Polystyrene nanoplastics and cadmium co-exposure aggravated cardiomyocyte damage in mice by regulating PANoptosis pathway.

Micro/nanoplastics (M/NPs) are the novel contaminants ubiquitous in the environment. Cadmium (Cd), a kind of heavy metal pollutant widely distributed, could potentially co-exist with PS-NPs in the environment. However, their combined effects on cardiomyocyte and its molecular mechanism in mammals remained ambiguous. Here, we examined whether PANoptosis, an emerging and complicated kind of programmed cell death, was involved in PS-NPs and Cd co-exposure-elicited cardiac injury. In this study, 60 male mice were orally subjected to environmentally relevant concentrations of PS-NPs (1 mg/kg) and/or CdCl2 (1.5 mg/kg) for 35 days. As we speculated, PS-NPs and Cd co-exposure affected the expression of pyroptosis(Caspase-1, Cleaved-Caspase-1, GSDMD, N-GSDMD, AIM2, Pyrin, NLRP3, IL-18, IL-1ß)-, apoptosis(Caspase-3, Cleaved-Caspase-3, Caspase-8, Cleaved-Caspase-8, Caspase-7, BAX)- and necroptosis (t-RIPK3, p-RIPK3, t-RIPK1, p-RIPK1, t-MLKL, p-MLKL, ZBP1)-related genes and protein, resulting in growth restriction and damaged myocardial microstructure in mice. Notably, the combined effects on Cd and PS-NPs even predominantly aggravated the toxic damage. Intriguingly, we fortuitously discovered PS-NPs and/or Cd exposure facilitated linear ubiquitination of certain proteins in mice myocardium. In summation, this study shed light toward the effects of Cd and PS-NPs on cardiotoxicity, advanced the understanding of myocardial PANoptosis and provided a scientific foundation for further exploration of the combined toxicological effects of PS-NPs and heavy metals.

N-acetylcysteine combined with insulin attenuates myocardial injury in canines with type 1 diabetes mellitus by modulating TNF-α-mediated apoptotic pathways and affecting linear ubiquitination.

The exact pathogenesis of type 1 diabetes mellitus (DM) is still unclear. Numerous organs, including the heart, will suffer damage and malfunction as a result of long-term hyperglycemia. Currently, insulin therapy alone is still not the best treatment for type 1 DM. In order to properly treat and manage patients with type 1 DM, it is vital to seek a combination that includes both insulin and additional medications. This study aims to explore the therapeutic effect and mechanism of N-acetylcysteine (NAC) combined with insulin on type 1 DM. By giving beagle canines injections of streptozotocin (STZ) and alloxan (ALX) (20 mg/kg each), a model of type 1 DM was created. The results showed that this combination could effectively control blood sugar level, improve heart function, avoid the damage of mitochondria and myocardial cells, and prevent the excessive apoptosis of myocardial cells. Importantly, the combination can activate nuclear factor kappa-B (NF-κB) by promoting linear ubiquitination of receptor-interacting protein kinase 1 (RIPK1) and NF-κB-essential modulator (NEMO) and inhibitor of NF-κB (IκB) phosphorylation. The combination can increase the transcription and linear ubiquitination of Cellular FLICE (FADD-like IL-1ß-converting enzyme) -inhibitory protein (c-FLIP), diminish the production of cleaved-caspase-8 p18 and cleaved-caspase-3 to reduce apoptosis. This study confirmed that NAC combined with insulin can promote the linear ubiquitination of RIPK1, NEMO and c-FLIP and regulate the apoptosis pathway mediated by TNF-α to attenuate the myocardial injury caused by type 1 DM. Meanwhile, the research served as a resource when choosing a clinical strategy for DM cardiac complications.

Ginsenoside Rb1 inhibits free fatty acids­induced oxidative stress and inflammation in 3T3­L1 adipocytes.

Free fatty acids (FFAs) increase in visceral fat and are inferred to be one of the underlying inducers of adipose tissue inflammation. In our previous study, it was demonstrated that ginsenoside Rb1 stimulates endothelial nitric oxide synthase (eNOS) and Sirtuin 1 to protect against endothelial cell senescence. In the present study, 3T3­L1 adipocytes were exposed to 0.5 mM FFAs with or without Rb1 (10­40 µM). Monocyte chemotactic protein­1 (MCP­1) and interleukin­6 (IL­6) secretion was measured using ELISA. Tumor necrosis factor­α (TNF­α) expression and nuclear factor­κB (NF­κB) p65 phosphorylation were detected using western blot analysis. Oxidative stress was determined via measuring intracellular reactive oxygen species (ROS) and nitric oxide (NO) production. The results demonstrated that MCP­1 and IL­6 secretion, as well as TNF­α expression, were significantly increased following FFA treatment, which was attenuated by Rb1 in a dose­dependent manner. Furthermore, Rb1 attenuated FFA­induced NF­κB phosphorylation, suggesting that the inhibitory effect of Rb1 on inflammatory cytokines was partially mediated through blockade of NF­κB phosphorylation. Further experiments demonstrated that Rb1 ameliorated FFA­induced ROS generation and NO reduction through upregulation of superoxide dismutase 2 and eNOS expression. Taken together, these results demonstrate proinflammatory and pro­oxidant effects of FFA on 3T3­L1 adipocytes, which are effectively ameliorated by Rb1. Suppression of inflammatory responses and oxidative stress may be a novel mechanism for attenuating the effect of Rb1 on adipocyte dysfunction.

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

Relationship between arterial stiffness assessed by brachial-ankle pulse wave velocity and coronary artery disease severity assessed by the SYNTAX score.

AIM: The SYNTAX score is a semi-quantitative angiographic tool used to determine the extent and severity of coronary artery disease (CAD). Automatic computer-assisted measurement of the brachial-ankle pulse wave velocity (baPWV) is a reproducible and valid method by which to assess arterial stiffness. Limited information is available for the association between the SYNTAX score and arterial stiffness in a CAD patient. We aim to assess the association between arterial stiffness determined by PWV and CAD severity assessed by angiography and the SYNTAX score. METHODS: 321 subjects underwent measurement of both baPWV and angiography from 2010 to 2011. BaPWV was divided into tertiles. Multiple logistic and linear regression analyses were used to evaluate the relationship between the SYNTAX score and baPWV. RESULTS: After adjusting for age, body mass index, smoking habits, family history of CAD, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), total cholesterol, triglycerides, LDL-cholesterol, fasting glucose, serum creatinine, uric acid and cysteine proteinase, analyses revealed that baPWV groups were significantly associated with the SYNTAX score. Compared with the lowest baPWV tertile, the adjusted odds ratios (ORs) of having a SYNTAXHIG for the MID and HIG baPWV tertiles were 4.76 (95% confidence interval: 1.71-6.33) and 4.13 (95% confidence interval: 1.12-5.27), respectively. We also used multiple linear regression analyses to assess the association between baPWV and the SYNTAX score, which showed that baPWV was associated with the SYNTAX score. CONCLUSION: Arterial stiffness determined by PWV is related to CAD severity assessed by angiography and the SYNTAX score.

Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in microglia cells.

Microglial activation plays an important role in neuroinflammation, which contributes to neuronal damage, and inhibition of microglial activation may have therapeutic benefits that could alleviate the progression of neurodegeneration. Recent studies have indicated that the antimalarial agent artemisinin has the ability to inhibit NF-κB activation. In this study, the inhibitory effects of artemisinin on the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated primary microglia. Our results show that artemisinin significantly inhibited LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO). Artemisinin significantly decreased both the mRNA and the protein levels of these pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) and increased the protein levels of IκB-α, which forms a cytoplasmic inactive complex with the p65-p50 heterodimeric complex. Artemisinin treatment significantly inhibited basal and LPS-induced migration of BV-2 microglia. Electrophoretic mobility shift assays revealed increased NF-κB binding activity in LPS-stimulated primary microglia, and this increase could be prevented by artemisinin. The inhibitory effects of artemisinin on LPS-stimulated microglia were blocked after IκB-α was silenced with IκB-α siRNA. Our results suggest that artemisinin is able to inhibit neuroinflammation by interfering with NF-κB signaling. The data provide direct evidence of the potential application of artemisinin for the treatment of neuroinflammatory diseases.

Predictors of clinical SYNTAX score in coronary artery disease: serum uric acid, smoking, and Framingham risk stratification.

BACKGROUND: High serum uric acid (SUA) has been well demonstrated to be associated with morbidity and mortality in the general population as well as in patients with coronary artery disease (CAD). Recent studies show that the clinical SYNTAX score (CSS) is a new tool for the risk stratification of patients with complex CAD. In this study, we aimed to evaluate whether SUA was associated with the complexity of CAD as evaluated by the CSS. METHODS: The study population consisted of 451 patients (69% male) who underwent coronary angiography for the assessment of CAD. A lesion was defined as significant if it caused a 50% reduction of the luminal diameter by visual estimation in vessels ≥1.5 mm. CSS was calculated by multiplying the SYNTAX score by a modified value of age, creatinine, and ejection fraction (ACEF) score (age/ejection fraction +1 for each 10 mL the creatinine clearance <60 mL/min per 1.73 m²). RESULTS: All subjects were divided into three groups according to CSS tertiles: CSSLOW (CSS 2-11; n = 147), CSSMID (CSS 12-21; n = 152), and CSSHIGH (CSS 22-68; n = 152). The SUA level was prominently related with CSS (5.29 ± 1.23 mg/dL, 6.92 ± 1.23 mg/dL, and 8.31 ± 1.46 mg/dL; P<.001). SUA was a significant predictor of CSS after adjustment for other risk factors (OR, 2.68; P<.001). CONCLUSION: SUA level was significantly associated with the severity and complexity of CAD evaluated by CSS. Further prospective clinical studies are needed to clarify the exact physiopathologic role of SUA in CAD.

Simvastatin prevents dopaminergic neurodegeneration in experimental parkinsonian models: the association with anti-inflammatory responses.

BACKGROUND: In addition to their original applications to lowering cholesterol, statins display multiple neuroprotective effects. N-methyl-D-aspartate (NMDA) receptors interact closely with the dopaminergic system and are strongly implicated in therapeutic paradigms of Parkinson's disease (PD). This study aims to investigate how simvastatin impacts on experimental parkinsonian models via regulating NMDA receptors. METHODOLOGY/PRINCIPAL FINDINGS: Regional changes in NMDA receptors in the rat brain and anxiolytic-like activity were examined after unilateral medial forebrain bundle lesion by 6-hydroxydopamine via a 3-week administration of simvastatin. NMDA receptor alterations in the post-mortem rat brain were detected by [³H]MK-801(Dizocilpine) binding autoradiography. 6-hydroxydopamine treated PC12 was applied to investigate the neuroprotection of simvastatin, the association with NMDA receptors, and the anti-inflammation. 6-hydroxydopamine induced anxiety and the downregulation of NMDA receptors in the hippocampus, CA1(Cornu Ammonis 1 Area), amygdala and caudate putamen was observed in 6-OHDA(6-hydroxydopamine) lesioned rats whereas simvastatin significantly ameliorated the anxiety-like activity and restored the expression of NMDA receptors in examined brain regions. Significant positive correlations were identified between anxiolytic-like activity and the restoration of expression of NMDA receptors in the hippocampus, amygdala and CA1 following simvastatin administration. Simvastatin exerted neuroprotection in 6-hydroxydopamine-lesioned rat brain and 6-hydroxydopamine treated PC12, partially by regulating NMDA receptors, MMP9 (matrix metalloproteinase-9), and TNF-a (tumour necrosis factor-alpha). CONCLUSIONS/SIGNIFICANCE: Our results provide strong evidence that NMDA receptor modulation after simvastatin treatment could partially explain its anxiolytic-like activity and anti-inflammatory mechanisms in experimental parkinsonian models. These findings contribute to a better understanding of the critical roles of simvastatin in treating PD via NMDA receptors.

Soluble vascular endothelial growth factor (VEGF) receptor-1 inhibits migration of human monocytic THP-1 cells in response to VEGF.

OBJECTIVE: We aimed to investigate the regulation and contribution of vascular endothelial growth factor (VEGF) and sFlt-1(1-3) to human monocytic THP-1 migration. MATERIALS AND METHODS: Ad-sFlt-1/FLAG, a recombinant adenovirus carrying the human sFlt-1(1-3) (the first three extracellular domains of FLT-1, the hVEGF receptor-1) gene, was constructed. L929 cells were infected with Ad-sFlt-1/FLAG and the expression of sFlt-1 was detected by immunofluorescent assay and ELISA. Corning(®) Transwell(®) Filter Inserts containing polyethylene terephthalate (PET) membranes with pore sizes of 3 µm were used as an experimental model to simulate THP-1 migration. Five VEGF concentrations (0, 0.1, 1, 10 and 100 ng/ml), four concentrations of sFlt-1(1-3)/FLAG expression supernatants (0.1, 1, 10 and 100 ng/ml), and monocyte chemoattractant protein-1 (MCP-1, 10 ng/ml) were used to test the ability of THP-1 cells to migrate through PET membranes. RESULTS: The sFlt-1(1-3) gene was successfully recombined into Ad-sFlt-1/FLAG. sFlt-1(1-3) was expressed in L929 cells transfected with Ad-sFlt-1/FLAG. THP-1 cell migration increased with increasing concentrations of VEGF, while cell migration decreased with increasing concentrations of sFlt1(1-3)/FLAG. sFlt1(1-3)/FLAG had no effect on MCP-1-induced cell migration. CONCLUSIONS: This study demonstrated that VEGF is able to elicit a migratory response in THP-1 cells, and that sFlt-1(1-3) is an effective inhibitor of THP-1 migration towards VEGF.

Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro.

AIM: To examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiac hypertrophy in vitro and to investigate the underlying molecular mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were treated with the specific AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the specific AMPK antagonist Compound C, and then stimulated with phenylephrine (PE). The Muscle RING finger 1 (MuRF1)-small interfering RNA (siRNA) was transfected into cardiomyocytes using Lipofectamine 2000. The surface area of cultured cardiomyocytes was measured using planimetry. The protein degradation was determined using high performance liquid chromatography (HPLC). The expression of beta-myosin heavy chain (beta-MHC) and MuRF1, as well as the phosphorylation levels of AMPK and Forkhead box O 1 (FOXO1), were separately measured using Western blot or real-time polymerase chain reaction. RESULTS: Activation of AMPK by AICAR 0.5 mmol/L inhibited PE-induced increase in cardiomyocyte area and beta-MHC protein expression and PE-induced decrease in protein degradation. Furthermore, AMPK activation increased the activity of transcription factor FOXO1 and up-regulated downstream atrogene MuRF1 mRNA and protein expression. Treatment of hypertrophied cardiomyocytes with Compound C 1 micromol/L blunted the effects of AMPK on cardiomyocyte hypertrophy and changes to the FOXO1/MuRF1 pathway. The effects of AICAR on cardiomyocyte hypertrophy were also blocked after MuRF1 was silenced by transfection of cardiomyocytes with MuRF1-siRNA. CONCLUSION: The present study demonstrates that AMPK activation attenuates cardiomyocyte hypertrophy by modulating the atrophy-related FOXO1/MuRF1 signaling pathway in vitro.

Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) inhibits experimental autoimmune encephalomyelitis in dark Agouti (DA) rats.

Previous studies have shown that vascular endothelial growth factor (VEGF) expression is up-regulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a model for MS, and may exacerbate the disease. However, it remains unknown whether anti-VEGF modalities could serve as a potential treatment for such central nervous system (CNS) autoimmune diseases. We constructed a recombinant adenoviral vector carrying FLAG-tagged sFlt-1(1-3) (the first three extracellular domains of Flt-1, the hVEGF receptor-1). Intramuscular transfection of the recombinant adenoviral vector suppressed VEGF-induced inflammatory cell infiltration in matrigel plugs. When given intracerebrally to EAE rats, recombinant sFlt-1(1-3) adenoviral vector significantly reduced disease severity compared to untreated rats. sFlt-1(1-3) gene transfer blocked VEGF and greatly reduced the number of cells that express VEGF and ED1-positive cells in CNS in EAE rats. This study demonstrates that sFlt-1(1-3) gene transfer into the brain ameliorates the severity of EAE by inhibiting monocyte recruitment in the CNS of dark Agouti rats.