SP1 mediates OGD/R-induced cardiomyocyte injury via enhancing the transcription of USP46.

BACKGROUND: One of the mechanisms responsible for the high mortality rate of acute myocardial infarction is myocardial ischemia-reperfusion injury (MI-RI). The present study focused on the role and regulatory mechanisms of specificity protein 1 (SP1) and ubiquitin-specific protease 46 (USP46) in oxygen-glucose deprivation/reperfusion (OGD/R)-induced cardiomyocyte injury. METHODS: OGD/R was used to treat cardiomyocytes AC16 to mimic ischemia-reperfusion in vitro. Cell viability, proliferation, and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine, and flow cytometry assays. Enzyme-linked immunosorbent assays analyzed the concentrations of TNF-α and IL-1ß. Several protein levels were analyzed by western blotting. The levels of iron (Fe2+), reactive oxygen species, malondialdehyde, and the activities of superoxide dismutase were analyzed by commercial kits. Chromatin immunoprecipitation and dual-luciferase report assays assessed the relationship between USP46 and SP1. RESULTS: USP46 and SP1 were upregulated in serum from MI patients and they had a positive correlation. OGD/R stimulation suppressed cardiomyocyte viability and proliferation, as well as induced cardiomyocyte inflammation, oxidative stress (OxS) injury, apoptosis, and ferroptosis, but these effects were impaired by USP46 or SP1 knockdown. SP1 could enhance the transcription of USP46, and USP46 overexpression reversed SP1 silencing-mediated effects on OGD/R-induced cardiomyocytes. SP1 mediated the AMPK signaling via USP46. CONCLUSION: SP1 mediated OGD/R-induced cardiomyocyte inflammation, OxS injury, apoptosis, and ferroptosis by inactivating the AMPK signaling via enhancing the transcription of USP46.

Central blockage of sympathetic nerves inhibits the abnormal vital signs and disturbance of the gut microbiota caused by continuous light exposure.

Background: Continuous light exposure increases sympathetic excitation in rats, leading to hypertension, left ventricular hypertrophy, and fibrosis. This study was aimed to investigate whether continuous light exposure causes destabilization of vital signs and gut microbiota (GM) in Sprague Dawley (SD) rats and whether clonidine hydrochloride (CH), a central sympathetic depressant drug, could prevent these changes. Methods: Eight-week-old male SD rats were divided into three groups with different interventions for 14 weeks: control group (CG), 2-mL pure water gavaged daily while on a normal 12-h light/dark cycle; continuous illumination group (CI), 2-mL pure water gavaged daily while receiving continuous exposure to light (300 lx); and drug administration group (DA), CH (10 µg/kg) gavaged daily while receiving continuous exposure to light (300 lx). Results: The results showed that blood pressure, heart rate, and body weight were significantly higher in the CI group than in the CG and DA groups (P < 0.05). Moreover, the Shannon index was higher in the DA group than in the CI group (P = 0.012). The beta diversity index in the CG group was significantly higher in the CI group (P = 0.039). The pairwise comparison results of the linear discriminant analysis effect size showed that Oscillospirales were enriched in the DA group, whereas the Prevotellaceae lineage (family level) > Prevotella (genus level) > Prevotellaceae_bacterium (species level) were enriched in the CI group. The Muribaculaceae family was more abundant in the CG group than in the CI group. Conclusion: Sympathetic nerve inhibition restored the abnormal vital signs and GM changes under continuous light exposure.

Lupeol Alleviates Myocardial Ischemia-Reperfusion Injury in Rats by Regulating NF-[Formula: see text]B and Nrf2 Pathways.

Cardiovascular disease is a global health problem. Previous studies revealed that it involves acute myocardial infarction and ischemia-reperfusion (I/R) injury. The mechanism of myocardial I/R injury is complex. But recognizing its mechanisms will bring important clinical significance. Lupeol is widely found in Chinese medicinal herbs and has been shown to have a variety of bio-activities. However, the pharmacological action of lupeol in the progress of myocardial ischemia-reperfusion injury (MIRI) is unclear. This study used a rat myocardial I/R model and the morphological changes in myocardium were determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The expression levels of IL-10, IL-1[Formula: see text], TNF-[Formula: see text], and IL-6 were assessed by quantitative real-time PCR (qRT-PCR) and ELISA. The expression levels of MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) level and inflammatory cytokines were quantified using ELISA. The cellular apoptotic rate was determined by TUNEL staining. The findings showed that lupeol significantly decreased myocardial infarction after I/R and ameliorated I/R-induced myocardial inflammation, apoptosis, and oxidative stress. Furthermore, our results suggested that lupeol protected against MIRI-induced myocardial infarction through modulation of NF-[Formula: see text]B and Nrf2 signaling pathways. In summary, this study first clarified the cardioprotective effects of lupeol against I/R-induced myocardial infarction in rats, which could be due to its anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Our study also highlighted a mechanism of NF-[Formula: see text]B and Nrf2 signaling, through which lupeol could be a promising agent in protecting against I/R-induced myocardial infarction.

Protocatechualdehyde inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclastogenesis and attenuates lipopolysaccharide-induced inflammatory osteolysis.

Inflammatory osteolysis as a consequence of chronic bacterial infection underlies several lytic bone conditions, such as otitis media, osteomyelitis, septic arthritis, periodontitis, periprosthetic infection, and aseptic loosening of orthopedic implants. In consideration of the lack of effective preventive or treatments options against infectious osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. The present study assessed the effect of protocatechualdehyde (PCA), a natural occurring polyphenolic compound with diverse biological activities including but not limited to antibacterial and antiinflammatory properties, on nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone loss in vivo. In the present study, it was found that PCA potently inhibited RANKL-induced osteoclast formation, fusion, and activation toward bone resorption in a dose-dependent manner via the suppression of the ERK/c-Fos/nuclear factor of activated T-cells, cytoplasmic 1 signaling axis. It was further demonstrated that the in vivo administration of PCA could effectively protect mice against the deleterious effects of LPS-induced calvarial bone destruction by attenuating osteoclast formation and activity in a dose-dependent manner. Collectively, these findings provided evidence for the potential therapeutic application of PCA in the prevention and treatment of infectious osteolytic conditions, and potentially other osteoclast-mediated bone diseases.

GST null polymorphisms may affect the risk of coronary artery disease: evidence from a meta-analysis.

BACKGROUND: Whether glutathione S-transferase (GST) null polymorphisms, namely GSTM1 null, GSTP1 null and GSTT1 null polymorphisms, influence the risk of coronary artery disease (CAD) or not remains unclear. Thus, the authors performed a meta-analysis to more robustly estimate associations between GST null polymorphisms and the risk of CAD by integrating the results of previous publications. METHODS: Medline, Embase, Wanfang, VIP and CNKI were searched comprehensively for eligible studies, and 45 genetic association studies were finally selected to be included in this meta-analysis. RESULTS: We found that GSTM1 null polymorphism was significantly associated with the risk of CAD in overall population (OR = 1.37, p = 0.003) and mixed population (OR = 1.61, p = 0.004), GSTP1 null polymorphism was significantly associated with the risk of CAD in overall population (OR = 1.23, p = 0.03), whereas GSTT1 null polymorphism was significantly associated with the risk of CAD in overall population (OR = 1.23, p = 0.02), Caucasians (OR = 1.23, p = 0.02) and East Asians (OR = 1.38, p < 0.0001). CONCLUSIONS: This meta-analysis demonstrated that GSTM1 null, GSTP1 null and GSTT1 null polymorphisms were all significantly associated with an increased risk of CAD.

ETS2 and microRNA-155 regulate the pathogenesis of heart failure through targeting and regulating GPR18 expression.

Heart failure (HF) is a global pandemic cardiovascular disease with increasing prevalence, but the pathogenesis remains to be elucidated. The present study aimed to investigate the underlying mechanism in heart failure (HF) using bioinformatics and experimental validation. A HF-associated dataset GSE84796 was downloaded from the Gene Expression Omnibus database and differentially expressed genes (DEGs) were screened for using Bayes method in the Limma package. Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to perform pathway enrichment analysis of these DEGs using The Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network of DEG-encoded proteins was subsequently constructed using the Search tool for the Retrieval of Interacting Genes/Proteins, and a transcription factor (TF)/miRNA-target network was constructed according to the WEB-based Gene SeT AnaLysis Tookit. The expression levels of microRNA (miRNA/miR)-155, G-protein coupled receptor 18 (GRP18) and E26 transformation-specific transcription factor 2 (ETS2) were analyzed in clinical HF samples, and functional validations were performed in H9c2 (2-1) cells. A total of 419 DEGs were identified, including 366 upregulated genes and 53 downregulated genes. The upregulated DEGs were significantly enriched in the pathways of 'cytokine-cytokine receptor interaction', 'natural killer cell mediated cytotoxicity' and 'primary immunodeficiency'. A total of two functional modules were identified in the PPI network: Module A was enriched in 3 KEGG pathways and module B was enriched in 15 KEGG pathways. Furthermore, a total of three miRNAs and eight TFs were identified in the TF/miRNA-target network. Specifically, GPR18 was discovered to be targeted by both ETS2 and miR-155. Clinical validation revealed that the expression levels of miR-155 were significantly decreased in the HF samples, whereas the expression levels of ETS2 and GPR18 were significantly increased in HF samples. In conclusion, the present study suggested that GPR18 may be a target of ETS2 and miR-155, and miR-155 may regulate cell viability and apoptosis in H9c2 (2-1) cells through targeting and regulating GPR18.

Novel amphiphilic polymeric ionic liquid-solid phase micro-extraction membrane for the preconcentration of aniline as degradation product of azo dye Orange G under sonication by liquid chromatography-tandem mass spectrometry.

A novel amphiphilic polymeric ionic liquid membrane containing a hydrophilic bromide anion and a hydrophobic carbonyl group was synthesized in dimethylformamide (DMF) systems using the ionic liquid 1-butyl-3-vinylimidazolium bromide (BVImBr) and the methylmethacrylate (MMA) as monomers. The prepared amphiphilic ploy-methylmethacrylate-1-butyl-3-vinylimidazolium bromide (MMA-BVImBr) was characterized by a scanning electron microscope and an infrared spectrum instrument. The results of solid-phase micro-extraction membrane (SPMM) experiments showed that the adsorption capacity of membrane was about 0.76µgµg(-1) for aniline. Based on this, a sensitive method for the determination of trace aniline, as a degradation product of azo dye Orange G under sonication, was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The calibration curve showed a good linearity ranging from 0.5 to 10.0µgL(-1) with a correlation coefficient value of 0.9998. The limit of quantification was 0.5µgL(-1). The recoveries ranged from 90.6% to 96.1%. The intra- and inter-day relative standard deviations were less than 8.3% and 10.9%. The developed SPMM-LC-MS/MS method was used successfully for preconcentration of trace aniline produced during the sonication of Orange G solution.

[Effects of FOXP3 on the progression of atherosclerosis plaque in ApoE-knock out mice].

AIM: To explore effects of FOXP3 on the progression of atherosclerosis plaque in hypercholesterolemic apoliprotein(apo)E-/- mice. METHODS: At 8 weeks of age, 32 male ApoE-/- mice were randomly divided into four groups of eight. Labeled: negative control group, positive control group, small interfering RNA (siRNA) group, and regulatory T cells transfer (Tregs) group. Lentivirus-mediated (siRNA) identified its function by Western blot was used to knock down FOXP3 and Foxp3(high+);CD4(+); CD25(+); Tregs acquired through magnetic activated cell sorting adoptive transfer assays in high fat diet ApoE-/- mice were done. The resulting atherosclerotic lesions were assessed by determining the number and function of CD4(+);CD25(+); Tregs, FOXP3 transcript levels and investigating the expression of Foxp3 protein in different tissues. Inflammatory cytokines were determined by ELISA. RESULTS: Animals treated with siRNA of FOXP3 showed a significant increase in atherosclerotic lesion formation and a reduction in the number and function of Foxp3(+);CD4(+);CD25(+); Tregs compared with other groups. Transfer of Foxp3(high+);CD4(+);CD25(+); Tregs significantly decreased atherosclerotic plaque formation and increased the number and function of Foxp3(+); CD4(+); CD25(+); Tregs. Foxp3 protein levels and FOXP3 transcript levels were lowest in the siRNA group, and were highest in tissues from the Tregs transfer group. CONCLUSION: FOXP3 plays an important role in regulating the inflammatory response within the atherosclerotic lesion. It can inhibit significant the progression of the atherosclerosis plaque in ApoE-/- mice.

CD4+CD25+Foxp3+ regulatory T cells protect the proinflammatory activation of human umbilical vein endothelial cells.

OBJECTIVE: To investigate the role of CD4(+)CD25(+)forkhead box P 3 (Foxp3)(+) T-regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and the mechanisms of it. METHODS AND RESULTS: ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4(+)CD25(-) T cells in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule, such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide. Moreover, Tregs downregulate proinflammatory factor nuclear factor-κB activation and induce resistance to suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus. Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth factor [TGF]-ß). CONCLUSIONS: Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving cytotoxic T-lymphocyte antigen-4-dependent cell-to-cell contact and also requiring soluble factors (mainly interleukin 10 and TGF-ß).

Oxidized low-density lipoprotein-induced proinflammatory cytokine response in macrophages are suppressed by CD4CD25(+)Foxp3(+) regulatory T cells through downregulating toll like receptor 2-mediated activation of NF-kappaB.

CD4(+)CD25(+) regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect oxLDL-induced proinflammatory response in macrophages. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxLDL for 48 hours to induce proinflammatory response. Our data showed that with oxLDL challenge, the Treg-modulated macrophages have decreased NO production and iNOS expression, decreased HLA-DR and CD86 expression, and down-regulated proinflammatory cytokine/chemokine production. Tregs can inhibit the pro-inflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that Treg-mediated suppression of the monocyte response to oxLDL was reflected by a reduction in the up-regulation of NF-kappaB activity accompanied by a decreased expression of TLR2 but not TLR4 at the transcriptional level. These results suggest that CD4(+)CD25(+)Foxp3(+) regulatory T cells may exert its suppressive functions on pro-inflammatory properties of OxLDL induced-macrophages partly through TLR2-NF-kappaB signaling pathway.

The role of CD4+CD25+ regulatory T cells in macrophage-derived foam-cell formation.

Cluster of differentiation (CD)4+CD25+ regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis, but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect macrophages foam-cell formation. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxidized LDL (oxLDL) for 48 h to transform foam cells. After co-culture with Tregs, macrophages showed a decrease in lipid accumulation, which was accompanied by a significantly downregulated expression of CD36 and SRA but no obvious difference in ABCA1 expression. Tregs can inhibit the proinflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that both cell-to-cell contact and soluble factors are required for Treg-mediated suppression on macrophage foam-cell formation. Cytokines, interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta) are the key factors for these suppressive functions.