ICAM-1 Gene Expression Research Articles

UPLC-QTOF-MS characterized, herbal medicine, pancogrit modulates NF-κB and JAK2/STAT3 signaling pathways in cell-based model of TNF-α dependent acute pancreatitis

Acute pancreatitis develops as a local inflammatory condition of the pancreas due to a wide range of factors including overdose of alcohol or drugs, gall stones, etc. At present, medications based on herbal formulations have gained importance due to their better safety profile. Current study assessed the pharmacological effects of Pancogrit, an herbal medicine, in caerulein and TNF-α-induced in vitro pancreatitis models. A comprehensive evaluation of phytometabolites present in Pancogrit was conducted and 85 phytometabolites were identified through UPLC-QTOF-MS and UPLC-UV. The bioactivity and mode of action of Pancogrit were assessed in caerulein-induced AR42J (rat pancreatic acinar) cells, TNF-α-induced PANC-1 (human pancreatic duct) cells, and NF-κB reporter cells. Pancogrit was found to decrease the elevated amylase and lipase activity in caerulein-induced AR42J cells. It ameliorated the increased ROS levels, IL-6, IL-8 release and NF-κB activity in TNF-α-induced PANC-1 and NF-κB reporter cells. Pancogrit also downregulated IL-6, IL-1β, ICAM-1, and STAT3 gene expression in PANC-1 cells. Moreover, Pancogrit inhibited the protein expression levels of phosphorylated STAT3, phosphorylated and total JAK2 in TNF-α-induced PANC-1 cells. Taken together, Pancogrit is a clinically relevant therapeutic agent for mitigation of pancreatitis.

Immunogenic effects of enamel matrix derivative on human alveolar ridge mucosa-derived vascular endothelial cells under lipopolysaccharide stimulation.

Early peri-implant disease detection remains difficult. Enamel matrix derivative (EMD), which is used for periodontal tissue regeneration, promotes leukocyte chemotactic factor and adhesion molecule expression in vascular endothelial cells. We hypothesized that stimulating vascular endothelial cells with EMD would induce an inflammatory response in the peri-implant mucosa, enabling early peri-implant infection detection. To verify this hypothesis, we assessed the intercellular adhesion between human alveolar ridge mucosa-derived vascular endothelial cells (ARMEC) stimulated with lipopolysaccharide (LPS) and EMD and human periodontal ligament-derived vascular endothelial cells (PDLEC). Leukocyte chemotactic factors and cell adhesion molecules were investigated and we established an experimental model of peri-implant disease by stimulating ARMEC (representing the peri-implant mucosa) with Porphyromonas gingivalis-derived LPS. ARMEC and PDLEC were obtained from patients (n = 6) who visited the Nippon Dental University Niigata Hospital. The cells were divided into four subcategories, each cultured with: LPS (1µg/mL), EMD (100µg/mL), LPS + EMD, and pure medium. Cell viability, leukocyte chemotactic factor (interleukin-8: IL-8), adhesion molecules (intercellular adhesion molecule-1: ICAM-1), tight junction protein gene expression (zonula occludens-1: ZO-1 and Occludin), and transendothelial electrical resistance (TEER) was then determined. LPS reduced ARMEC viability, whereas simultaneous stimulation with EMD improved it. LPS and EMD stimulation enhanced IL-8 and ICAM-1 gene expression, suppressed TEER, and decreased ZO-1 and Occludin expression levels compared to that with stimulation with LPS alone. EMD stimulates leukocyte migration, increase vascular permeability, and trigger an immune response in the peri-implant mucosa, thus facilitating the early detection and treatment of peri-implant disease.

Ubiquinol attenuates γ-radiation induced coronary and aortic changes via PDGF/p38 MAPK/ICAM-1 related pathway

Endothelial vascular injury is one of the most pivotal disorders emerging during radiotherapy. It is crucial to rely on strong antioxidants to defend against vascular damage. The current study was carried out to investigate the ameliorative effect of ubiquinol (Ubq) against gamma (γ)-radiation induced aortic and coronary changes, with highlighting its role in suppression of p38 mitogen activated protein kinase (MAPK). Exposure to γ-radiation was adopted as a potent detrimental model that induces vascular tissue damage. Concisely, male albino rats were irradiated at a dose level of 7 Gy and treated daily with Ubq (10 mg/kg/day, p.o.) for 7 days pre-and post-irradiation. At the end of the experiment, lipid profile, 8-hydroxydeoxyguanosine (8-OHdG), gene expression of intercellular adhesion molecule (ICAM-1), platelet derived growth factor (PDGF), p38 MAPK and matrix metalloproteinase-9 (MMP-9) were estimated. Exposure to radiation significantly deteriorates aortic and coronary tissues. Conversely, administration of Ubq significantly reduced serum t-cholesterol, LDL and triglycerides (p = 0.001). In addition, Ubq prevented oxidative DNA damage (8-OHdG) (p = 0.1) and reduced serum MMP-9 (p = 0.001) which contributed to the endothelial cells damage. The positive impact of Ubq was more apparent in suppression of both PDGF (p = 0.001) and p38 MAPK (p = 0.1) protein concentrations, leading subsequently in reduction of ICAM-1 (p = 0.001) gene expression. As a conclusion, vascular endothelial damage brought on by γ-radiation is one of the leading causes of coronary and aortic deteriorations which could be successfully mitigated by Ubq.

Effects of grape juice intake on the cell migration properties in overweight women: Modulation mechanisms of cell migration in vitro by delphinidin-3-O-glucoside

Overweight and obesity are typical conditions of chronic low-intensity systemic inflammatory responses, and both have become more common in recent decades, which emphasizes the necessity for healthier diet intake. Fruits such as grapes are rich in anthocyanins, one of which is delphinidin, a promising chemopreventive agent with anti-inflammatory properties. Considering that polymorphonuclear cells (PMNs) are rapidly mobilized to tissues when the inflammatory process is initiated, this study aimed to understand the impact of grape juice intake and delphinidin on the migration properties of PMNs. Overweight women ingested 500 mL of grape juice for 28 days, and then lipid and inflammatory profiles, as well as the white blood cell count (WBC), were evaluated. Additionally, the gene expression of inflammatory markers and quantified migration molecules such as CD11/CD18, ICAM-1 and VCAM-1 were evaluated in PMNs. The influence of delphinidin-3-O-glucoside in vitro on some migration properties was also evaluated. Grape juice intake did not influence the lipid profile or affect the WBC. However, NFκB gene expression was reduced in PMNs, also reducing the circulating values of IL-8, sICAM-1, and sVCAM-1. The in vitro results demonstrated that delphinidin significantly reduced the migration potential of cells and reduced CD11-/CD18-positive cells, the gene expression of ICAM-1, and the phosphorylation and gene expression of NFκB. Additionally, delphinidin also reduced the production of IL-6, IL-8, and CCL2. Grape juice, after 28 days of intervention, influenced some properties related to cell migration, and delphinidin in vitro can modify the cell migration properties.

Biological profiling in the spectrum of acute coronary syndrome: characterizing patients from MINOCA to NSTEMI with different features of the culprit plaque

Abstract Background In the last two decades, the development of procedures and tools such as coronary angiography and optical coherence tomography (OCT) have made it possible to highlight several aspects of acute coronary syndromes (ACS), identifying multiple subgroups such as patients with myocardial infarction with non-obstructive coronary arteries (MINOCA) and patients with Non-ST-Elevation Myocardial Infarction (NSTEMI) and ruptured fibrous cap (RFC) or intact fibrous cap (IFC) culprit lesions. The aim of the study is to better characterize the ACS spectrum by evaluating the biological profile of non-characterized NTSEMI, RFC-NSTEMI, IFC-NSTEMI, and MINOCA patients. Methods We enrolled 56 consecutive patients presenting with ACS, specifically 16 patients with non-characterized NSTEMI, 12 patients with RFC-NSTEMI, 8 patients with IFC-NSTEMI, and 20 patients with MINOCA. Biological samples were collected and stored. We performed gene expression analysis through qRT-PCR technique for 21 genes. Results Our data displayed several differences in gene expression levels involved in inflammation, cellular adhesion, extracellular matrix turnover, and oxidative stress between the groups under examination. We observed no significant difference between non-characterized NSTEMI and MINOCA for all the studied genes. Alongside, MINOCA displayed significantly lower levels of the following genes than RFC and IFC patients: CD44, GPX1, ICAM1, PLA2G7, SOD1, and VEGFA. Furthermore, when compared with IFC patients, MINOCA showed lower levels of EDN1, LGALS8, MMP1, and TNF, and non-characterized NSTEMI showed a lower gene expression of CD31, ICAM1, and PI16. Finally, CD44, EDN1, GPX1, LGALS8, NOS3, SOD1, TFRC, and VEGFA were more expressed in RFC and IFC compared to non-characterized NSTEMI patients. Conclusions Our preliminary data, besides exploring new pathways in MINOCA patients, reflects the need for characterization and patients’ stratifications in the clinical management of ACS, since no differences were observed between non-characterized NSTEMI and MINOCA. This result may indicate that a one size fits all approach is not the most suitable since a plaque could be an innocent bystander and should be treated accordingly. A precision medicine approach that recognizes the presence of obstructive plaque and defines the type of culprit plaque (i.e., RFC or IFC) could identify better treatment, for a tailored therapy, allowing better prognosis and quality of life.Figure

ОЦЕНКА ЛОКАЛЬНОГО ПРОФИЛЯ ЦИТОКИНОВ, ЭКСПРЕССИРУЕМЫХ НАТИВНЫМИ КЛАПАНАМИ СЕРДЦА, ПОЛУЧЕННЫМИ ОТ ПАЦИЕНТОВ С ПРИОБРЕТЕННЫМИ ПОРОКАМИ СЕРДЦА

Doing. The pathogenesis of acquired heart defects is a complex process involving many different factors. One of the triggers for the development of dysfunctions of native heart valves can be an inflammatory process caused by various types of pathogens. The aim of the study was to evaluate the local profile of cytokines expressed by native heart valves obtained from patients with acquired heart defects during cardiac surgery. Materials and Methods: As a material for the study, biopsies of native mitral heart valves were obtained from 4 patients with infective endocarditis and from 10 patients with rheumatic heart disease. RNA was isolated using a commercial RNeasy® Plus Universal Mini Kit (Qiagen, Germany), protein extraction was performed using T-PER™ Tissue Protein Extraction Reagent lysis buffer (ThermoScientific, USA) with the addition of protease inhibitors Halt™ Protease Inhibitor Cocktail (ThermoScientific, USA) and 0.5M EDTA solution (ThermoScientific, USA). The level of cytokines expressed by native valves was determined by dot blotting using the Proteome Profiler™ Human Cytokine Array Kit (ARY005B) (R&D Systems, USA). Cytokine gene expression was assessed by qPCR using SYBR Green probes (JSC Evrogen, Russia) on a ViiA7 Real-Time PCR System amplifier (Applied Biosystems, USA). Statistical analysis of the results obtained was performed using the GraphPad Prism 8 program (GraphPad Software, USA). Results: The proteins MIF (macrophage migration inhibitory factor), PAI-1 (plasminogen activator inhibitor-1), ICAM-1 (intercellular adhesion molecule-1) and CXCL12 (stromal growth factor-1) were found to be registered in native valves, obtained from both patients with IE and patients with RHD. Semi-quantitative analysis showed that native valves obtained from patients with IE are characterized by a four-fold increase in the level of PAI-1 secretion and a two-fold decrease in the level of ICAM-1 and CXCL12 compared with the control. MIF was expressed at the same level in both studied groups. IL-1ra (interleukin 1 receptor antagonist), IL-6 (interleukin 6), IL-8 (interleukin 8), IL-16 (interleukin 16), CCL4 (chemokine ligand 4), CCL5 (chemokine ligand 5), and CXCL1 ( chemokine ligand 1) were found only in valves affected by IE. The expression of the MIF, IL6, and IL8 genes was increased in the experimental group relative to the control, and the expression of the PAI1, IL1RA, and CXCL1– genes was decreased in the experiment relative to the control. The gene expression of ICAM1, CXCL12, CCL4, CCL5 and IL16 was the same in both studied groups. Conclusions: The results of the study show that native heart valves affected by IE are characterized by a unique cytokine profile compared to valves obtained from patients with non-infectious endocardial pathology. Native heart valves affected by IE show nonspecific local inflammation, active neovascularization, and decreased resistance to pathogenic bacteremia.

Cyclic di-AMP Rescues Porphyromonas gingivalis–Aggravated Atherosclerosis

Growing evidence demonstrates the relationship between periodontitis and atherosclerotic cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis (Pg) has been shown to contribute to the progression of atherosclerosis. Cyclic diadenylate monophosphate (c-di-AMP) has been widely studied as an immune adjuvant for tumor immunotherapy, given its ability to activate the stimulator of interferon genes (STING) and regulate trained immunity. This study sought to elucidate the role of c-di-AMP in Pg-associated atherosclerosis. Periodontitis and atherosclerosis mouse models were established by ligature application around maxillary second molars and feeding ApoE knockout mice with a high-fat diet. We found that periodontitis and atherosclerosis were more severe in mice exposed to Pg than mice that underwent ligature placement only, while prophylactic treatment with c-di-AMP activated trained immunity and elicited significant alleviation of alveolar bone resorption, as well as reduced blood lipid levels and atherosclerotic plaque accumulation. After 3 mo of intervention, c-di-AMP limited the elevation of cytokines interleukin (IL)–6, IL-1β, tumor necrosis factor α, and interferon β; extracellular matrix remodeling enzymes MMP-2 and MMP-9; and adhesion molecules ICAM-1 and VCAM-1 gene expression. The mechanism underlying Pg-aggravated atherosclerosis may be attributed to changes in microbiota composition in oral and aortic plaques and excess inflammatory response, whereas c-di-AMP could prevent the effects of Pg infection due to its potential ability to activate trained immunity and regulate microecological balance. Our findings suggest a positive role of c-di-AMP in alleviating Pg-aggravated atherosclerosis by regulating the immune response and influencing the local microenvironment.

Abstract TP222: Aging-associated Medin Induces Human Brain Microvascular Endothelial Cell Pro-inflammatory And Pro-thrombotic Activation Partly Via NFκB

Background: Vascular aging is characterized by endothelial dysfunction and proinflammatory & prothrombotic changes. It is responsible for aging-associated pathologies including stroke, vascular dementia (VaD) and Alzheimer’s disease (AD). The underlying mechanisms of vascular aging are poorly understood. Medin is an amyloidogenic protein that accumulates in aging vasculature, is associated with VaD and AD, and is a candidate mediator of vascular aging through unknown mechanisms. Our aim is to test the hypothesis that medin induces proinflammatory and prothrombotic changes in human brain microvascular endothelial cells (HBMVECs) through nuclear factor kappa B (NFκB). Methods: HBMVECs were exposed to vehicle, recombinant medin (5 μM, a physiologically relevant dose) without or with RO106-9920 (a small molecule selective NFκB inhibitor, 10 μM) or RO106-9920 alone for 20 hours (N=3-17/treatment). Cell lysates were measured for gene expression of inflammatory cytokines/chemokines (IL-8, IL-6, ICAM-1 and VCAM-1) and thrombotic factors (thrombomodulin (TM), a transmembrane glycoprotein and potent inhibitor of coagulation, plasminogen activator inhibitor-1 (PAI-1) which promotes thrombosis and tissue factor (TF) which initiates the coagulation cascade), using real time PCR. Results: See Figure. Medin increased gene expression of IL-8, IL-6, ICAM-1, VCAM-1 and PAI-1, reduced TM with no change in TF. Co-treatment of medin with RO106-9920 attenuated the increases in IL-8, IL-6, ICAM-1, VCAM-1 and the reduction in TM, with no change in PAI-1. Conclusions: Medin caused increased pro-inflammatory gene expression in HBMVECs. It also caused reduced TM and increased PAI-1 gene expression, signifying prothrombotic activation. Co-treatment with NFκB inhibitor prevented medin’s proinflammatory effect, and effect on TM, but not PAI-1. The effect of inducing endothelial cell activation supports medin as a potential novel target to treat vascular aging.

Development of bilayered porous silk scaffolds for thymus bioengineering

The thymus coordinates the development and selection of T cells. It is structured into two main compartments: the cortex and the medulla. The replication of such complex 3D environment has been challenged by bioengineering approaches. Nevertheless, the effect of the scaffold microstructure on thymic epithelial cell (TEC) cultures has not been deeply investigated. Here, we developed bilayered porous silk fibroin scaffolds and tested their effect on TEC co-cultures. The small and large pore scaffolds presented a mean pore size of 84.33 ± 21.51 μm and 194.90 ± 61.38 μm, respectively. The highly porous bilayered scaffolds presented a high water absorption and water content (> 94 %), together with mechanical properties in the range of the native tissue. TEC (i.e., medullary (mTEC) and cortical (cTEC) cell lines) proliferation is increased in scaffolds with larger pores. The co-culture of both TEC lines in the bilayered porous silk scaffolds presents enhanced cell proliferation and metabolic activity when compared with mTEC in single culture. Also, when the co-culture occurred with cTEC in the small pores layer and mTEC in the large pores layer, a 9.2- and 18.9-fold increase in Foxn1 and Icam1 gene expression in cTEC is evident. These results suggest that scaffold microstructure and the co-culture influence TEC's behaviour. Bilayered silk scaffolds with adjusted microstructure are a valid alternative for TEC culture, having possible applications in advanced thymus bioengineering strategies.

Bio-Functional Activities of Tuscan Bee Pollen.

Bee pollen represents one of the most complete natural foods playing an important role in the diet for its health qualities and therapeutic properties. This work aimed to characterize a Tuscan bee pollen by evaluating its phytochemical profile and the in vitro and ex vivo antioxidant activities. The isolation and taxonomic and functional characterization of yeasts in the sample has been also conducted. Finally, the pollen anti-inflammatory potential has been assessed on a TNFα-inflamed human colorectal adenocarcinoma cell line (HT-29). Our results highlighted a good phytochemical composition in terms of polyphenols, flavonoids, flavonols, monomeric anthocyanins, and carotenoids. In addition, we detected good antioxidant activity and radical scavenging capacity by in vitro and ex vivo assays, as well as good antioxidant activity by isolated yeasts. Data showed no cytotoxic effects of bee pollen extracts, with average viability values >80% at each tested dose. Moreover, TNFα treatment did not affect HT-29 viability while upregulating IL-8, COX-2, and ICAM-1 gene expression, otherwise reduced by both doses of bee pollen. In conclusion, our sample represents an interesting functional food and a potential probiotic product, having high phytochemical compound levels and good antioxidant activities, as well as anti-inflammatory effects on the TNFα-inflamed HT-29 cell line.

Insufficient Oxygen Supplementation During Cardiopulmonary Resuscitation Leads to Unfavorable Biological Response While Hyperoxygenation Contributes to Metabolic Compensation.

Sudden cardiac arrest (CA) is the third leading cause of death. Immediate reoxygenation with high concentrations of supplemental oxygen (O2) during cardiopulmonary resuscitation (CPR) is recommended according to the current guidelines for adult CA. However, a point in controversy exists because of the known harm of prolonged exposure to 100% O2. Therefore, there have been much debate on an optimal use of supplemental O2, yet little is known about the duration and dosage of O2 administration. To test whether supplying a high concentration of O2 during CPR and post resuscitation is beneficial or harmful, rats subjected to 10-minute asphyxia CA were administered either 100% O2 (n=8) or 30% O2 (n=8) for 2hours after CPR. Two hours after initiating CPR, the brain, lung, and heart tissues were collected to compare mRNA gene expression levels of inflammatory cytokines, apoptotic and oxidative stress-related markers. The 100% O2 group had significantly shorter time to return of spontaneous circulation (ROSC) than the 30% O2 group (62.9±2.2 and 77.5±5.9seconds, respectively, P<0.05). Arterial blood gas analysis revealed that the 100% O2 group had significantly higher PaCO2 (49.4±4.9mmHg and 43.0±3.0mmHg, P<0.01), TCO2 (29.8±2.7 and 26.6±1.1mmol/L, P<0.05), HCO3- (28.1±2.4 and 25.4±1.2mmol/L, P<0.05), and BE (2.6±2.3 and 0.1±1.4mmol/L, P<0.05) at 2hours after initiating CPR, but no changes in pH (7.37±0.03 and 7.38±0.03, ns). Inflammation- (Il6, Tnf) and apoptosis- (Casp3) related mRNA gene expression levels were significantly low in the 100% O2 group in the brain, however, oxidative stress moderator Hmox1 increased in the 100% O2 group. Likewise, mRNA gene expression of Icam1, Casp9, Bcl2, and Bax were low in the 100% O2 group in the lung. Contrarily, mRNA gene expression of Il1b and Icam1 were low in the 30% O2 group in the heart. Supplying 30% O2 during and after CPR significantly delayed the time to ROSC and increased inflammation-/apoptosis- related gene expression in the brain and lung, indicating that insufficient O2 was associated with unfavorable biological responses post CA, while prolonged exposure to high-concentration O2 should be still cautious in general.

EFFECTS OF PALM OIL DERIVED TOCOTRIENOL RICH FRACTION AND VITAMIN E ISOMERS ON BIOMARKERS OF EARLY ATHEROGENESIS IN STIMULATED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

This study was conducted to investigate the effects of tocotrienol rich fraction (TRF), α-TOC, and pure TCT isomers (α-. γ- &amp; δ-TCT) on inflammation, endothelial activation, nuclear factor kappa B (NFκB), endothelial nitric oxide synthase (eNOS) and monocyte binding activity (MBA) in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of α-TOC, pure TCT isomers and TRF (0.3-10 µM) together with lipopolysaccharides (LPS) for 16 h. Culture medium and cells were collected and measured for the protein and gene expression of IL-6, TNF-α, NFκB, ICAM-1, VCAM-1, e-selectin, and eNOS. Monocyte binding activity (MBA) was measured by Rose Bengal staining. Area under the curve (AUC) analysis revealed that TRF and pure TCT particularly γ- and δ- isomers, showed better inhibition of inflammation and endothelial activation, MBA and greater eNOS increment than α-TOC. These suggest that TRF and pure TCT isomers have potential as preventive anti-atherogenic agents by attenuating the release of early biomarkers of atherogenesis which is better than α-TOC in LPS-stimulated human endothelial cells.

Effects of nicotine and lipopolysaccharide stimulation on adhesion molecules in human gingival endothelial cells.

Smoking is a risk factor for periodontitis, and the immune response of periodontal tissues in patients with periodontitis may be strongly affected by smoking. The purpose of this study was to elucidate the bioactivity and signal transduction of human gingival endothelial cells (HGECs) due to nicotinic stimulation using a cultured medium supplemented with lipopolysaccharide (LPS) as a model of periodontitis. HGECs were cultured in medium supplemented with LPS, nicotine, nicotine + LPS, and medium supplemented without nicotine or LPS (control). Cell proliferation was assessed using Alamar blue. Cytotoxicity was assessed by lactate dehydrogenase leakage. The expression of adhesion molecule-1 (ICAM-1, VCAM-1) was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay. The expression of nicotinic acetylcholine receptor (nAChR) subunits (α3, α5, α7, β2 and β4) was evaluated by RT-PCR. The involvement of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C (PKC) cell signaling pathways in ICAM-1 and VCAM-1 expression was investigated by RT-qPCR with specific inhibitors. HGECs stimulated with LPS, nicotine and nicotine + LPS showed inhibition of cell proliferation, increase of cell death, and increase of gene and protein expression of ICAM-1. Moreover, HGECs showed the presence of α5 and α7 nAChR subunits. The expression of ICAM-1 in HGECs stimulated with LPS, nicotine, and nicotine + LPS was significantly suppressed by p38MAPK inhibitor, but not by a PKC inhibitor. The nAChR subunits of HGECs are α5 and α7, and that HGECs stimulated with nicotine and LPS express ICAM-1 via p38MAPK pathway.

In Vitro Atheroprotective Effects of Trigonella Foenum Graecum (TFG) and its Saponins in LPS-Stimulated Human Coronary Artery Endothelial Cells

There has been a shift towards utilizing natural products as an adjunct therapy to standard treatment in the prevention of coronary artery disease, and Trigonella foenum graecum (TFG) is one of the potential natural products of interest. In the present study, we attempted to determine the effects of TFG and its saponins on atherosclerosis related biomarkers in vitro. Protein expression of markers of inflammation, endothelial activation and transcription factors were measured by Procarta™ and ELISA assays. Gene expression of the same markers were determined by qPCR and the interaction between monocytes and HCAECs were evaluated through monocyte binding assay following 16 h of treatment with TFG and saponins. Both TFG and its saponins exhibited reducing effects on atherosclerosis-related markers. Based on the area under the curve (AUC) analysis, TFG reduced protein and gene expressions of ICAM-1 and VCAM-1 better than the saponins, while saponins reduced E-selectin expression better than TFG. Saponins showed a reduction of gene and protein expressions of IL-6, IL-8, NF-κB p50 and p65 better than TFG. TFG is more effective in reducing binding of monocytes to endothelial cells than saponins. TFG better reduced endothelial activation but exerted weaker anti-inflammatory effects than saponins, suggesting the possible synergism with other compounds in the crude extract which enhances attenuation of endothelial activation while inhibiting anti-inflammatory properties of saponins in the crude extract.&#x0D; HIGHLIGHTS&#x0D; &#x0D; This research determined the effects of Trigonella foenum graecum and its saponins on atherosclerosis-related biomarkers in-vitro using human coronary artery endothelial cells&#x0D; Trigonella foenum graecum can reduce endothelial activation better than its saponins&#x0D; Saponins appears to exert better anti-inflammatory effects in its single form&#x0D; &#x0D; GRAPHICAL ABSTRACT

Hepatocyte Nuclear Factor 1α Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor-κB in Experimental Model of Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with low-grade inflammation that activates nuclear factor–κB (NF–κB), which upregulates the expression of numerous NF–κB responsive genes, including the genes encoding IL-6, ICAM-1, VCAM-1, and MCP-1. Herein, we found the coordinated overexpression of genes encoding RelA/p65 (a subunit of NF–κB) and HNF1α in the livers of chronic renal failure (CRF) rats—an experimental model of CKD. The coordinated overexpression of RelA/p65 and HNF1α was associated with a significant increase in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. A positive correlation between liver RelA/p65 mRNA levels and a serum concentration of creatinine and BUN suggest that RelA/p65 gene transcription is tightly related to the progression of renal failure. The knockdown of HNF1α in the HepG2 cell line by siRNA led to a decrease in Rel A/p65 mRNA levels. This was associated with a decrease in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. The simultaneous repression of HNF-1α and RelA/p65 by clofibrate is tightly associated with the downregulation of IL-6, ICAM-1, VCAM-1, and MCP-1 gene expression. In conclusion, our findings suggest that NF–κB could be a downstream component of the HNF1α-initiated signaling pathway in the livers of CRF rats.

Gene expression profile of epithelial-mesenchymal transition in tumours of patients with nonsmall cell lung cancer: the influence of COPD.

Epithelial–mesenchymal transition (EMT) is involved in the pathophysiology of lung cancer (LC) and COPD, and the latter is an important risk factor for LC. We hypothesised that the EMT gene expression profile and signalling cascade may differ in LC patients with COPD from those with no respiratory diseases. In lung tumour specimens obtained through video-assisted thoracoscopic surgery from LC (n=20, control group) and LC-COPD patients (n=30), gene expression (quantitative real-time PCR amplification) of EMT markers SMAD3, SMAD4, ZEB2, TWIST1, SNAI1, ICAM1, VIM, CDH2, MMP1 and MMP9 was detected. In lung tumours of LC-COPD compared to LC patients, gene expression of SMAD3, SMAD4, ZEB2 and CDH2 significantly declined, while no significant differences were detected for the other analysed markers. A significant correlation was found between pack-years (smoking burden) and SMAD3 gene expression among LC-COPD patients. LC-COPD patients exhibited mild-to-moderate airway obstruction and a significant reduction in diffusion capacity compared to LC patients. In lung tumour samples of patients with COPD, several markers of EMT expression, namely SMAD3, SMAD4, ZEB2 and CDH2, were differentially expressed suggesting that these markers are likely to play a role in the regulation of EMT in patients with this respiratory disease. Cigarette smoke did not seem to influence the expression of EMT markers in this study. These results have potential clinical implications in the management of patients with LC, particularly in those with underlying respiratory diseases.

Human umbilical cord-derived mesenchymal stem cells not only ameliorate blood glucose but also protect vascular endothelium from diabetic damage through a paracrine mechanism mediated by MAPK/ERK signaling

BackgroundEndothelial damage is an initial step of macro- and micro-vasculature dysfunctions in diabetic patients, accounting for a high incidence of diabetic vascular complications, such as atherosclerosis, nephropathy, retinopathy, and neuropathy. However, clinic lacks effective therapeutics targeting diabetic vascular complications. In field of regenerative medicine, mesenchymal stem cells, such as human umbilical cord-derived MSCs (hucMSCs), have great potential in treating tissue damage.MethodsTo determine whether hucMSCs infusion could repair diabetic vascular endothelial damage and how it works, this study conducted in vivo experiment on streptozotocin-induced diabetic rat model to test body weight, fasting blood glucose (FBG), serum ICAM-1 and VCAM-1 levels, histopathology and immunohistochemical staining of aorta segments. In vitro experiment was further conducted to determine the effects of hucMSCs on diabetic vascular endothelial damage, applying assays of resazurin staining, MTT cell viability, wound healing, transwell migration, and matrigel tube formation on human umbilical vein endothelial cells (HUVECs). RNA sequencing (RNAseq) and molecular experiment were conducted to clarify the mechanism of hucMSCs.ResultsThe in vivo data revealed that hucMSCs partially restore the alterations of body weight, FBG, serum ICAM-1 and VCAM-1 levels, histopathology of aorta and reversed the abnormal phosphorylation of ERK in diabetic rats. By using the conditioned medium of hucMSCs (MSC-CM), the in vitro data revealed that hucMSCs improved cell viability, wound healing, migration and angiogenesis of the high glucose-damaged HUVECs through a paracrine action mode, and the altered gene expressions of IL-6, TNF-α, ICAM-1, VCAM-1, BAX, P16, P53 and ET-1 were significantly restored by MSC-CM. RNAseq incorporated with real-time PCR and Western blot results clarified that high glucose activated MAPK/ERK signaling in HUVECs, while MSC-CM reversed the abnormal phosphorylation of ERK and overexpressions of MKNK2, ERBB3, MYC and DUSP5 in MAPK/ERK signaling pathway.ConclusionsHucMSCs not only ameliorated blood glucose but also protected vascular endothelium from diabetic damage, in which MAPK/ERK signaling mediated its molecular mechanism of paracrine action. Our findings provided novel knowledge of hucMSCs in the treatment of diabetes and suggested a prospective strategy for the clinical treatment of diabetic vascular complications.

Lipophagy-ICAM-1 pathway associated with fatty acid and oxygen deficiencies is involved in poor prognoses of ovarian clear cell carcinoma.

Serum starvation and hypoxia (SSH) mimics a stress condition in tumours. We have shown that intercellular adhesion molecule-1 (ICAM-1) protein is synergistically expressed in ovarian clear cell carcinoma (CCC) cells under SSH in response to an insufficient supply of fatty acids (FAs). This ICAM-1 expression is responsible for resistance against the lethal condition, thereby promoting tumour growth. However, the underlying mechanisms that link SSH-driven ICAM1 gene expression to impaired FA supply and its clinical relevance are unclear. The underlying mechanisms of how FA deficiency induces ICAM-1 expression in cooperation with hypoxia were analysed in vitro and in vivo. Clinical significance of CCC cell-derived ICAM-1 and the mechanism associated with the transcriptional synergism were also investigated. ICAM-1 expression was mediated through lipophagy-driven lipid droplet degradation, followed by impaired FA-lipid droplet flow. Lipophagy induced ICAM1 expression through stabilisation of NFκB binding to the promoter region via Sam68 and hTERT. Analyses of clinical specimens revealed that expression of ICAM-1 and LC3B, an autophagy marker associated with lipophagy, significantly correlated with poor prognoses of CCC. The lipophagy-ICAM-1 pathway induced under a tumour-like stress conditions contributes to CCC progression and is a potential therapeutic target for this aggressive cancer type.

Inhibition of galectin-3 ameliorates high-glucose-induced oxidative stress and inflammation in ARPE-19 cells

Purpose Retinal pigment epithelium (RPE) has been found to be participated in the pathogenesis of DR in recent years. Galectin-3 (Gal-3) is involved in many diabetic complications and ophthalmological diseases. However, the role of Gal-3 in RPE cells in DR remains unknown. This study aims to investigate the role of Gal-3 in ARPE-19 cells under high glucose treatment. Materials and methods ARPE-19 cells were cultured under normal or high glucose (HG) for 48 h. Expression of Gal-3 was inhibited by Si-Gal-3 transfection. Apoptosis was checked by flow cytometry. Oxidative stress was checked by measuring ROS, MDA levels, and SOD activities. Occludin and ZO-1 expression were checked by immunofluorescence staining. Genes involved in inflammatory response were measured by real-time PCR and Western blot. Results Gal-3 expression could be increased by HG treatment in ARPE-19 cells. Gal-3 knockdown might reduce oxidative stress, apoptosis, and gene expression of VCAM-1, ICAM-1, and integrin-β1 induced by HG treatment. The gene expression of IL-1β could be markedly promoted by HG treatment and this increasement was partly alleviated by Gal-3 knockdown only at the mRNA level. The reduced expression of ZO-1 and occludin caused by HG could also be improved by Gal-3 knockdown. Conclusion Gal-3 participated in increased oxidative stress and inflammatory response caused by HG in ARPE-19 cells.

Upregulation of microRNA-223 expression in gingival crevicular blood of women with gestational diabetes mellitus and periodontitis

Background/PurposeMicroRNA-223 (miR-223) is involved in several inflammatory diseases, including gestational diabetes mellitus (GDM) and periodontitis. We first described a procedure for purifying miR-223 from gingival crevicular blood (GCB) of pregnant women with or without GDM and periodontitis. This study aimed to determine whether GDM and/or periodontitis modifies miR-223 expression in pregnant women and to analyze miR-223-targeted messenger RNA (mRNA) expression levels in GCB compared to peripheral blood (PB).Materials and methodsPregnant women were allocated to 4 groups: 10 women with GDM and periodontitis (GDM/P), 10 women with GDM without periodontitis (GDM/NP), 9 women with periodontitis and without GDM (NGDM/P) and 10 women without either condition (NGDM/NP). Clinical parameters of GDM and periodontal status were examined. GCB and PB were collected to assess miR-223, ICAM-1, IL-1β and β1-integrin gene expression by quantitative real-time polymerase chain reaction.ResultsThe GDM/P group demonstrated the highest miR-223 expression levels among the 4 groups in GCB. A significant difference was found between GDM/P and GDM/NP group (P = 0.04). In contrast, the GDM/P showed the lowest miR-223 expression level in PB among the 4 groups. Moreover, ICAM-1 and IL-1β mRNA expression exhibited the opposite trend of miRNA-223, indicating that miRNA-223 might regulate the mRNA function of those genes by epigenetic events.ConclusionThe upregulation of miR-223 expression in GCB but downregulation in PB, ICAM-1 and IL-1β genes expression in women with GDM and periodontitis suggest a promising role of miR-223 in the association between GDM and periodontitis.