RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

BACKGROUNDS: Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. METHODS: Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. RESULTS: The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7 days and sustained for 28 days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. CONCLUSIONS: In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

Reduced DMPC and PMPC in lung surfactant promote SARS-CoV-2 infection in obesity.

OBJECTIVE: Obesity is an established risk factor for higher SARS-CoV-2 viral loads, severe COVID-19 pneumonia requiring hospitalization, and worse outcomes. However, the underlying mechanisms for the increased risk are not well understood. SARS-CoV-2 is a respiratory virus with the primary route of entry through the lungs, where the Spike protein of SARS-CoV-2 binds to the ACE2 receptor on pneumocytes. Lung surfactant produced by type II pneumocytes plays a major role in respiratory defense against infections. Surfactant predominantly contains lipids, especially phosphatidylcholines (PC), and obesity is characterized by aberrant lipid metabolism. We hypothesized that altered lipid composition in lung surfactant in obesity may promote SARS-CoV-2 infection, leading to severe COVID-19 disease. METHODS: Lipidomic analysis of lung tissue and bronchoalveolar lavage fluid (BALF) was performed using LC-MS/MS. The effects of PCs on SARS-CoV-2 pseudovirus infection were studied in HEK293T cells with ACE2 overexpression and in Vero-E6 cells with endogenous ACE2 expression. For the cell-cell fusion assay, HEK293T-ACE2 and HEK293T expressing SARS-CoV-2 Spike/eGFP were used as the target and effector cells, respectively. RESULTS: Lipidomic analysis revealed that myristic acid-containing dimyristoyl-PC (DMPC) and palmitoylmyristoyl-PC (PMPC) were reduced in lung tissue and BALF from high fat diet-induced obese mice. DMPC and PMPC markedly inhibited wild type and D614G mutant SARS-CoV-2 infection in HEK293T-ACE2 and Vero-E6 cells. Feeding obese mice with trimyristin, the triglycerides of myristic acid, increased DMPC and PMPC levels in lung surfactant. Lipid extract from BALF of trimyristin-treated obese mice mitigated the elevated wild type and D614G mutant SARS-CoV-2 infection. The inhibitory effects of DMPC and PMPC on SARS-CoV-2 infection were reversed by cholesterol. CONCLUSIONS: The reduced DMPC and PMPC in lung surfactant may promote SARS-CoV-2 infection. Increasing DMPC and PMPC in lung surfactant could be an innovative strategy for preventing and treating severe COVID-19 disease in obesity.

Retinol-Binding Protein 4 Promotes Cardiac Injury After Myocardial Infarction Via Inducing Cardiomyocyte Pyroptosis Through an Interaction With NLRP3.

Background Acute myocardial infarction (AMI) is one of the leading causes of cardiovascular morbidity and mortality worldwide. Pyroptosis is a form of inflammatory cell death that plays a major role in the development and progression of cardiac injury in AMI. However, the underlying mechanisms for the activation of pyroptosis during AMI are not fully elucidated. Methods and Results Here we show that RBP4 (retinol-binding protein 4), a previous identified proinflammatory adipokine, was increased both in the myocardium of left anterior descending artery ligation-induced AMI mouse model and in ischemia-hypoxia‒induced cardiomyocyte injury model. The upregulated RBP4 may contribute to the activation of cardiomyocyte pyroptosis in AMI because overexpression of RBP4 activated NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome, promoted the precursor cleavage of Caspase-1, and subsequently induced GSDMD (gasdermin-D)-dependent pyroptosis. In contrast, knockdown of RBP4 alleviated ischemia-hypoxia‒induced activation of NLRP3 inflammasome signaling and pyroptosis in cardiomyocytes. Mechanistically, coimmunoprecipitation assay showed that RBP4 interacted directly with NLRP3 in cardiomyocyte, while genetic knockdown or pharmacological inhibition of NLRP3 attenuated RBP4-induced pyroptosis in cardiomyocytes. Finally, knockdown of RBP4 in heart decreased infarct size and protected against AMI-induced pyroptosis and cardiac dysfunction in mice. Conclusions Taken together, these findings reveal RBP4 as a novel modulator promoting cardiomyocyte pyroptosis via interaction with NLRP3 in AMI. Therefore, targeting cardiac RBP4 might represent a viable strategy for the prevention of cardiac injury in patients with AMI.

Gold nanoparticles alleviates the lipopolysaccharide-induced intestinal epithelial barrier dysfunction.

Nanotechnology is used in the immune response manipulation to treat various human diseases. In the present study, we explored the effects of Au nanoparticles (AuNPs) on the lipopolysaccharide (LPS)-induced epithelial barrier dysfunction and inflammatory response of colonic epithelial NCM460 cells. According to the results of cell counting kit-8 and flow cytometry analysis, the viability of NCM460 cells was inhibited, and the apoptosis was increased after LPS treatment, and AuNPs reversed these changes in a dose-dependent way. The permeability was evaluated by detecting the flux of fluorescein isothiocyanate-dextran and transepithelial electrical resistance. LPS enhanced the permeability and promoted barrier dysfunction of NCM460 cells. Enzyme-linked immunosorbent sorbent assay results revealed that the concentrations of pro-inflammatory factors and nitric oxide were elevated by LPS treatment and decreased by the AuNPs. LPS aggravated the inflammatory response, which was rescued by the AuNPs. Moreover, LPS promoted the activation of the nuclear factor kappa-B and extracellular signal-regulated kinase/c-Jun NH-terminal kinase signaling pathways, which were inhibited by AuNPs.

Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis.

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can improve skeletal muscle performance both in humans and mice. We here showed that dexamethasone-induced atrophy, as evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression, and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and gasdermin-D (GSDMD). Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine treatment ameliorated dexamethasone-induced muscle pyroptosis and atrophy both in vivo and in vitro. Activation of NLRP3 using LPS and ATP not only increased the cleavage and activation of Caspase-1 and GSDMD, but also increased the expression levels of atrophy markers MuRF1 and Atrogin-1 in trimetazidine-treated C2C12 myotubes. Mechanically, dexamethasone inhibited the phosphorylation of PI3K/AKT/FoxO3a, which could be attenuated by trimetazidine. Conversely, co-treatment with a PI3K/AKT inhibitor, picropodophyllin, remarkably increased the expression of NLRP3 and reversed the protective effects of trimetazidine against dexamethasone-induced C2C12 myotube pyroptosis and atrophy. Taken together, our study suggests that NLRP3/GSDMD-mediated pyroptosis might be a novel mechanism for dexamethasone-induced skeletal muscle atrophy. Trimetazidine might be developed as a potential therapeutic agent for the treatment of dexamethasone-induced muscle atrophy.

Serum retinol-binding protein 4 as a predictor of cardiovascular events in elderly patients with chronic heart failure.

AIMS: RBP4 is an adipokine with adverse effects on cardiovascular system. Increased circulating retinol-binding protein 4 (RBP4) has been linked to chronic heart failure (CHF). However, whether elevated RBP4 is correlated with a poor prognosis in elderly patients with CHF remains unclear. The aim of this study was to evaluate the prognostic value of serum RBP4 in elderly patients with CHF. METHODS AND RESULTS: We enrolled 934 consecutive elderly patients (aged 60 years and older) with CHF and 138 age-matched and sex-matched control subjects in a prospective cohort study and explored the association of serum RBP4 levels with the clinical outcomes using multivariate Cox regression analyses. Serum RBP4 levels were elevated in CHF patients when compared with controls (46.66 ± 12.38 µg/mL vs. 40.71 ± 7.2 µg/mL, P < 0.001). Patients with the highest RBP4 concentrations had higher N terminal pro brain natriuretic peptide (NT-proBNP) levels but lower left ventricular eject fraction (LVEF) and estimated glomerular filtration rate (P < 0.001). Serum RBP4 levels were increased as the New York Heart Association functional class increased and LVEF decreased (P < 0.001) and were negatively correlated with LVEF (r = -0.154, P < 0.001) but positively correlated with NT-proBNP levels (r = 0.074, P = 0.023). Multivariate Cox regression analysis suggested that log RBP4 was an independent predictor for major adverse cardiac event(s) [hazard ratio (HR) = 2.61, 95% confidence interval (CI) = 1.19-5.70], together with age, male, LVEF, log NT-proBNP, and estimated glomerular filtration rate. Moreover, log RBP4 was also an independent predictor for cardiovascular mortality (HR = 2.24, 95% CI = 1.35-5.39) and CHF rehospitalization (HR = 2.54, 95% CI = 1.09-5.60) even after adjustment for the established adverse prognostic factors for CHF. The Kaplan-Meier survival curves showed that high concentration of RBP4 was a prognostic indicator of major adverse cardiac event(s) in patients with CHF. CONCLUSIONS: Our findings demonstrate for the first time that elevated serum RBP4 is correlated with worse outcome in elderly patients with CHF.

Retinol-binding protein 4 is closely correlated to blood pressure level and E/A in untreated essential hypertension patients.

BACKGROUND: Hypertension, a common chronic disease, is a leading cause of death and other cardiovascular diseases. Recent studies show that an inflammatory factor named retinol binding protein 4 (RBP4) was increased with cardiovascular diseases. However, the relationship between RBP4 and hypertension in patients remains unclear. METHODS: The study cohort was composed of patients with essential hypertension (EH) and healthy control (HC) subjects from the Second Affiliated Hospital of Nanjing Medical University [2017-2019]. The levels of RBP4 and echocardiography were compared in the current study. Statistical differences between two groups were analyzed using unpaired Student's t-tests and the correlation between the two variables adopts Pearson correlation analysis. SPSS 23.0 was used for all statistical analysis. RESULTS: Analysis of patient plasma samples revealed that RBP4 in EH group was greater than HC group (P<0.05). The area under the ROC curve was 0.717. Specificity and sensitivity were 80.4% and 60.8%, respectively. RBP4 had positive correlation with left ventricular systolic diameter (LVDs), interventricular septal thickness (IVS) and left ventricular posterior wall thickness (LVPW), negative correlation with left ventricular shortening fraction (FS) and ejection fraction (EF) (P<0.05), and no correlation with left ventricular end-diastolic diameter (LVDd) (P>0.05). RBP4 was closely related with E/A, evaluation method of left ventricular diastolic function, in patients with EH. CONCLUSIONS: RBP4 levels are closely correlated with blood pressure (BP) levels and might be involved in the regulation of left ventricular diastolic function in patients with EH.

MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1.

Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.

Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells.

BACKGROUND: Thermoresponsive nanoparticles have become an attractive candidate for designing combined multimodal therapy strategies because of the onset of hyperthermia and their advantages in synergistic cancer treatment. In this paper, novel cetuximab (C225)-encapsulated core-shell Fe3O4@Au magnetic nanoparticles (Fe3O4@Au-C225 composite-targeted MNPs) were created and applied as a therapeutic nanocarrier to conduct targeted magneto-photothermal therapy against glioma cells. METHODS: The core-shell Fe3O4@Au magnetic nanoparticles (MNPs) were prepared, and then C225 was further absorbed to synthesize Fe3O4@Au-C225 composite-targeted MNPs. Their morphology, mean particle size, zeta potential, optical property, magnetic property and thermal dynamic profiles were characterized. After that, the glioma-destructive effect of magnetic fluid hyperthermia (MFH) combined with near-infrared (NIR) hyperthermia mediated by Fe3O4@Au-C225 composite-targeted MNPs was evaluated through in vitro and in vivo experiments. RESULTS: The inhibitory and apoptotic rates of Fe3O4@Au-C225 composite-targeted MNPs-mediated combined hyperthermia (MFH+NIR) group were significantly higher than other groups in vitro and the marked upregulation of caspase-3, caspase-8, and caspase-9 expression indicated excellent antitumor effect by inducing intrinsic apoptosis. Furthermore, Fe3O4@Au-C225 composite-targeted MNPs-mediated combined hyperthermia (MFH+NIR) group exhibited significant tumor growth suppression compared with other groups in vivo. CONCLUSION: Our studies illustrated that Fe3O4@Au-C225 composite-targeted MNPs have great potential as a promising nanoplatform for human glioma therapy and could be of great value in medical use in the future.

Preparation and characterization of Fe3O4@Au-C225 composite targeted nanoparticles for MRI of human glioma.

OBJECTIVE: To study the characterization of Fe3O4@Au-C225 composite targeted MNPs. METHODS: Fe3O4@Au-C225 was prepared by the absorption method. The immunosorbent assay was used to evaluate its absorption efficiency at C225 Fc. ZETA SIZER3000 laser particle size analyzer, ultraviolet photometer and its characteristics were analyzed by VSM. the targeting effect of Fe3O4@Au-C225 composite targeted MNPs on U251 cells in vitro were detected by 7.0 Tesla Micro-MR; and subcutaneous transplanted human glioma in nude mice were performed the targeting effect in vivo after tail vein injection of Fe3O4@Au-C225 composite targeted MNPs by MRI. RESULTS: The self-prepared Fe3O4@Au composite MNPs can adsorb C225 with high efficiency of adsorption so that Fe3O4@Au-C225 composite targeted MNPs were prepared successfully. Fe3O4@Au-C225 composite targeted MNPs favorably targeted human glioma cell line U251 in vitro; Fe3O4@Au-C225 composite targeted MNPs have good targeting ability to xenografted glioma on nude mice in vivo, and can be traced by MRI. CONCLUSION: The Fe3O4@Au-C225 composite targeted MNPs have the potential to be used as a tracer for glioma in vivo.

Interaction Between Hydrophobic Au Nanoparticles and Pulmonary Surfactant (DPPC) Monolayers.

Hydrophobic nanoparticles from the air get into the lungs through inhalation and firstly interact with pulmonary surfactant, which is essential for normal lung function. 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) is the major component of pulmonary surfactant. Interaction between Langmuir monolayer of DPPC and hydrophobic Au nanoparticles (Au NPs) were investigated in this study by atomic force microscope. The experimental results indicated that incorporation of hydrophobic Au NPs into the monolayer hindered the phase transition, reduced the compressibility of monolayer and disrupted the large micro-domains in liquid-condensed (LC) phase into smaller ones. Moreover, Au NPs that modulated by DPPC domains formed wandering wires, which were enclosed LC phase domains, at low concentration and relatively large islands at high concentration. The large islands still remained on the water surface after five cycles of compression-expansion at high concentration of Au NPs, but single NPs or wires with 1-2 particle width escaped into the water during the compression through the help of DPPC coating. The interaction between hydrophobic NPs and pulmonary surfactant provides an insight into the toxicological effects of inhaled NPs from the atmosphere to the function of pulmonary surfactant in the lung.

Functionalized Au@Ag-Au nanoparticles as an optical and SERS dual probe for lateral flow sensing.

Lateral flow assay strips (LFASs) with Au nanoparticles (NPs) have been widely used as a probe for biomarkers in point-of-care testing; however, there still remain challenges in detection sensitivity and quantitative analysis. In this study, we developed a surface-enhanced Raman scattering (SERS)-based LFAS for quantitative analysis of a biomarker in the low concentration range. Moreover, apart from conventional Au NPs, three other types of citrate-capped Au-Ag bimetallic NPs: Au core with Ag shell NPs (Au@Ag NPs), rattle-like Au core in Ag-Au shell NPs (Au@Ag-Au NPs) and Ag-Au NPs were prepared and functionalized, and their solution-based SERS activities were comprehensively studied by experimental measurement and theoretical analysis. The results clearly indicated that the citrate-capped Au@Ag-Au NPs exhibited the highest SERS activity among the probes tested. Au@Ag-Au NPs were used as both optical and SERS probes in a SERS-based LFAS. In the presence of the analyte at high concentrations, a purple color appeared in the test zone. Highly sensitive and quantitative analysis was realized by measurement of SERS signals from the test lines. One of the most specific markers for cardiac injury, cardiac troponin I (cTnI), was chosen as the detection model. The detection limit of the SERS-based LFAS for cardiac troponin I was 0.09 ng/mL, lowered by nearly 50 times compared with visual results, and could be further lowered by optimization. These results demonstrated that the SERS-based LFAS using citrate-capped Au@Ag-Au NPs as probes can be a powerful tool for highly sensitive and quantitative detection of biomarkers. Graphical abstract A surface-enhanced Raman scattering (SERS)-based lateral flow assay strip using rattle-like Au core in Ag-Au shell (Au@Ag-Au) nanoparticles as probes was developed for quantitative analysis of a biomarker, with a detection limit nearly 50 times lower than that of visual assessment. C control line, T test line.