXS253E gene methylation levels were associated with the favorable prognosis of CRC patients. Furthermore, DXS253E levels were linked to the expression levels of several immunomodulatory genes and an abundance of immune cells. Mechanistically, the overexpression of DXS253E enhanced proliferation, migration, invasion, and the aerobic glycolysis of CRC cells through the AKT/mTOR pathway. Conclusions: We demonstrated that DXS253E functions as a potential role in CRC progression and may serve as an indicator of outcomes and a therapeutic target for regulating the AKT/mTOR pathway in CRC. [ABSTRACT FROM AUTHOR]

The present study aimed to assess the anti-leishmanial effects of curcumin nanoemulsion (CUR-NE) against Leishmania major (MRHO/IR/75/ER) in both in vitro and in vivo experiments. CUR-NE was successfully prepared via the spontaneous emulsification method. The in vitro effect of various concentrations of CUR-NE against L. major promastigotes was assessed using the flow cytometry method. In vivo experiments were carried out in BALB/c mice inoculated subcutaneously with 2 × 106 L. major promastigotes. Mice were treated with topical CUR-NE (2.5 mg/ml), intra-lesion injection of CUR-NE (2.5 mg/ml), topical CUR suspension (CUR-S, 2.5 mg/ml), topical NE without CUR (NE-no CUR), amphotericin B as the positive control group, and infected untreated mice as the negative control group. In vitro exposure of promastigotes to CUR-NE showed a dose-dependent anti-leishmanial effect, with a 67.52 ± 0.35% mortality rate at a concentration of 1250 µg/ml and an IC50 of 643.56 µg/ml. In vivo experiments showed that topical CUR-NE and CUR-S significantly decreased the mean lesion size in mice after four weeks from 4.73 ± 1.28 to 2.78 ± 1.28 mm and 4.45 ± 0.88 to 3.23 ± 0.59 mm, respectively (p = 0.001). Furthermore, CUR-NE significantly decreased the parasite load in treated mice compared with the negative control group (p = 0.001). Results from the current study demonstrated the promising activity of CUR-NE against L. major in both in vitro and in vivo experiments. Moreover, CUR-NE was more efficient than CUR-S in healing and reducing parasite burden in mouse models. Future studies should aim to identify molecular mechanisms as well as the pharmacologic and pharmacokinetic aspects of CUR-NE. [ABSTRACT FROM AUTHOR]

Background: This study explored the impact of predicted miRNAs on DNA methyltransferases (DNMTs) and the PODXL gene in Nalm6 cells, revealing the significance of these miRNAs in acute lymphocytic leukemia (ALL). Methods: A comprehensive approach was adopted, integrating bioinformatic analyses encompassing protein structure prediction, molecular docking, dynamics, and ADMET profiling, in conjunction with evaluations of gene and miRNA expression patterns. This methodology was employed to elucidate the therapeutic potential of catechin compounds in modulating the activity of DNA methyltransferases (DNMTs) and the PODXL gene. Results: The findings from our investigation indicate that catechins possess the capability to inhibit DNMT enzymes. This inhibitory effect is associated with the upregulation of microRNAs miR-200c and miR-548 and a concurrent downregulation of PODXL gene expression. These molecular interactions culminate in an augmented apoptotic response within ALL (Nalm6) cells. Conclusion: The study posits that catechins may represent a viable therapeutic avenue for inducing apoptosis in ALL cells. This is achieved through the modulation of epigenetic mechanisms and alterations in gene expression profiles, highlighting the potential of catechins as agents for cancer therapy. [ABSTRACT FROM AUTHOR]

Background: Pulmonary embolism (PE) is a life-threatening thromboembolic disease for which there is limited evidence for effective prevention and treatment. Our goal was to determine whether genetically predicted circulating blood cell traits could influence the incidence of PE. Methods: Using single variable Mendelian randomization (SVMR) and multivariate Mendelian randomization (MVMR) analyses, we identified genetic associations between circulating blood cell counts and lymphocyte subsets and PE. GWAS blood cell characterization summary statistics were compiled from the Blood Cell Consortium. The lymphocyte subpopulation counts were extracted from summary GWAS statistics for samples from 3757 individuals that had been analyzed by flow cytometry. GWAS data related to PE were obtained from the FinnGen study. Results: According to the SVMR and reverse MR, increased levels of circulating white blood cells (odds ratio [OR]: 0.88, 95% confidence interval [CI]: 0.81-0.95, p = 0.0079), lymphocytes (OR: 0.90, 95% CI: 0.84-0.97, p = 0.0115), and neutrophils (OR: 0.88, 95% CI: 0.81–0.96, p = 0.0108) were causally associated with PE susceptibility. MVMR analysis revealed that lower circulating lymphocyte counts (OR: 0.84, 95% CI: 0.75-0.94, p = 0.0139) were an independent predictor of PE. According to further MR results, this association may be primarily related to HLA-DR+ natural killer (NK) cells. Conclusions: Among European populations, there is a causal association between genetically predicted low circulating lymphocyte counts, particularly low HLA-DR+ NK cells, and an increased risk of PE. This finding supports observational studies that link peripheral blood cells to PE and provides recommendations for predicting and preventing this condition. [ABSTRACT FROM AUTHOR]

Context: Bu-shen-yi-jing-fang (BSYJF) has been reported to reduce amyloid-β (Aβ)1–42 deposition in the brain of APP/PS1 mice and ameliorate cognitive function. However, its neuroprotective mechanism remains unclear. Objective: This study aims to investigate whether BSYJF exerts a protective effect on Aβ1–42-induced oxidative stress injury and explore its possible mechanism. Materials and methods: The platform databases TCMSP, Swiss, TTD, DrugBank, and GeneCards were used to mine the targets of Alzheimer's disease (AD) and BSYJF. The platform databases STRING and Metascape were used to build the interaction network of the target protein, and Cytoscape software was used to analyze this network and screen out the key pathways. Aβ1–42-treated SKNMC cells were established to verify the mechanism of BSYJF and the key proteins. The downstream proteins and antioxidants as well as apoptosis and ferroptosis of the PI3K/AKT/Nrf2 signaling pathway were validated using an in vitro SKNMC cell model experiment. The expression levels of related proteins were detected using Western blotting. Flow cytometry and immunofluorescence staining were used to analyze apoptosis and ferroptosis. Results: Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis considered the key signal pathways, mainly involving the PI3K/AKT signaling pathway. Experimental validation demonstrated that BSYJF treatment markedly increased the activity of the PI3K/AKT pathway, which could exert anti-AD effects. Conclusions: Our data provided compelling evidence that the protective effects of BSYJF might be associated with their regulation of the PI3K/AKT/Nrf2 signaling pathway. These studies offered a potential therapy for natural herbal medicine treatment of AD. [ABSTRACT FROM AUTHOR]

Aims of this study: This study aims to investigate the potential of Huangqin Tang (HQT), a traditional Chinese medicine formulation, in the treatment of breast cancer (BC) through a comprehensive approach integrating network pharmacology, molecular docking, and experimental validation. Methods: Chemical composition and target information of HQT were collected using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Disease-related target genes were obtained from the GeneCards database. Network pharmacological analysis, including construction of compound-disease-target networks and protein-protein interaction networks, was performed. Molecular docking simulations were conducted to evaluate the binding affinity between HQT components and key targets. Experimental validation was carried out using cell viability assays, clone formation assays, flow cytometry, Western blotting, and pathway analysis. Results: A total of 210 candidate targets were identified. Network analysis revealed STAT3, AKT1, MAPK3 etc. as central targets. Enrichment analysis suggested HQT may exert anti-tumor effects through regulating lipid metabolism and inflammation related pathways. Molecular docking showed that the key compounds baicalein, wogonin, kaempferol and quercetin all bound effectively to MAPK1. The binding of baicalein to IL6 and naringenin to TNF-α was also relatively stable. The experimental results demonstrated that HQT effectively inhibited the proliferation of breast cancer cells, with IC50 values of 2.334 mg/mL and 1.749 mg/mL in MCF-7 cells at 24 h and 48 h, and IC50 values of 1.286 mg/mL and 1.496 mg/mL in MDA-MB-231 cells at 24 h and 48 h, respectively. Furthermore, HQT induced cell cycle arrest at the G2/M phase in breast cancer cells and downregulated the expression of related proteins including CDK1, Cyclin B1, CDK2, and Cyclin E. Additionally, HQT promoted apoptosis in breast cancer cells by upregulating the expression of Bak and CC-3, while downregulating the expression of Bcl-2. Notably, HQT also exhibited regulatory effects on the HIF-1 signaling pathway. Conclusions: This study provides insights into the potential multi-component and multi-target mechanisms of HQT against BC, suggesting it may achieve therapeutic effects through regulating inflammatory response and cancer-related pathways via the identified active compounds and targets. The findings highlight the importance of integrating traditional medicine with modern approaches for the development of novel cancer therapies. [ABSTRACT FROM AUTHOR]

Background: Type 2 diabetes (T2D) has become an epidemic. Delays in diagnosis and as a consequent late treatment has resulted in high prevalence of complications and mortality. Secreted frizzled-related protein 4 (SFRP4), has been recently identified as a potential early biomarker of T2D related to obesity, due to its association with low grade inflammation in adipose tissue and impaired glucose metabolism. We aimed to evaluate the role of SFRP4 in prediabetes and T2D in a Mexican population. Methods: This was a cross-sectional study that included 80 subjects with T2D, 50 subjects with prediabetes and 50 healthy individuals. Fasting SFRP4 and insulin concentrations were measured by ELISA. Human serum IL-10, IL-6, IL-1β and IL-8 levels were quantified by flow cytometry. Genotyping was performed by TaqMan® probes. Results: Prediabetes and T2D patients had significantly higher SFRP4 levels than controls (P < 0.05). In turn, prediabetes subjects had higher SFRP4 concentrations than control subjects (P < 0.05). Additionally, the prediabetes and T2D groups had higher concentrations of proinflammatory molecules such as IL-6, IL-1β and IL-8, and lower concentrations of IL-10, an anti-inflammatory cytokine, than controls (P < 0.001). The serum SFRP4 concentrations were positively correlated with parameters that are elevated in prediabetes and T2D states, such as, HbA1c and homeostasis model assessment insulin resistance (HOMA-IR), (r = 0.168 and 0.248, respectively, P < 0.05). Also, serum SFRP4 concentrations were positively correlated with concentrations of pro-inflammatory molecules (CRP, IL-6, IL-1β and IL-8) and negatively correlated with the anti-inflammatory molecule IL-10, even after adjusting for body mass index and age (P < 0.001). The genetic variant rs4720265 was correlated with low HDL concentrations in T2D (P < 0.05). Conclusions: SFRP4 correlates positively with the stage of prediabetes, suggesting that it may be an early biomarker to predict the risk of developing diabetes in people with high serum concentrations of SFRP4, although further longitudinal studies are required. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer is one of the most common gynecological malignancies. Previous studies have shown that the ethanol extract of Sophora moorcroftiana seeds (EESMS) possesses an antiproliferative effect on several tumors in vitro. Therefore, in this study, we assessed the impact of EESMS on human cervical carcinoma (HeLa) cell proliferation. Methods: The proliferation and apoptotic effects of HeLa cells treated with EESMS were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, dual acridine orange/ethidium bromide double staining, flow cytometry, and western blotting. Single-cell level atomic force microscopy (AFM) was conducted to detect the mechanical properties of HeLa cells, and proteomics and bioinformatics methods were used to elucidate the molecular mechanisms of EESMS. Results: EESMS treatment inhibited HeLa cell proliferation by blocking the G0/G1 phase, increasing the expression of Caspase-3 and affecting its mechanical properties, and the EESMS indicated no significant inhibitory effect on mouse fibroblasts L929 cell line. In total, 218 differentially expressed proteins were identified using two-dimensional electrophoresis, and eight differentially expressed proteins were successfully identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The differentially expressed proteins were involved in various cellular and biological processes. Conclusion: This study provides a perspective on how cells change through biomechanics and a further theoretical foundation for the future application of Sophora moorcroftiana as a novel low-toxicity chemotherapy medication for treating human cervical cancer. [ABSTRACT FROM AUTHOR]

Background: Myocardial infarction (MI) poses a global public health challenge, often associated with elevated mortality rates and a grim prognosis. A crucial aspect of the inflammatory injury and healing process post-MI involves the dynamic differentiation of macrophages. A promising strategy to alleviate myocardial damage after MI is by modulating the inflammatory response and orchestrating the shift from pro-inflammatory (M1) to anti-inflammatory (M2) macrophages, aiming to achieve a reduced M1/M2 ratio. Nuanxinkang (NXK), a simplified herbal decoction, has demonstrated noteworthy cardioprotective, inflammation-regulating, and myocardial energy metabolism-regulating properties. Methods: In this study, we constructed an MI model by ligating coronary arteries to investigate the efficacy of NXK in improving ventricular remodeling and cardiac function. Mice were administered NXK (1.65 g/kg/d) or an equivalent volume of regular saline via gavage for 28 consecutive days, commencing the day after surgery. Then, we conducted echocardiography to assess the cardiac function, Masson staining to illustrate the extent of myocardial fibrosis, TUNEL staining to reveal myocardial apoptosis, and flow cytometry to analyze the polarization of M1 and M2 macrophages in the hearts. Besides, a lipopolysaccharide (LPS)-induced pro-inflammatory macrophage (M1) polarization model was implemented in RAW264.7 cells to elucidate the underlying mechanism of NXK in regulating macrophage polarization. RAW264.7 cells were pre-treated with or without NXK-containing serum. Oxidative stress was detected by MitoSox staining, followed by Seahorse energy metabolism assay to evaluate alterations in mitochondrial metabolic patterns and ATP production. Both In vivo and in vitro, HIF-1α and PDK1 were detected by fluorescent quantitative PCR and Western blotting. Results: In vivo, MI mice exhibited a decline in cardiac function, adverse ventricular remodeling, and an increase in glycolysis, coupled with M1-dominant polarization mediated by the HIF-1α/PDK1 axis. Notably, robust responses were evident with high-dose NXK treatment (1.65 g/kg/day), leading to a significant enhancement in cardiac function, inhibition of cardiac remodeling, and partial suppression of macrophage glycolysis and the inflammatory phenotype in MI mice. This effect was achieved through the modulation of the HIF-1α/PDK1 axis. In vitro, elevated levels of mitochondrial ROS production and glycolysis were observed in LPS-induced macrophages. Conversely, treatment with NXK notably reduced the oxidative stress damage induced by LPS and enhanced oxidative phosphorylation (OXPHOS). Furthermore, NXK demonstrated the ability to modify the energy metabolism and inflammatory characteristics of macrophages by modulating the HIF-1α/PDK1 axis. The influence of NXK on this axis was partially counteracted by the HIF-1α agonist DMOG. And NXK downregulated PDK1 expression, curtailed glycolysis, and reversed LPS-induced M1 polarization in macrophages, similar to the PDK1 inhibitor DCA. Conclusion: In conclusion, NXK protects against MI-induced cardiac remodeling by inducing metabolic reprogramming and phenotypic differentiation of macrophages, achieved through the modulation of the HIF-1α/PDK1 axis. This provides a novel and promising strategy for the treatment of MI. Highlights: Balancing the coordination between M1 and M2 macrophages emerges as a promising therapeutic strategy for addressing myocardial infarction. Metabolic reprogramming plays a crucial role in the activation and differentiation of macrophages. NXK formulation intricately regulates macrophage polarization through the HIF-1α/PDK1 axis within a metabolic-inflammatory network. [ABSTRACT FROM AUTHOR]

Background: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. Results: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. Conclusion: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship. [ABSTRACT FROM AUTHOR]

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle. [ABSTRACT FROM AUTHOR]

Background: Galangin, a flavonoid compound, is derived from Alpinia officinarum Hance. Previous studies have shown that galangin can inhibit the proliferation of hepatocellular carcinoma (HCC), but its mechanism is still unclear. This study aims to investigate the potential targets and molecular mechanisms of galangin on HCC through network pharmacology, bioinformatics, molecular docking, and experimental in vitro validation. Methods: In this study, network pharmacology was used to investigate the targets and mechanisms of galangin in the treatment of HCC. AutoDockTools software was used to simulate and calculate the binding of galangin to its core targets. GO and KEGG enrichment analyses were conducted in the DAVID database to explore the main biological functions and signaling pathways impacted by galangin intervention. In addition, bioinformatics was applied to examine the correlation between the differential expressions of the anti-HCC core targets of galangin and the survival of patients with HCC. Finally, the findings obtained from network pharmacology and bioinformatics were verified in cell experiments. Results: A total of 67 overlapping target genes of galangin and HCC were identified. Through the analysis of the protein-protein interaction (PPI) network, 10 hub genes with the highest degree of freedom were identified, including SRC, ESR1, MMP9, CDK4, CCNB1, MMP2, CDK2, CDK1, CHK1, and PLK1. These genes were found to be closely related to the degradation of the extracellular matrix, signal transduction, and the cell cycle. GO and KEGG enrichment analyses revealed that galangin exerts an anti-HCC role by affecting various signaling pathways, including the cell cycle, pathways in cancer, and the PI3K-Akt signaling pathway. The results of molecular docking indicated a significant interaction between galangin and CCNB1, CDK4, CDK1, and PLK1. Bioinformatics analysis revealed that CCNB1, CDK4, CDK1, and PLK1 were upregulated in the liver of patients with HCC at both the mRNA and protein levels. Flow cytometry analysis showed that galangin induced G0/G1 phase arrest and cell apoptosis in HepG2 and Huh7 cells. Additionally, galangin suppressed the expression of key proteins and mRNAs involved in the cell cycle pathway. Conclusions: These results suggest that galangin inhibits the growth of HCC cells by arresting the cell cycle at the G0/G1 phase. [ABSTRACT FROM AUTHOR]

Background: Luteolin (3,4,5,7-tetrahydroxy flavone) is reported to strongly protect from acute carbon tetrachloride (CCl4) -induced liver injury or fibrosis. Ferroptosis can be induced by hepatic injury, and contributes to liver fibrosis development. The exact functional mechanism underlying luteolin inhibition of hepatic injury and whether ferroptosis is involved are unclear. Methods: Mice model and cell model of liver injury were constructed or induced to explore the effect and molecular mechanisms of Luteolin in the treatment of hepatic injury using CCl4. Cell Counting Kit-8 (CCK-8) and flow cytometry were used to evaluate HepG2 cell viability and apoptosis. The differential expressed genes involved in liver injury were scanned using RNA-seq and confirmed using functional study. Western blot was used to detect the indicators related to ferroptosis. Results: Luteolin attenuated hepatic injury by alleviating cell morphology and decreasing serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels in vivo mice models, and increasing cell viability, downregulating arachidonate 12-lipoxygenase (ALOX12), cyclooxygenase-2 (COX-2) and P21 protein expression, suppressing apoptosis in vitro cell models. Luteolin also inhibited ferroptosis by stimulating glutathione peroxidase 4 (GPX4) and mitochondrial ferritin (FTMT) protein expression, increasing glutathione (GSH) content, and minimizing Fe2+ and malondialdehyde (MDA) levels. Solute carrier family 7a member 11 (SLC7A11) was identified to be a key regulatory gene that participated in luteolin attenuation of CCl4-induced hepatic injuries in HepG2 cells using Microarray assay. Functional study showed that SLC7A11 can alleviate hepatic injury and ferroptosis. Conclusion: Luteolin attenuated CCl4-induced hepatic injury by inhibiting ferroptosis via SLC7A11. SLC7A11 may serve as a novel alternative therapeutic target for hepatic injury. [ABSTRACT FROM AUTHOR]

Background: Myocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis. Methods: Oral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function. Results: Acacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells. Conclusion: Acacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis. [ABSTRACT FROM AUTHOR]

The lncRNA NUTM2A-AS1 has been shown to be dysregulated in gastric cancer, while the roles in glioma is unclear. The aim of this study was to investigate the roles and potential mechanisms of lncRNA NUTM2A-AS1 in the proliferation and apoptosis of glioma cells. The StarBase software and dual luciferase reporter assay were used to identify the relationship between lncRNA NUTM2A-AS1 and miR-376a-3p, and miR-376a-3p and YAP1. The expression of lncRNA NUTM2A-AS1, miR-376a-3p, and YAP1 in human glioma cell lines was detected by qRT-PCR. MTT and flow cytometry were used to detect the effects of lncRNA NUTM2A-AS1 or miR-376a-3p on the proliferation and apoptosis of U251 and A172 cells, respectively. In addition, changes of Bax and Bcl-2 expression in glioma cells were further verified by western blotting and qRT-PCR. The results showed that the expression of lncRNA NUTM2A-AS1 was elevated in glioma cell lines, while miR-376a-3p was decreased. LncRNA NUTM2A-AS1 was negatively correlated with miR-376a-3p. Silencing of lncRNA NUTM2A-AS1 enhanced the levels of miR-376a-3p, leading to reduced cell proliferation and increased apoptosis in glioma cells. YAP1 was a direct target of miR-376a-3p, and it was negatively regulated by miR-376a-3p in U251 and A172 cells. Further mechanistic studies suggested that miR-376a-3p reduced glioma cell proliferation and increased apoptosis by inhibiting YAP1 expression. In addition, lncRNA NUTM2A-AS1 positively regulated of YAP1 expression in glioma cells. In conclusion, silencing of lncRNA NUTM2A-AS1 inhibited proliferation and induced apoptosis in human glioma cells via the miR-376a-3p/YAP1 axis. [ABSTRACT FROM AUTHOR]

Background: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. Methods: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. Results: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 μM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 μM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 μM of CEP did not influence P-glycoprotein expression. 0.03~0.3 μM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 μM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. Conclusions: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP. [ABSTRACT FROM AUTHOR]

Background: After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom. Results: To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots. Conclusions: DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly. CZiF6AaEp7jbjKaYRmvD15 Video Abstract [ABSTRACT FROM AUTHOR]

Background: Cancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative. Hypothesis/Purpose: In this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7. Study design: An integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene. Methods: We applied a systems-based analysis to introduce a drug–target–pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis. Results: The mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22). Conclusion: Our data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes. [ABSTRACT FROM AUTHOR]

Objective: Explore the therapeutic mechanism of Coptidis Rhizome (CR) in periodontitis using network pharmacology, and validate it through molecular docking and in vitro experiments. Methods: Screened potential active components and target genes of CR from TCMSP and Swiss databases. Identified periodontitis-related target genes using GeneCards. Found common target genes using Venny. Conducted GO and KEGG pathway analysis. Performed molecular docking and in vitro experiments using Berberine, the main active component of CR, on lymphocytes from healthy and periodontitis patients. Assessed effects on inflammatory factors using CCK-8, flow cytometry, and ELISA. Results: Fourteen active components and 291 targets of CR were identified. 30 intersecting target genes with periodontitis were found. GO and KEGG analysis revealed oxidative stress response and IL-17 signaling pathway as key mechanisms. Molecular docking showed strong binding of Berberine with ALOX5, AKT1, NOS2, and TNF. In vitro experiments have demonstrated the ability of berberine to inhibit the expression of Th17 + and other immune related cells in LPS stimulated lymphocytes, and reduce the secretion of IL-6, IL-8, and IL-17. Conclusion: CR treats periodontitis through a multi-component, multi-target, and multi-pathway approach. Berberine, its key component, acts through the IL-17 signaling pathway to exert anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers. [ABSTRACT FROM AUTHOR]

Background: Gastrointestinal cancer (GIC) ranks as the highest cause of cancer-related deaths globally. GIC patients are often diagnosed at advanced stages, limiting effective treatment options. Chemotherapy, the common GIC recommendation, has significant disadvantages such as toxicity and adverse effects. Natural products contain substances with diverse pharmacological characteristics that promise for use in cancer therapeutics. In this study, the flower of renowned Asian medicinal plant, Shorea roxburghii was collected and extracted to investigate its phytochemical contents, antioxidant, and anticancer properties on GIC cells. Methods: The phytochemical contents of Shorea roxburghii extract were assessed using suitable methods. Phenolic content was determined through the Folin-Ciocalteu method, while flavonoids were quantified using the aluminum chloride (AlCl3) method. Antioxidant activity was evaluated using the FRAP and DPPH assays. Cytotoxicity was assessed in GIC cell lines via the MTT assay. Additionally, intracellular ROS levels and apoptosis were examined through flow cytometry techniques. The correlation between GIC cell viability and phytochemicals, 1H-NMR analysis was conducted. Results: Among the four different solvent extracts, ethyl acetate extract had the highest phenolic and flavonoid contents. Water extract exhibited the strongest reducing power and DPPH scavenging activity following by ethyl acetate. Interestingly, ethyl acetate extract demonstrated the highest inhibitory activity against three GIC cell lines (KKU-213B, HepG2, AGS) with IC50 values of 91.60 µg/ml, 39.38 µg/ml, and 35.59 µg/ml, while showing less toxicity to normal fibroblast cells. Ethyl acetate extract induced reactive oxygen species and apoptosis in GIC cell lines by downregulating anti-apoptotic protein Bcl-2. Metabolic profiling-based screening revealed a positive association between reduced GIC cell viability and phytochemicals like cinnamic acid and its derivatives, ferulic acid and coumaric acid. Conclusions: This study highlights the potential of natural compounds in Shorea roxburghii in the development of more effective and safer anticancer agents as options for GIC as well as shedding light on new avenues for cancer treatment. [ABSTRACT FROM AUTHOR]