mRNA Expression Of Molecules Research Articles

Expression of mRNA for molecules that regulate angiogenesis, endothelial cell survival, and vascular permeability is altered in endothelial cells isolated from db/db mouse hearts

Metabolic syndrome (MetS) is a condition that includes symptoms, such as obesity, hyperglycemia, and hypertension, which elevate cardiovascular risk. An impaired angiogenic response of endothelial cells (ECs) in heart and peripheral organs has been proposed in MetS, but the mechanisms of this phenomenon have not been thoroughly explored. Results obtained from evaluating the whole myocardium are inconsistent, since different types of cells react differently to MetS environment and a variety of molecular pathways are involved in the angiogenic response. Therefore, the aim of this paper was to study one selected pathway—the VEGF/VEGFR pathway, which regulates the angiogenic response and microvascular permeability in ECs isolated from db/db mouse hearts. The expression of mRNAs for VEGF/VEGFR axis proteins was assessed with RT-PCR in ECs isolated from control and db/db mouse myocardium. The density of CD31-, VEGFR2-, and VE-cadherin-positive cells was examined with confocal microscopy, and the ultrastructure of ECs was analyzed with transmission electron microscopy. The aortic ring assay was used to assess the capacity of ECs to respond to angiogenic stimuli. Our results showed a decreased number of microvessels, diminished expression of VE-cadherin and VEGFR2 and widened gaps between the ECs of microcapillaries. The aortic ring assay showed a diminished number of sprouts in db/db mice. These results may indicate that ECs in MetS enhance the production of mRNA for VEGF/VRGFR axis proteins, yet sprout formation and vascular barrier maintenance are limited. These novel data may provide a foundation for further studies on ECs dysfunction in MetS.

Treatment with APAC, a dual antiplatelet anticoagulant heparin proteoglycan mimetic, limits early collar-induced carotid atherosclerotic plaque development in Apoe−/− mice

Background and aimsMast cell-derived heparin proteoglycans (HEP-PG) can be mimicked by bioconjugates carrying antithrombotic and anti-inflammatory properties. The dual antiplatelet and anticoagulant (APAC) construct administered, either locally or intravenously (i.v.), targets activated endothelium, its adhesion molecules, and subendothelial matrix proteins, all relevant to atherogenesis. We hypothesized that APAC influences cellular interactions in atherosclerotic lesion development and studied APAC treatment during the initiation and progression of experimental atherosclerosis. MethodsMale western-type diet-fed Apoe−/− mice were equipped with perivascular carotid artery collars to induce local atherosclerosis. In this model, mRNA expression of adhesion molecules including ICAM-1, VCAM-1, P-Selectin, and Platelet Factor 4 (PF4) are upregulated upon lesion development. From day 1 (prevention) or from 2.5 weeks after lesion initiation (treatment), mice were administered 0.2 mg/kg APAC i.v. or control vehicle three times weekly for 2.5 weeks. At week 5 after collar placement, mice were sacrificed, and lesion morphology was microscopically assessed. ResultsAPAC treatment did not affect body weight or plasma total cholesterol levels during the experiments. In the prevention setting, APAC reduced carotid artery plaque size and volume by over 50 %, aligning with decreased plaque macrophage area and collagen content. During the treatment setting, APAC reduced macrophage accumulation and necrotic core content, and improved markers of plaque stability. ConclusionsAPAC effectively reduced early atherosclerotic lesion development and improved markers of plaque inflammation in advanced atherosclerosis. Thus, APAC may have potential to alleviate the progression of atherosclerosis.

Unraveling the unphosphorylated STAT3-unphosphorylated NF-κB pathway in loss of function STAT3 Hyper IgE syndrome.

Patients with loss of function signal transducer and activator of transcription 3-related Hyper IgE Syndrome (LOF STAT3 HIES) present with recurrent staphylococcal skin and pulmonary infections along with the elevated serum IgE levels, eczematous rashes, and skeletal and facial abnormalities. Defective STAT3 signaling results in reduced Th17 cells and an impaired IL-17/IL-22 response primarily due to a compromised canonical Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that involves STAT3 phosphorylation, dimerization, nuclear translocation, and gene transcription. The non-canonical pathway involving unphosphorylated STAT3 and its role in disease pathogenesis, however, is unexplored in HIES. This study aims to elucidate the role of unphosphorylated STAT3-unphosphorylated NF-κB (uSTAT3-uNF-κB) activation pathway in LOF STAT3 HIES patients. The mRNA expression of downstream molecules of unphosphorylated STAT3-unphosphorylated NF-κB pathway was studied in five LOF STAT3 HIES patients and transfected STAT3 mutants post-IL-6 stimulation. Immunoprecipitation assays were performed to assess the binding of STAT3 and NF-κB to RANTES promoter. A reduced expression of the downstream signaling molecules of the uSTAT3-uNF-κB complex pathway, viz., RANTES, STAT3, IL-6, IL-8, ICAM1, IL-8, ZFP36L2, CSF1, MRAS, and SOCS3, in LOF STAT3 HIES patients as well as the different STAT3 mutant plasmids was observed. Immunoprecipitation studies showed a reduced interaction of STAT3 and NF-κB to RANTES in HIES patients. The reduced expression of downstream signaling molecules, specially RANTES and STAT3, confirmed the impaired uSTAT3-uNF-κB pathway in STAT3 LOF HIES. Decreased levels of RANTES and STAT3 could be a significant component in the disease pathogenesis of Hyper IgE Syndrome.

Acetylcholine regulates the melanogenesis of retinal pigment epithelia cells via a cAMP-dependent pathway: A non-neuronal function of cholinergic system in retina

The turnover rate of melanogenesis in retinal pigment epithelium (RPE) and its molecular signaling remain unclear. This study aimed to investigate the role of cholinergic signaling in the process of melanogenesis of cultured RPE cells. Here, a human retinal pigment epithelia cell line, ARPE-19 cell, was used to study the process of melanogenesis. The mRNA and protein expressions of cholinergic molecules, e.g., acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and melanogenic molecules i.e., tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), and melanin pigment were measured during melanogenesis of cultured ARPE-19 cells. Forskolin (a cAMP inducing agent), acetylcholine (ACh) and bethanechol (Bch; a muscarinic AChR agonist) were used to induce melanogenesis in the cultures. Muscarinic acetylcholine receptor (mAChR) antagonists were employed to identify the receptor subtype. During melanogenesis of ARPE-19 cells, the mRNA and protein expressions of cholinergic molecules, e.g., AChE and BChE, were increased along with melanogenic molecules, i.e., TYR, MITF and melanin pigment. Forskolin, ACh, and Bch induced an upregulation of melanogenesis in cultured ARPE-19 cultures: the induction was parallel to an increase of AChE expression. The Bch-induced enzymatic activities and mRNA levels of AChE and TYR were fully blocked by the treatments of gallamine (a M2 specific antagonist), tropicamide (a M4 specific antagonist) and atropine (non-specific antagonist for mAChRs). Cholinergic signaling via M2/M4 mAChRs regulates melanogenesis in cultured ARPE-19 cells through a cAMP-dependent pathway. This study provides insights into the regulation of RPE cell melanogenesis via a non-neuronal function of cholinergic system.

Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts.

In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied. Experimental research. 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D). Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures. The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.

Immunomodulatory Effect of Cordyceps militaris Polysaccharide on RAW 264.7 Macrophages by Regulating MAPK Signaling Pathways.

Polysaccharide is one of the principal bioactive components found in medicinal mushrooms and has been proven to enhance host immunity. However, the possible mechanism of immunomodulatory activity of Cordyceps militaris polysaccharide is not fully understood. Hot water extraction and alcohol precipitation, DEAE-Sephadex A-25 chromatography, and Sephadex G-100 chromatography were used to isolate polysaccharide from C. militaris. A high-molecular-weight polysaccharide isolated from C. militaris was designated as HCMP, which had an Mw of 6.18 × 105 Da and was composed of arabinose, galactose, glucose, mannose, and xylose in a mole ratio of 2.00:8.01:72.54:15.98:1.02. The polysaccharide content of HCMP was 91.2% ± 0.16. The test in vitro showed that HCMP activated mouse macrophage RAW 264.7 cells by enhancing phagocytosis and NO production, and by regulating mRNA expressions of inflammation-related molecules in RAW 264.7 cells. Western blotting revealed that HCMP induced the phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, using inhibitors of MAPKs decreased the mRNA levels of inflammation-related molecules induced by HCMP. These data evidenced that the immunomodulatory effect of HCMP on RAW 264.7 macrophages was mediated via the MAPK signaling pathway. These findings suggested that HCMP could be developed as a potent immunomodulatory agent for use in functional foods and dietary supplements.

The stimulation of TH2 cells results in increased IL-5 and IL-13 production via the H4 receptor.

Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. Total CD4+ T cells obtained from healthy or AD individuals and invitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in invitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of invitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in invitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.

WTAP/YTHDF1-mediated m6A modification amplifies IFN-γ-induced immunosuppressive properties of human MSCs

IntroductionThe immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the “license” of several pro-inflammatory factors to express immunosuppressive molecular profiles, which determines the therapeutic efficacy of MSCs in immune-mediated inflammatory diseases. Of those, interferon-γ (IFN-γ) is a key inducer for the expression of immunosuppressive molecular profiles; however, the mechanism underlying this effect is unknown. ObjectivesTo elucidate the regulation mechanism and biological functions of N6-methyladenosine (m6A) modification in the immunosuppressive functions by the IFN-γ-licensing MSCs. MethodsEpitranscriptomic microarray analysis and MeRIP-qPCR assay were performed to identify the regulatory effect of WTAP in the IFN-γ-licensing MSCs. RIP-qPCR, western blot, qRT-PCR and RNA stability assays were used to determine the regulation of WTAP/m6A/YTHDF1 signaling axis in the expression of immunosuppressive molecules. Further, functional capacity of T cells was tested using flow cytometry, and both DSS-induced colitis mice and CIA mice were constructed to clarify the effect of WTAP and YTHDF1 in MSC-mediated immunosuppression. ResultsWe identified that IFN-γ increased the m6A methylation levels of immunosuppressive molecules, while WTAP deficiency abolished the IFN-γ-induced promotion of m6A modification. IFN-γ activated ERK signaling, which induced WTAP phosphorylation. Additionally, the stabilization of WTAP post-transcriptionally increased the mRNA expression of immunosuppressive molecules (IDO1, PD-L1, ICAM1, and VCAM1) in an m6A-YTHDF1-dependent manner; this effect further impacted the immunosuppressive capacity of IFN-γ licensing MSCs on activated T cells. Notably, WTAP/YTHDF1 overexpression enhanced the therapeutic efficacy of IFN-γ licensing MSCs and restructures the ecology of inflammation in both colitis and arthritis models. ConclusionOur results showed that m6A modification of IDO1, PD-L1, ICAM1, and VCAM1 mRNA mediated by WTAP-YTHDF1 is involved in the regulation of IFN-γ licensing MSCs immunosuppressive abilities, and shed a light to enhance the clinical therapeutic potential of IFN-γ-licensing MSCs.

Antioxidant Vitamins (Vit C and E) Protect the Kidney from Glyphosate- Exposed Renal Damage in Experimental Rats - A Molecular Study

Glyphosate is a widely used herbicide and the active ingredient in Monsanto's Roundup, among other herbicide formulations. Vitamin C (ascorbic acid) and vitamin E (tocopherol) are both antioxidants that play important roles in reducing oxidative DNA damage and protecting cells from the harmful effects of free radicals. The study aimed to analyse the effect of antioxidant vitamins (vitamins C and E) on the expression of IGF-1 and KIM-1 molecules in the kidneys of glyphosate-exposed rats. Adult male Albino Wistar rats were grouped into 3. Group I: Control; Group II: Glyphosate treated (100mg); Group III: Glyphosate+ vitamin E & C treated. Serum, insulin, and mRNA expression of insulin-like growth factor (IGF-1) and kidney injury molecules (KIM-1) mRNA expression by RT-PCR. Serum insulin was measured by the ELISA method. Glyphosate-exposed rats developed hyperglycaemia and hyper compared to control but antioxidant vitamins (Vitamin C and E) supplementation, reduced the glyphosate mediate increased fasting blood glucose and insulin near to that of the control level. Similarly, mRNA expression of both IGF-1 and KIM-1 were also significantly altered due to glyphosate induction whose effects were found to be reduced when vitamins C and E were treated. It is concluded that glyphosate exposure causes renal damage and develops diabetic nephropathy by modulating the expression of IGF-1 and KIM-1 molecules in the kidney. Vitamin C and E potentially were able to reduce the detrimental changes caused by glyphosate. Hence, vitamin C and E supplements could be considered therapeutic drugs for diabetic nephropathy.

Effects of porcine epidemic diarrhea virus infection on CD21+ B cells activation

Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high morbidity and mortality among neonatal piglets. Humoral immunity plays a vital role in the host anti-PEDV infection process, but the mechanism of PEDV-induced B-cell immune response remains unknown. In this study, the effects of PEDV infection on CD21+ B cell activation were systematically analyzed through animal experiments. Enzyme-linked immunosorbent assays (ELISA) revealed that low levels of serum-specific IgA, IgM, or IgG were detected in piglets after PEDV infection, respectively. Serum interleukin (IL)-6 levels increased significantly at 4 d after infection, and the levels of IL-4, B-cell activating factor (BAFF), interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10 decreased at 7 d after infection. Fluorescence-activated cell sorting (FACS) showed that expression levels of CD21, MHC Ⅱ, CD40, and CD38 on B cell surfaces were significantly higher. In contrast, the proportions of CD21+IgM+ B cells were decreased in peripheral blood mononuclear cells (PBMCs) from the infected piglets. No differences were found in the percentage of CD21+CD80+ and CD21+CD27+ B cells in PBMCs from the infected piglets. In addition, the number of CD21+B cells in PBMCs stimulated with PEDV in vitro was significantly lower. No significant change in the mRNA expression of BCR molecules was found while the expression levels of paired immunoglobulin-like receptor B (PIR-B), B cell adaptor molecule of 32 kDa (Bam32) and BAFF were decreased. In conclusion, our research demonstrates that virulent strains of PEDV profoundly impact B cell activation, leading to alterations in phenotypic expression and BCR signaling molecules. Furthermore, this dysregulation results in compromised specific antibody secretion and perturbed cytokine production, highlighting the intricate immunological dysfunctions induced by PEDV infection.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

SRPK1 Promotes Glioma Proliferation, Migration, and Invasion through Activation of Wnt/β-Catenin and JAK-2/STAT-3 Signaling Pathways.

Currently, the treatment of gliomas still relies primarily on surgery and radiochemotherapy. Although there are various drugs available, including temozolomide, the overall therapeutic effect is unsatisfactory, and the prognosis remains poor. Therefore, the in-depth study of the mechanism of glioma development and a search for new therapeutic targets are the keys to improving the therapeutic treatment of gliomas and improving the prognosis of patients. Immunohistochemistry is used to detect the expression of relevant molecules in tissues, qPCR and Western blot are used to detect the mRNA and protein expression of relevant molecules, CCK-8 (Cell Counting Kit-8) is used to assess cell viability and proliferation capacity, Transwell is used to evaluate cell migration and invasion ability, and RNA transcriptome sequencing is used to identify the most influential pathways. SRPK1 (SRSF protein kinase 1) is highly expressed in gliomas but is not expressed in normal tissues. Its expression is positively correlated with the grades of gliomas and negatively correlated with prognosis. SRPK1 significantly promotes the occurrence and development of gliomas. Knocking down SRPK1 leads to a significant decrease in the proliferation, migration, and invasion abilities of gliomas. Loss of SRPK1 expression induces G2/M phase arrest and mitotic catastrophe, leading to apoptosis in cells. Overexpression of SRPK1 activates the Wnt/β-catenin (wingless-int1/β-catenin) and JAK-2/STAT-3 (Janus kinase 2/signal transducer and activator of transcription 3) signaling pathways, promoting the proliferation, migration, and invasion of gliomas. Overexpression of SRPK1 rescues the reduced cell proliferation, migration, and invasion abilities caused by the silencing of β-catenin or JAK-2. A stable shRNA-LN229 cell line was constructed, and using a nude mouse model, it was found that stable knockout of SRPK1 significantly reduced the tumorigenic ability of glioma cells, as evidenced by a significant decrease in the subcutaneous tumor volume and weight in nude mice. We have demonstrated that SRPK1 is highly expressed in gliomas. Overexpression of SRPK1 activates the Wnt/β-catenin and JAK-2/STAT-3 signaling pathways, promoting the proliferation, migration, and invasion of gliomas. Silencing SRPK1-related signaling pathways may provide potential therapeutic options for glioma patients.

Nutritional and physiological responses of female and male largemouth bass (Micropterus salmoides) to different dietary starch levels

In this study, we adopted a 2 × 2 factorial design incorporating different dietary starch levels and sexes to assess the impact of dietary starch on the growth performance, feeding response, and glucolipid metabolism of female and male largemouth bass. Two isonitrogenous (49.6%) and isolipidic (10.4%) diets were formulated with 10% starch (low-starch level) and 20% starch (high-starch level). The results indicated that male largemouth bass exhibited superior growth performance compared to females regardless of the dietary starch levels. Furthermore, males demonstrated enhanced feed utilization of the high-starch diet compared to females. Males also exhibited significantly lower mRNA expressions of feeding inhibition molecules (cholecystokinin, leptin b, leptin b receptor, or cocaine and amphetamine-regulated transcript 2) in the brain compared to females, a phenomenon unrelated to dietary starch levels. Compared to females, males fed the high-starch diet displayed elevated mRNA expression of genes associated with glycolysis (hexokinase, pyruvate kinase, and phosphofructokinase) in the liver, as well as lower expression of gluconeogenesis-related genes (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase), which coincided with lower hepatic glycogen in males. Furthermore, males fed with the high-starch diet exhibited higher mRNA expression of genes involved in hepatic lipid catabolism (carnitine palmitoyl transferase 1, hormone-sensitive lipase, and adipose triglyceride lipase). In contrast, males exhibited lower expression of hepatic lipid synthesis regulation (acetyl-CoA carboxylase 1, peroxisome proliferator-activated receptor γ, and fatty acid synthase) in the liver compared with females fed with the low-starch diet. Additionally, upon comparing the hepatic transcriptomes of males and females fed with the high-starch diet, the males exhibited a significant up-regulation of glucokinase2 and hexokinase, coupled with down-regulation of glucose-6-phosphatase. In summary, male largemouth bass exhibited superior growth performance at this developmental stage compared to females regardless of dietary starch levels. Males also exhibited higher feed utilization when fed the high-starch diet, which was likely due to increased activity in glycolytic processes and enhanced fatty acid decomposition compared to females.

Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

Euphorbia factorL2 suppresses the generation of liver metastatic ascites in breast cancer via inhibiting NLRP3 inflammasome activation.

Metastasis is the leading cause of death in patients with breast cancer, in part due to the lack of effective treatments. Euphorbia factorL2 (EFL2) is a diterpenoid extracted from Euphorbia lathyris L. seeds, which has attracted increasing attention in recent years due to its anticancer effect. However, the role and molecular mechanism of EFL2 in breast cancer liver metastasis remain unclear. In the present study, a breast cancer liver metastasis model was constructed and the effect of EFL2 on ascites generation in mice was examined. H&E staining detected inflammatory cells and tumor cells in the liver, small intestine and tumor tissues. Western blotting and reverse transcription‑quantitative PCR were used to detect the protein and mRNA expression of NLR family pyrin domain containing‑3 (NLRP3) and related molecules in tumor tissues. Immunohistochemistry was used to detect the levels of CD4 and CD8 Tcells in tumor tissue and immunofluorescence was used to further detect the expression level of NLRP3. Finally, the aforementioned experiments were further verified by overexpressing NLPR3. It was found that EFL2 inhibited generation of ascites in the model in a dose‑dependent manner. Furthermore, EFL2 inhibited tumor cell metastasis and enhanced immune cell infiltration. Meanwhile, EFL2 dose‑dependently downregulated the mRNA and protein expression of NLRP3 and related molecules in the model, and overexpression of NLRP3 abolished these beneficial effects of EFL2. Taken together, the present experimental data suggested that EFL2 has a significant inhibitory effect on ascites of breast cancer liver metastasis invivo, which may inhibit tumor cell metastasis by downregulating NLRP3 expression, providing an experimental basis for treating breast cancer metastasis.

Non-canonical NFKB signaling endows suppressive function through FOXP3-dependent regulatory T cell program

Regulatory T cells (Tregs) play a central role in modulating adaptive immune responses in humans and mice. The precise biological role of non-canonical nuclear factor ‘κ-light-chain-enhancer’ of activated B cells (NFKB) signaling in human Tregs has yet to be fully elucidated. To gain insight into this process, a Treg-like cell line (MT-2) was genetically modified using CRISPR/Cas9. Interestingly, NFKB2 knockout MT-2 cells exhibited downregulation of FOXP3, while NFKB1 knockout did not. Additionally, mRNA expression of FOXP3-dependent molecules was significantly reduced in NFKB2 knockout MT-2 cells. To better understand the functional role of the NFKB signaling, the NFKB1/NFKB2 loci of human primary Tregs were genetically edited using CRISPR/Cas9. Similar to MT-2 cells, NFKB2 knockout human Tregs displayed significantly reduced FOXP3 expression. Furthermore, NFKB2 knockout human Tregs showed downregulation of FOXP3-dependent molecules and a diminished suppressive function compared to wild-type and NFKB1 knockout Tregs. These findings indicate that non-canonical NFKB signaling maintains a Treg-like phenotype and suppressive function in human Tregs through the FOXP3-dependent regulatory T cell program.

Steamed Ginseng Berry Powder Ameliorates Skeletal Muscle Atrophy via Myogenic Effects.

Sarcopenia is an age-related loss of muscle mass and function for which there is no approved pharmacological treatment. We tested direct efficacy by evaluating grip strength improvement in a sarcopenia mouse model rather than drug screening, which inhibits specific molecular mechanisms. Various physiological functions of ginseng berries are beneficial to the human body. The present study aimed to evaluate the efficacy and safety of steamed ginseng berry powder (SGBP). SGBP administration increased myotube diameter and suppressed the mRNA expression of sarcopenia-inducing molecules. SGBP also reduced the levels of inflammatory transcription factors and cytokines that are known to induce sarcopenia. Oral administration of SGBP improved muscle mass and physical performance in a mouse model of sarcopenia. In summary, our data suggest that SGBP is a novel therapeutic candidate for the amelioration of muscle weakness, including sarcopenia.

Effects of chitinase-1 inhibitor in obesity-induced and -aggravated asthma in a murine model

AimsDespite recent investigations on the role of chitinase in asthma, its role in obesity-induced asthma has not been evaluated. Therefore, we investigated the roles of chitin, chitinase-1, and a chitinase-1 inhibitor (compound X, CPX) in a murine model. Main methodsWe assigned C57BL/6 mice to the ovalbumin (OVA) model or obesity model group. In the OVA model, mice received intraperitoneal OVA twice within a 2-week interval and intranasal OVA for 3 consecutive days. Additionally, chitin was intranasally administered for 3 consecutive days, and CPX was intraperitoneally injected three times over 5 days. In the obesity model, a high-fat diet (HFD) was maintained for 13 weeks, and CPX was intraperitoneally injected eight times over 4 weeks. Key findingsIn the OVA model, chitin aggravated OVA-induced airway hyper-responsiveness (AHR), increased bronchoalveolar lavage fluid (BALF) cell proliferation, increased fibrosis, and increased the levels of various inflammatory cytokines (including chitinase-1, TGF-β, TNF-α, IL-1 β, IL-6, IL-4, and IL-13). CPX treatment significantly ameliorated these effects. In the obesity model, HFD significantly increased AHR, BALF cell proliferation, fibrosis, and the levels of various inflammatory cytokines. Particularly, compared to the control group, the mRNA expression of chitinase, chitinase-like molecules, and other molecules associated with inflammation and the immune system was significantly upregulated in the HFD and HFD/OVA groups. Immunofluorescence analysis also showed increased chitinase-1 expression in these groups. CPX significantly ameliorated all these effects in this model. SignificanceThis study showed that CPX can be an effective therapeutic agent in asthma, especially, obesity-induced and -aggravated asthma to protect against the progression to airway remodeling and fibrosis.

Dienogest does not augment the gene expression of adhesion molecules, MCP-1, and monocyte adherence in human endothelial cells

Objective Medroxyprogesterone acetate (MPA) may increase the risk of atherosclerosis during hormone replacement therapy (HRT); therefore, the effect of progestogens other than MPA on atherosclerotic lesions requires evaluation. Adhesion of monocytes to vascular endothelial cells is an important early step in atherosclerosis progression. MCP-1 is a key chemokine that promotes monocyte migration and adhesion to vascular endothelial cells. In this study, we investigated the effects of dienogest (DNG), an alternative progestogen, on monocyte adhesion and cytokine expression in human umbilical vein endothelial cells (HUVECs). Study Design HUVECs were treated with DNG, natural progesterone, or MPA, followed by interleukin (IL)-1β stimulation. The mRNA expression of adhesion molecules (E-selectin and ICAM-1) and cytokines (MCP-1 and IL-6) was examined using real-time PCR. A flow chamber system was used to examine the effect of DNG on the adhesion of U937 monocytic cells to monolayer HUVECs. Results Unlike MPA, DNG did not alter the mRNA expression of E-selectin, ICAM-1, MCP-1, and IL-6 in HUVECs. Moreover, it did not increase the number of monocytes adhering to HUVECs in the flow chamber system. However, MPA treatment significantly enhanced monocyte adhesion to HUVECs (p < 0.05). Conclusions DNG had no effect on the mRNA expression of adhesion molecules and cytokines in HUVECs, as well as the monocyte adhesion to HUVECs, suggesting that DNG can be explored as an alternative to MPA for HRT.

Inhibitory checkpoint molecule mRNA expression in canine soft tissue sarcoma.

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy.