Analysis Of Protein Expression Research Articles

Cisplatin resistance alters ovarian cancer spheroid formation and impacts peritoneal invasion

Epithelial ovarian cancer (EOC) is an aggressive and lethal gynaecologic malignancy due to late diagnosis and acquired resistance to chemotherapeutic drugs, such as cisplatin. EOC metastasis commonly occurs through the extensive dissemination of multicellular aggregates, formed of cells originally shed from the primary ovarian tumour, within the peritoneal cavity. However, little is known about how cisplatin resistance (CR) alters the biophysical properties of EOC multicellular aggregates and how this impacts metastasis. In this interdisciplinary study, light and atomic force microscopy was used, alongside quantitative gene and protein expression analysis, to reveal distinct differences in the biophysical properties of CR spheroids, which correlated with altered protein expression of plasminogen activator inhibitor-1 (PAI-1) and Tenascin-C. CR SKOV3 spheroids (IC50: 25.5 µM) had a significantly greater area and perimeter and were less spherical, with a reduced Young’s modulus, (p < 0.01) compared to parental (P) SKOV3 spheroids (IC50: 5.4 µM). Gene expression arrays revealed upregulation of genes associated with cell adhesion, extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) in CR spheroids, while immunofluorescence assays demonstrated increased protein expression of PAI-1 (p < 0.05; implicated in cell adhesion) and reduced protein expression of Tenascin-C (p < 0.01; implicated in elasticity) in CR spheroids compared to P spheroids. Furthermore, the CR spheroids demonstrated altered interactions with a surface that mimics the peritoneal lining post mesothelial clearance (Matrigel). CR spheroids were significantly less adhesive with reduced disaggregation on Matrigel surfaces, compared to P spheroids (p < 0.05), while CR cells were more invasive compared to P cells. The combined characterisation of the biophysical and biological roles of EOC multicellular aggregates in drug resistance and metastasis highlight key proteins which could be responsible for altered metastatic progression that may occur in patients that present with cisplatin resistance.

Astaxanthin Prevents Glucocorticoid-Induced Femoral Head Osteonecrosis by Targeting Ferroptosis through the JAK2/STAT3 Signaling Pathway.

Glucocorticoid (GC) is extensively used in clinical practice, and the osteonecrosis of the femoral head caused by them is a common issue in orthopedic surgery, yet the underlying mechanisms remain unclear. Astaxanthin (AST), a potent natural antioxidant, has an unexplored impact on GC-induced osteonecrosis of the femoral head (GIONFH). This study explores the effects and mechanisms of AST in counteracting dexamethasone (Dex)-induced ferroptosis and GIONFH. We developed a rat model of GIONFH using intraperitoneal Dex injections and conducted in vitro analysis by culturing osteoblasts (OBs) with Dex treatment. We assessed the impact of AST on Dex-treated OBs using C11-BODIPY and FerroOrange staining, mitochondrial functionality tests, and protein expression analyses through Western blot and immunofluorescence. The influence of AST on bone microarchitecture of femoral head in rat was assessed using micro-CT, hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry at imaging and histological levels. Our findings suggest that AST exerts an inhibitory effect on Dex-induced ferroptosis and GIONFH. In vitro, AST treatment increased glutathione and decreased malondialdehyde, lipid peroxidation, and mitochondrial-reactive oxygen species. Additionally, AST treatment also enhances the phosphorylation of STAT3, upregulates glutathione peroxidase 4 and osteogenic-related proteins, and stimulates bone formation. To delve deeper into the mechanism, the findings revealed that AST triggered activation of JAK2/STAT3 signaling. Moreover, the use of siRNA-STAT3 blocked the beneficial effect of AST in OBs cultivated with Dex. In brief, AST combats GIONFH by activating the JAK2/STAT3 pathway to inhibit ferroptosis.

Benzimidazole Fungicide Carbendazim Induces Gut Inflammation through the TLR5/NF-κB Pathway in Grass Carp.

Fungicides have been increasingly used across various sectors, including agriculture and textiles. The biocidal properties of fungicides may negatively impact the stability of intestinal microbiota, thereby posing a threat to intestinal health. In this study, we investigated the mechanisms of intestinal damage and functional abnormalities in grass carp following a 42-day exposure to the widely used fungicide carbendazim at environmentally relevant concentrations (0.2 to 20 μg/L). Histopathological observations, mRNA and protein expression analyses, biochemical analysis, quantification of short-chain fatty acids (SCFAs), cytokines, lipopolysaccharide (LPS), and 16S ribosomal ribonucleic acid (rRNA), as well as internal transcribed spacer (ITS) sequencing, were performed. At environmentally relevant concentrations, carbendazim strongly induced intestinal inflammation, leading to increased transcriptional and translational levels of genes involved in the toll-like receptor five (TLR5)/nuclear factor kappa B (NF-κB) pathway, including TLR5, NF-κB, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNFα). Additionally, carbendazim damaged intestinal barriers and reduced the expression of tight junction proteins (e.g., occludin and zonula occludens-1/2), goblet cells, and immunoglobulin M levels, while also disrupting the gut microbiome, leading to intestinal metabolic disorders, particularly decreases in SCFAs and increases in LPS. Treatment with the TLR5 antagonist TH1020 mitigated intestinal inflammation caused by carbendazim, subsequently improving mechanical barrier function. Overall, our findings provide new insights into the toxicological mechanisms underlying intestinal damage caused by carbendazim in grass carp, indicating that carbendazim poses a significant threat to nontarget organisms. Given its widespread detection in the environment, these results underscore the substantial ecological risks to the gut health of fish living in carbendazim-contaminated water bodies.

METTL14/IGF2BP2-MEDIATED M6A MODIFICATION OF STEAP1 AGGRAVATES ACUTE LUNG INJURY INDUCED BY SEPSIS.

Background: Acute lung injury (ALI) is a severe complication of sepsis, characterized by inflammation, edema, and injury to alveolar cells, leading to high mortality rates. Septic ALI is a complex disease involving multiple factors and signaling pathways. STEAP family member 1 (STEAP1) has been reported to be upregulated in a sepsis-induced ALI model. However, the role of STEAP1 in the regulation of septic ALI is not yet fully understood. Methods: The study stimulated human pulmonary microvascular endothelial cells (HPMECs) using lipopolysaccharides (LPS) to establish an in vitro ALI model. The study used quantitative real-time polymerase chain reaction to measure mRNA expression, and western blotting assay or immunohistochemistry assay to analyze protein expression. Cell Counting Kit-8 assay was performed to assess cell viability. Flow cytometry was conducted to analyze cell apoptosis. Tube formation assay was used to analyze the tube formation rate of human umbilical vein endothelial cells. Enzyme-linked immunosorbent assays were used to measure the levels of interleukin 1beta and tumor necrosis factor alpha. The levels of Fe 2+ and reactive oxygen species were determined using colorimetric and fluorometric assays, respectively. The glutathione level was also determined using a colorimetric assay. m6A RNA immunoprecipitation assay, dual-luciferase reporter assay, and RNA immunoprecipitation assay were performed to identify the association of STEAP1 with methyltransferase 14, N6-adenosine-methyltransferase noncatalytic subunit (METTL14) and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). The transcript half-life of STEAP1 was analyzed by actinomycin D assay. Finally, a rat model of polymicrobial sepsis was established to analyze the effects of STEAP1 knockdown on lung injury in vivo . Results: We found that the mRNA expression levels of STEAP1 and METTL14 were upregulated in the blood of ALI patients induced by sepsis compared to healthy volunteers. LPS treatment increased the protein levels of STEAP1 and METTL14 in HPMECs. STEAP1 depletion attenuated LPS-induced promoting effects on HPMECs' apoptosis, inflammatory response, and ferroptosis, as well as LPS-induced inhibitory effect on tube formation. We also found that METTL14 and IGF2BP2 stabilized STEAP1 mRNA expression through the m6A methylation modification process. Moreover, METTL14 silencing attenuated LPS-induced effects by decreasing STEAP1 expression in HPMECs, and STEAP1 silencing ameliorated cecal ligation and puncture-induced lung injury of mice. Conclusion: METTL14/IGF2BP2-mediated m6A modification of STEAP1 aggravated ALI induced by sepsis. These findings suggest potential therapeutic targets for the treatment of this disease.

Comparative analysis of p53 protein expression in early-onset and later-onset colorectal cancers

Background: The global incidence of early onset colorectal cancer (CRC) is rapidly rising. However the reason for this and genomic characteristics of early onset CRC is largely unknown. p53 mutation is common in CRC and has been linked to an increased risk of early onset CRC. Methods: All CRC patients operated at Department of Surgical Oncology, NICRH, between October 1, 2021 and September 30, 2022 were included in this study. Demographic and clinical data and surgical findings were recorded. Histopathology and p53 immunohistochemistry (IHC) analysis was done. Patients were categorized in two groups – ‘Early onset’ (diagnosed before 50 years of age) and ‘Late onset’ (diagnosed at or after 50 years of age). p53 protein IHC status (Mutant or Wild type) and other clinicopathological features were compared between the two groups. Result: Out of 129 patients operated, 75 (58.14%) patients were in the early onset group. Compared to the late onset group, early onset patients had longer duration of symptoms (112.4 days vs. 88.7 days; p = .032), more peritoneal seedlings during surgery (14.7% vs. 1.9%, p = .011), and more poor differentiation (p = .062) and higher grades (p = .017) on histopathological analysis. Early onset patients had significantly more p53 mutation on IHC analysis than elderly ones (34.7% vs. 16.7%, p = .018). Age of onset showed no significance on either disease free survival (DFS) or overall survival (OS) in this study. On comparison of patients with either mutant (MT) or wild type (WT) p53 protein, those with MTp53 had more positive family history (20.0% vs. 3.2%, p=.004), higher levels of CEA (6.4 vs. 4.3, p = .028), higher pathological stage (p = .029), more chance of disease progression or recurrence during follow up (p=.035). Short term survival analysis showed patients with p53 mutation had significantly less DFS (p < .001) and OS (p = .001). Conclusion: Early onset CRC was more prevalent in this study, and was associated with significantly more p53 mutation than the elderly patients. The clinical application of p53 mutation analysis by IHC in all CRC cases, particularly in the early onset patients, should be evaluated. J Bangladesh Coll Phys Surg 2025; 43: 5-12

P-2232. Tracheal aspirate and plasma proteomics reveals the local and systemic host immune response to severe pediatric lower respiratory viral infections

Abstract Background Viral lower respiratory tract infections (LRTI) are a leading cause of child mortality worldwide. Improved understanding of local and systemic host immune responses to severe viral LRTI could reveal insights into pathophysiology, lead to novel host-based diagnostic tests, and inform personalized treatment. To date, no studies have yet employed proteomics to simultaneously characterize the lower airway and systemic proteome in pediatric viral LRTI. Figure 1 Methods We used SomaScan® to assess relative expression of 1,305 proteins from tracheal aspirate (TA) and plasma in 63 patients with acute respiratory failure, a subset of a larger prospective multicenter cohort. We performed differential protein expression and pathway enrichment analyses between viral LRTI (n=41; n=24 with bacterial superinfection) and non-infectious respiratory failure (n=22), developed a diagnostic classifier using LASSO regression, and comparatively analyzed TA and plasma samples. Subanalyses were performed to investigate the impact of bacterial superinfection and viral load. Figure 2 Comparison of host protein expression between Viral LRTI and No LRTI cohorts in tracheal aspirate. (A) Volcano plot of the differentially expressed proteins, with proteins significantly upregulated in LRTI shown in red, and proteins significantly downregulated in LRTI shown in blue. The top ten proteins based on adjusted P-value are labeled. (B) Heat map showing differential expression of the top 20 proteins based on adjusted P-value (rows) across all patients (columns). Dendrogram clustering (top) highlights the proteomic differences between the two groups. (C) Pathway enrichment analysis showing the top ten pathways (all upregulated) ordered by Normalized Enrichment Score. Dot color indicates the false discovery rate (FDR) adjusted P-value, and size indicates the number of proteins included in the pathway. (D) Receiver operator characteristic (ROC) curve of the proteomic classifier to distinguish Viral LRTI from No LRTI. Results We identified a distinct proteomic signature of severe viral LRTI in TA with 200 differentially expressed proteins (adjusted P-value< 0.05), with upregulation of proteins key for type I interferon response, NK cells, and cytotoxic T cells and downregulation of inflammation-modulating proteins. A parsimonious diagnostic classifier achieved an AUC of 0.96 (95% CI 0.90-1.00) for diagnosing LRTI. In contrast, the systemic host response to viral LRTI in plasma was more subtle with 56 differentially expressed proteins. Correlation between TA and plasma proteomics was limited, although the interferon-stimulated protein ISG15 demonstrated increased expression across both compartments. Bacterial superinfection showed upregulation of TNF-stimulated protein TSG-6, C-reactive protein, and interferon signaling compared to viral infection alone. Viral load exhibited positive correlation with interferon proteins and negative correlation with neutrophil aggregation proteins. Conclusion We characterized the lower airway and systemic proteomic host response of severe pediatric viral LRTI and identified proteins with potential mechanistic role in disease severity that could be targeted with novel interventions. Disclosures Jack Kamm, PhD, Genentech: Employee (started there after leaving this study group) Nadir Yehya, MD, AstraZeneca: Advisor/Consultant Lilliam Ambroggio, PhD, Pfizer Inc.: Grant/Research Support

Proteomics profiling of inflammatory responses to elexacaftor/tezacaftor/ivacaftor in cystic fibrosis

BackgroundCFTR modulator therapies have positive clinical outcomes, yet chronic inflammation and bacterial infections persist in people with CF (pwCF). How elexacaftor–tezacaftor–ivacaftor (ETI) fails to improve innate immune signaling responsible for bacterial clearance and inflammation resolution remains unknown.MethodsWe used an unbiased proteomics approach to measure the effect of ETI on inflammatory proteins. Plasma from 20 pediatric pwCF and 20 non-CF (NCF) was collected during routine examination and 3 months after ETI initiation. Protein screening was performed with an inflammation panel (Target 96, Olink®). Bioinformatics analysis was used to determine changes in protein expression.ResultsThere were significantly fewer pulmonary exacerbations after ETI initiation, along with sustained improvement in lung function and reduced bacterial colonization. Unpaired analysis of CF pre-ETI and NCF resulted in 34 significantly different proteins. Of these, CCL20, MMP-10, EN-RAGE, and AXIN1 had a log2 fold change of 1.2 or more. There was a modest shift in overall CF protein profiles post-ETI toward the NCF cluster. Unpaired analysis of protein differential expression between NCF and CF post-ETI identified a total of 24 proteins significantly impacted by ETI therapy (p-value ≤ 0.05), with only CCL20 having a log2 fold change higher than 1.2. Paired analysis (CF pre- and CF post-ETI) of differential protein expression demonstrated significant expression changes of MMP-10, EN-RAGE, and IL-17A. Pathway analysis identified significantly impacted pathways such as the NF-κB pathway.ConclusionThis study showed that ETI in a pediatric cohort had a modest effect on several inflammatory proteins with potential as biomarkers. Pathways significantly impacted by ETI can be further studied for future therapies to combat persistent inflammation and dysregulated immunity.

Deep learning based analysis of G3BP1 protein expression to predict the prognosis of nasopharyngeal carcinoma.

Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) emerges as a pivotal oncogenic gene across various malignancies, notably including nasopharyngeal carcinoma (NPC). The use of automated image analysis tools for immunohistochemical (IHC) staining of particular proteins is highly beneficial, as it could reduce the burden on pathologists. Interestingly, there have been no prior studies that have examined G3BP1 IHC staining using digital pathology. Whole-slide images (WSIs) were meticulously collected and annotated by experienced pathologists. A model was intricately designed and rigorously tested to yield the quantitative data regarding staining intensity and extent. The collective output data was subjected multiplicative analysis, exploring its correlation with the prognosis. The G3BP1 molecular marker scoring model was successfully established utilizing deep learning methodologies, with a calculated threshold staining scores of 1.5. Notably, patients with NPC exhibiting higher expression levels of G3BP1 proteins displayed significantly lower for overall survival rates (OS). Multivariate analysis further validated that positive expression of G3BP1 stood as an independent poorer prognostic factors, indicating a poorer prognosis for NPC patients. Computational pathology emerges as a transformative tool capable of substantially reducing the burden on pathologists while concurrently enhancing and diagnostic sensitivity and specificity. The positive expression of G3BP1 protein serves as valuable, independent biomarker, offering predictive insights into a poor prognosis for patients with NPC.

Metabolic profiling of meningioma reveals novel subgroup-specific biologic insights and outcome dependencies.

Our group and others have recently identified four molecular groups of meningioma, with unique underlying biology and outcomes. The relevance of group-specific metabolite profiles (particularly among hypermetabolic tumours), has not been explored. We performed untargeted metabolic profiling of meningiomas representing each molecular group and WHO grade. Prognostic biochemicals were identified using Cox regression and their biological importance was explored using RNA and protein-based pathway analyses. Validation was performed using targeted high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Global metabolic profiling identified 560 unique biochemicals. We identified a 21-metabolite outcome signatures which is strongly predictive of outcome after adjusting for WHO grade, extent of resection, and receipt of adjuvant radiotherapy (HR 326.49, 95%CI 16.72-6375.48, p < 0.0001). The abundance of N6-trimethyllysine was associated with earlier time to recurrence on our whole cohort (log-rank p = 0.009) and within hypermetabolic and WHO grade 2 tumours specifically; this was validated using targeted HPLC-MS/MS on two cohorts. Consensus RNA and protein expression analysis demonstrated as association between N6-trimethyllysine abundance and activation of oxidative phosphorylation pathways, which portended worse outcomes in the hypermetabolic subgroup but, interestingly, better outcomes in the proliferative subgroup. By contrast, upregulated pyruvate and lactate transporters were associated with worse outcomes in proliferative meningiomas specifically. This is the first study to demonstrate a subgroup-specific prognostic role of N6-trimethyllysine in hypermetabolic meningiomas, offering increasingly granular outcome predictions using a widely accessible technique (HPLC-MS/MS). We also suggest fundamental differences in preferred energy utilization between and a potential need for subgroup-specific therapies.

Rapid human oogonia-like cell specification via transcription factor-directed differentiation.

The generation of germline cells from human induced pluripotent stem cells (hiPSCs) represents a milestone toward in vitro gametogenesis. Methods to recapitulate germline development beyond primordial germ cells in vitro have relied on long-term cell culture, such as 3-dimensional organoid co-culture for ~four months. Using a pipeline with highly parallelized screening, this study identifies combinations of TFs that directly and rapidly convert hiPSCs to induced oogonia-like cells (iOLCs). We demonstrate that co-expression of five TFs - namely, ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX, induces high efficiency DDX4-positive iOLCs in only four days in a feeder-free monolayer culture condition. We also show improved production of human primordial germ cell-like cells (hPGCLCs) from hiPSCs by expression of DLX5, HHEX, and FIGLA. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance human germ cell specification in vitro, and further establish unique computational and experimental tools for human in vitro oogenesis research.

P0200 Evaluating the efficacy of tonsil-derived mesenchymal stem cells and intestinal stem cells-like cells in modulating fibrosis and inflammation in murine colitis models

Abstract Background Current treatments in inflammatory bowel disease (IBD) primarily target inflammation, but effective anti-fibrotic treatments are limited. This study evaluates the therapeutic potential of tonsil derived mesenchymal stem cell (TMSCs) and TMSC induced intestinal stem cell-like cell (T-ISCs) in treating intestinal fibrosis and inflammation using two murine colitis models. Methods Two chronic colitis models were established using dinitrobenzene sulfonic acid (DNBS) and dextran sulfate sodium (DSS) in mice. TMSCs and T-ISCs were administered intraperitoneally, with mice divided into seven groups, including control and treatment groups. Colitis symptoms were assessed using disease activity index (DAI) and colon length. After euthanasia on day 30, colon tissues were collected for analysis. Fibrosis markers and inflammatory cytokines were assessed using real-time PCR, while fibrosis severity was evaluated through Masson’s Trichrome staining. In addition, immunofluorescence and western blotting was performed to confirm the fibrosis-related protein expression. Results The therapeutic effects of TMSCs and T-ISCs were confirmed in both DNBS and DSS colitis models. In the DNBS colitis model, both TMSCs and T-ISCs treatments significantly reduced DAI and restored colon length compared to the PBS group (colitis control group) (all p &amp;lt; 0.05). TMSCs treatment markedly decreased three fibrosis markers, including COL1A1, Vimentin, and TGF-β, with statistical significance (p &amp;lt; 0.01, p &amp;lt; 0.01, and p &amp;lt; 0.05, respectively), while T-ISCs significantly reduced only COL1A1 expression (p &amp;lt; 0.05). Histological analyses using Masson’s Trichrome staining showed decreased fibrosis scores in both TMSCs- and T-ISCs-treated groups, and protein expression analysis via immunofluorescence and western blot also confirmed reductions in a-SMA, Vimentin, COL1A1, and TGF-β in both TMSCs- and T-ISCs-treated groups, with a particularly pronounced reduction observed in the TMSCs-treated group. In the DSS colitis model, both TMSCs and T-ISCs treatments significantly decreased the colon shortening compared to the PBS group (all p &amp;lt; 0.05). Although fibrosis markers and scores showed no significant changes, inflammatory cytokine levels were markedly reduced in the DSS colitis model. Notably, T-ISCs demonstrated significant reductions in both IL-1β (p &amp;lt; 0.01) and IL-6 (p &amp;lt; 0.05). Conclusion TMSCs and T-ISCs demonstrate therapeutic effects in murine colitis models mimicking IBD, with fibrosis reduction in the DNBS model and inflammation reduction in the DSS model. These findings suggest that TMSC-based stem cell therapies are promising strategies for treating fibrosis and inflammation in IBD.

P0196 Metabolic dysregulation in Crohn’s disease fibrosis: Targeting WISP1 to restore ECM homeostasis

Abstract Background In patients with Crohn’s disease (CD), homeostasis of the extracellular matrix (ECM) is significantly disrupted, frequently leading to severe complications such as intestinal stenosis. The maintenance of organ functionality depends on a delicate balance between inflammatory mediators, ECM production, and degradation processes. However, these mechanisms remain poorly understood. This study investigates the dynamic interplay between ECM homeostasis and metabolic pathways in CD, providing novel insights into their role in disease pathogenesis and potential therapeutic intervention. Methods Bulk RNA sequencing was performed to compare fibrotic and non-fibrotic intestinal tissues from CD patients undergoing ileocecal resection, aiming to identify key metabolic and ECM-related pathways. Primary matrix-producing fibroblasts were studied in 2D cultures and spheroids to characterize their metabolic activity, with a focus on fatty acid oxidation (FAO) and glycolysis through gene and protein analysis, metabolic assays, and fatty acid profiling (GC-FID). Chronic intestinal fibrosis was modelled in vivo using dextran sodium sulfate (DSS)-treated mice, allowing therapeutic evaluation of WISP1 neutralisation. The in vivo model was characterized using histological and clinical scoring, as well as gene and protein expression analysis. Results Gene set enrichment analysis revealed increased WNT signaling coupled with a reduction in fatty acid metabolism. Additionally, fibrotic tissue exhibited elevated levels of oleic acid and palmitate, indicative of increased lipid accumulation. WISP1, a WNT mediator, was identified as an adipokine and a key regulator of the metabolic shift from fatty acid oxidation to glycolysis. The disruption of FAO-related genes, including PPARγ, CPT1A, CPT2, and ACOX1, demonstrated reciprocal effects on fibroblast activity: pharmacological inhibition of FAO increased matrix production by fibroblasts, whereas activation of FAO via the PPARα agonist fenofibrate induced a catabolic fibroblast phenotype. This phenotype was characterized by suppressed ECM expression and enhanced ECM degradation via MMP1. Importantly, therapeutic neutralization of WISP1 significantly reduced fibrosis in a chronic DSS-induced colitis model. Conclusion This study presents a novel therapeutic strategy for managing fibrosis in Crohn’s disease. By pharmacologically neutralizing WISP1, this approach aims to correct the underlying metabolic dysfunction, thereby restoring metabolic balance and ECM homeostasis.

P0070 Distinct Serum Proteome Alterations Following Vedolizumab and Ustekinumab Therapy in IBD Patients

Abstract Background Inflammatory bowel disease (IBD) is a chronic intestinal disorder characterized by relapse and remission. Dysregulated cytokine networks and excessive immune activation drive intestinal inflammation. Therapies targeting cytokines (e.g., TNF, IL-12/23) and immune cell trafficking (e.g., integrin inhibitors) have been effective. However, the impact of anti-integrin (vedolizumab) and anti-IL-12/23 (ustekinumab) therapies on systemic inflammation is poorly understood. This study compares serum proteome profiles of patients treated with vedolizumab or ustekinumab to elucidate their distinct effects on inflammatory pathways. Methods This study included patients with Crohn’s disease (CD), ulcerative colitis (UC), and age- and sex-matched healthy donors (HD), who received either ustekinumab or vedolizumab treatment. The ustekinumab cohort comprised 23 CD and 10 UC patients, while the vedolizumab cohort included 7 CD and 27 UC patients. Serum samples were collected from ustekinumab-treated patients at baseline (week 0) and 8 weeks post-treatment, and from vedolizumab-treated patients at baseline (week 0) and 6 weeks post-treatment. Additionally, serum samples were collected from 32 healthy donors. Proteomic analysis was performed using the Olink Inflammation Panel, measuring 92 inflammation-related proteins. Differential protein expression analysis was conducted to compare baseline and post-treatment samples within each cohort. Results Serum proteomic analysis using the Olink Inflammation panel revealed significant alterations in the levels of 15 proteins in IBD patients compared to healthy controls. Pro-inflammatory markers, including 4E-BP1, SIRT2 and TNF were significantly upregulated, indicating immune activation in IBD. Conversely, anti-inflammatory regulators, such as LIF-R and SCF, were significantly downregulated. Ustekinumab treatment led to upregulation of IL-12B and downregulation of 4E-BP1, SIRT2. Notably, IL-12B was upregulated in both CD and UC, while 4E-BP1 and SIRT2 were downregulated in CD (week 8 vs week 0). Conversely, vedolizumab treatment induced robust proteomic changes over time. Between week 0 and week 6, majority of proteins showed significant differential expression, more than the differences observed between IBD and HD groups. Conclusion Serum proteomic profiles of IBD patients differ from healthy donors. Ustekinumab induced minimal serum changes, indicating a limited systemic effect, while vedolizumab led to substantial proteomic shifts, suggesting broader modulation of inflammation. These distinct effects highlight the differing mechanisms of ustekinumab and vedolizumab on systemic inflammation in IBD, prompting further investigation to optimize therapeutic strategies.

Extracellular vesicle-mediated VEGF-A mRNA delivery rescues ischaemic injury with low immunogenicity.

Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the pre-clinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischaemic vascular disease. After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models of ischaemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays. VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by western blot, microscopy, and enzyme-linked immunosorbent assay. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischaemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with adeno-associated viral and lipid nanoparticle VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically. This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischaemic vascular disease.

Pine (Pinus koraiensis) Nut Oil Ameliorates Cholesterol Homeostasis and Inflammation via Modulating the miR-34a/122 Pathways in the Liver of Rats Fed a High-Cholesterol Diet.

Pine (Pinus koraiensis) nut oil (PNO) has been reported to have various beneficial effects on hepatic triglyceride accumulation and atherosclerosis in animal models. MicroRNAs (miRs) are involved in various diseases by modulating physiological processes. However, the mechanism underlying PNO's effects on the regulation of miRs involved in hepatic cholesterol homeostasis and inflammation remains unclear. This study investigated the effects of PNO on the regulation of the miR-34a/122 pathways involved in cholesterol homeostasis and inflammation in the liver using a high-cholesterol diet (HCD) rat model. Six-week-old male Sprague-Dawley rats were randomly divided into three groups (n = 8/group) and provided with (1) a cholesterol-free diet, (2) an HCD containing 1% cholesterol and 0.5% cholic acid, or (3) an HCD containing 5% PNO for 4 weeks. Lipid analysis of serum and liver, histological evaluation, and analysis of gene and protein expression were performed. PNO supplementation in HCD improved hepatic lipid profiles and elevated serum high-density lipoprotein cholesterol compared to the HCD group. PNO significantly upregulated hepatic gene expression levels of liver X receptor α and ATP-binding cassette transporter A1/G1, which are involved in cholesterol efflux (P < 0.05). Gene expressions of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, monocyte chemoattractant protein-1, and inducible nitric oxide synthase were downregulated by PNO (P < 0.05). PNO also suppressed TNF-α and IL-6 protein levels by 22.3% and 17.3%, respectively (P < 0.05). PNO reduced hepatic nuclear factor-kappa B activity by 16.4% and decreased nitric oxide production in the liver and serum (P < 0.05). Furthermore, hepatic miR-34a and miR-122 expressions decreased by 16.4% and 15.7% by PNO, respectively (P < 0.05). These results suggest that PNO may affect cholesterol homeostasis and inflammation, which are partially associated with the miR-34a/122 pathways in the liver under an HCD.

Upregulation of Piezo2 and increased extracellular matrix protein in diabetic kidney disease mice.

Mechanical forces such as glomerular hyperfiltration are crucial in the pathogenesis and progression of diabetic kidney disease. Piezo2 is a mechanosensitive cation channel and plays a major role in various biological and pathophysiological phenomena. We previously reported Piezo2 expression in mouse and rat kidneys and its alteration by dehydration and hypertension. To elucidate the alteration of Piezo2 expression and its consequences in a mouse model of diabetic kidney disease, we used high salt-fed male KK-Ay mice, an accelerated genetic model of diabetic kidney disease. KK-Ay mice exhibited marked obesity, hyperglycemia, elevated blood pressure, higher creatinine clearance, and overt albuminuria. Histopathological analysis revealed glomerular hypertrophy, mesangial expansion, macrophage infiltration, tubular vacuolization, and interstitial fibrosis. The mRNA and protein expression analyses revealed (1) increased fibronectin protein expression in the glomerular areas, (2) upregulated Piezo2 expression in the glomerular mesangial cells and interstitial region, (3) increased Piezo2 and the fibronectin-coding gene Fn1 mRNA, and (4) a strong correlation of Piezo2 expression with that of Fn1 in the kidneys of diabetic kidney disease mice. Piezo2 upregulation and fibronectin accumulation were alleviated by an angiotensin II receptor blocker. In accordance with these in vivo results, in vitro study demonstrated that Piezo2 overexpression increased fibronectin production in HEK293T cells. In conclusion, we demonstrated that Piezo2 expression was upregulated in glomerular mesangial cells in a mouse model of diabetic kidney disease. Our results suggest that Piezo2 contributes to the progression of diabetic kidney disease by mediating glomerular fibronectin production, leading to glomerulosclerosis. Hyperfiltration is crucial in the pathogenesis of diabetic kidney disease. We showed that Piezo2 expression is upregulated in mesangial cells of diabetic kidney disease mice with glomerular fibronectin accumulation. Piezo2 overexpression increased fibronectin production in HEK293T cells. Piezo2 may contribute to diabetic kidney disease progression by mediating glomerular fibronectin production.

HMGB1 secretion by resveratrol in NSCLC: A pathway to ferroptosis-mediated platelet activation suppression.

Cancer-associated venous thromboembolism (CAT) is a frequent and serious complication in cancer patients. Resveratrol, a natural compound with reported anti-tumor effects, is not fully understood in its role regarding CAT in lung cancer. This study aims to explore resveratrol's potential to diminish platelet activation induced by lung adenocarcinoma cells and uncover the underlying mechanisms. Clinical data on coagulation function in non-small cell lung cancer (NSCLC) patients were gathered. A549 human lung cancer cells and Lewis mouse lung cancer cells were employed to assess tumor-induced platelet activation and the impact of resveratrol on this process. Western blotting analyzed protein expression, electron microscopy examined extracellular vesicle (EV) morphology, flow cytometry measured platelet activation, reactive oxygen species (ROS), and exocrine protein expression, while ELISA quantified secretory proteins. Tumor control and platelet function were studied in tumor-bearing mice. We identified that hematological profiles of NSCLC patients frequently manifest a hypercoagulable state relative to healthy controls and lung cancer cells could instigate platelet activation, yet resveratrol could attenuate this phenomenon induced by lung cancer. Resveratrol stimulates lung cancer cells to release HMGB1-enriched EVs, promoting platelet ferroptosis and inhibiting platelet activation through increased ROS, lipid peroxidation, and disrupted cystine transporters. In vivo studies confirm that resveratrol inhibits lung cancer cell growth and suppresses tumor-induced platelet activation in mice. Our studies revealed that resveratrol alleviated lung cancer-induced ferroptosis associated platelet activation. This suggests a potential pharmacological approach for preventing and treating both lung cancer and CAT.

Ameliorative Effect of Glycyrrhizic Acid on Diosbulbin B-Induced Liver Injury and Its Mechanism.

This study aimed to clarify the protective effect of Glycyrrhizic acid (GL) against Diosbulbin B (DB) - induced liver injury in mice and investigate its mechanisms of action. A liver injury DB was established in mice through the oral administration of DB for 15 days. At the same time, GL was administered to the mice for treatment. After the experiment, the pharmacodynamics and mechanisms of GL in ameliorating DB-induced liver injury were explored using biochemical indexes, non-targeted metabolomics, targeted metabolomics, Western blotting analysis of protein expression, 16S rDNA sequencing, and Spearman correlation analysis. The results show reduced liver function indices and improved DB-induced hepatic pathological changes. It also attenuated DB-induced hepatic inflammation and oxidative stress. Hepatic metabolomics revealed that GL regulated ABC transporters and bile secretion. Targeted bile acid (BA) metabolomics and Western blotting demonstrated that GL improved DB-induced reduction in BA efflux by regulating FXR-mediated efflux transporters. Furthermore, analysis of 16S rDNA gene sequencing revealed that GL effectively restored the relative abundance of beneficial bacteria, reduced the relative abundance of harmful bacteria, and reinstated the structure of the intestinal flora. Additionally, correlation analyses between BA and intestinal flora indicated that Firmicutes, Bacteroidota, TDGA, DGA, UDGA, GDGA, THDGA, and HDGA could serve as major markers for DB-induced liver injury. In conclusion, GL significantly improved DB-induced liver injury by increasing the expression of Nrf2/FXR-BSEP/MRP2/P-gp/UGT1A1, promoting BA efflux, regulating intestinal flora, and alleviating inflammation and oxidative stress.

Long-term smoking contributes to aging frailty and inflammatory response.

In recent years, the health challenges linked to frailty in the elderly, particularly those worsened by cigarette smoke, have become more pronounced. However, quantitative studies examining the impact of smoking dosage on frailty in this population remain limited. To address this gap, we developed a model using smoke-exposed elderly mice. Fifteen-month-old C57BL/6J mice were exposed to smoke from two burning cigarettes for 15 min in a whole-body chamber. This exposure occurred 4, 6, and 8 times daily for 30 days, representing low, medium, and high smoking dosages, respectively. Frailty levels were assessed through rotation and grip strength tests, alongside lung histopathology and inflammatory factor protein expression analyses across the three dosage groups. Additionally, we used the Gene Expression Omnibus (GEO) database to validate the correlation between frailty and inflammation in elderly smokers, facilitating cross-comparisons between animal model findings and human sample data. Our results show that mice exposed to high-dose smoking were significantly more prone to frailty, with notable reductions in maximal grip strength (P < 0.01) and drop time (P < 0.001). Among human samples, 69.2% of elderly smokers exhibited a frailty phenotype, compared to just 15.4% of nonsmokers. Both smoking-exposed mice and elderly smokers demonstrated upregulation of tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β) in lung tissue and serum. Mechanistically, this upregulation activates the NF-κB signaling pathway. Our findings quantitatively link smoking-induced frailty to increased levels of TNF-α and IL-1β, providing experimental evidence for the diagnosis and prevention of frailty in elderly populations.

WDR62 mediates MAPK/ERK pathway to stimulate DNA damage repair and attenuate cisplatin sensitivity in lung adenocarcinoma.

Chemotherapy resistance has long stood in the way of therapeutic advancement for lung cancer patients, the malignant tumor with the highest incidence and fatality rate in the world. Patients with lung adenocarcinoma (LUAD) now have a dismal prognosis due to the development of cisplatin (DDP) resistance, forcing them to use more costly second-line therapies. Therefore, overcoming resistance and enhancing patient outcomes can be achieved by comprehending the regulatory mechanisms of DDP resistance in LUAD. WD repeat domain 62 (WDR62) expression in LUAD tissues and in DDP-resistant or sensitive LUAD patients was analyzed bioinformatically, and a K-M plot was utilized to assess survival status. Real-time quantitative PCR was employed for WDR62 expression detection, cell-counting kit-8 assay for half maximal inhibitory concentration determination, flow cytometry for cell apoptosis detection, immunofluorescence for γ-H2AX expression analysis, and western blot for nonhomologous end joining repair and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway-related protein expression analysis. Poor prognosis was linked to WDR62, which was overexpressed in LUAD tissues and cells. Compared to sensitive cells, DDP-resistant cells had increased WDR62 expression. WDR62 knockdown may enhance DDP-induced cell apoptosis while reducing cell proliferation and DNA damage repair. Functional investigations verified that overexpressed WDR62's encouraging impact on DNA damage repair in A549/DDP cells could be reversed by MAPK inhibitors, increasing the cells' susceptibility to DDP. LUAD cells became less sensitive to DDP when WDR62 activated the MAPK/ERK pathway, which promoted DNA damage repair, indicating that DDP resistance might be reversed by treating LUAD with inhibitors of the MAPK pathway.