Interferon-regulatory factor-1 boosts bevacizumab cardiotoxicity by the vascular endothelial growth factor A/14-3-3γ axis.

AIM: Myocardial injury is a significant cause of death. This study investigated the role and underlying mechanism of interferon-regulatory factor-1 (IRF1) in bevacizumab (BVZ)-induced cardiomyocyte injury. METHODS AND RESULTS: HL-1 cells and C57BL/6 mice receiving BVZ treatment were used to establish in vitro and in vivo models of myocardial injury. The relationship between VEGFA and 14-3-3γ was verified through co-immunoprecipitation and Glutathione S Transferase (GST) pull-down assay. Cell viability and apoptosis were analysed by MTT, propidium iodide (PI) staining and flow cytometry. The release of lactate dehydrogenase (LDH), cardiac troponins T (cTnT), and creatine kinase MB (CK-MB) was measured using the enzyme linked immunosorbent assay. The effects of knocking down IRF1 on BVZ-induced mice were analysed in vivo. IRF1 levels were increased in BVZ-treated HL-1 cells. BVZ treatment induced apoptosis, inhibited cell viability, and promoted the release of LDH, cTnT, and CK-MB. IRF1 silencing suppressed BVZ-induced myocardial injury, whereas IRF1 overexpression had the opposite effect. IRF1 regulated VEGFA expression by binding to its promoter, with the depletion of VEGFA or 14-3-3γ reversing the effects of IRF1 knockdown on the cell viability and apoptosis of BVZ-treated HL-1 cells. 14-3-3γ overexpression promoted cell proliferation, inhibited apoptosis, and reduced the release of LDH, cTnT, and CK-MB, thereby alleviating BVZ-induced HL-1 cell damage. In vivo, IRF1 silencing alleviated BVZ-induced cardiomyocyte injury by regulating the VEGFA/14-3-3γ axis. CONCLUSION: The IRF1-mediated VEGFA/14-3-3γ signalling pathway promotes BVZ-induced myocardial injury. Our study provides evidence for potentially new target genes for the treatment of myocardial injury.

Urinary Metabolite Signatures for Predicting Elderly Stroke Survivors with Depression.

BACKGROUND: Post-stroke depression (PSD) is a major complication in stroke survivors, especially in elderly stroke survivors. But there are still no objective methods to diagnose depression in elderly stroke survivors. Thus, this study was conducted to identify potential biomarkers for diagnosing elderly PSD subjects. METHODS: Elderly (60 years or older) stroke survivors with depression were assigned into the PSD group, and elderly stroke survivors without depression and elderly healthy controls (HCs) were assigned into the non-depressed group. Urinary metabolite signatures obtained from gas chromatography-mass spectrometry (GC-MS)-based metabolomic platform were collected. Both univariate and multivariate statistical analysis were used to find the differential urinary metabolites between the two groups. RESULTS: The 78 elderly HCs, 122 elderly stroke survivors without depression and 124 elderly PSD subjects were included. A set of 13 differential urinary metabolites responsible for distinguishing PSD subjects from non-depressed subjects were found. The Phenylalanine, tyrosine and tryptophan biosynthesis, Phenylalanine metabolism and Galactose metabolism were found to be significantly changed in elderly PSD subjects. The phenylalanine was significantly negatively correlated with age and depressive symptoms. Meanwhile, a biomarker panel consisting of 3-hydroxyphenylacetic acid, tyrosine, phenylalanine, sucrose, palmitic acid, glyceric acid, azelaic acid and α-aminobutyric acid was identified. CONCLUSION: These results provided candidate molecules for developing objective methods to diagnose depression in elderly stroke survivors, suggested that taking supplements of phenylalanine might be an effective method to prevent depression in elderly stroke survivors, and would be helpful for future revealing the pathophysiological mechanism of PSD.

miR-140-5p mediates bevacizumab-induced cytotoxicity to cardiomyocytes by targeting the VEGFA/14-3-3γ signal pathway.

Bevacizumab (BVZ) is the first recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGFA) approved by the FDA for the treatment of different kinds of cancers, especially colorectal cancer. Although the anti-tumor effects have been verified, the side effects of BVZ are also noteworthy, among which, cardiotoxicity may be the most serious side effect of BVZ. However, the exact mechanisms of cardiotoxicity induced by BVZ have been little explored. This study was conducted in vitro in a human cardiac myocyte (HCM) model. MTT assay was conducted to determine BVZ-stimulated cell viability. For testing the function and mechanism, the cells were transfected with miR-140-5p mimics, miR-140-5p inhibitor and/or VEGFA small interfering RNA (si-VEGFA). Then, apoptosis of the cells was detected via annexin V/propidium iodide (AV-PI) staining followed by flow cytometry. qRT-PCR and western blot assays were applied to measure gene expression (i.e. mRNA) and protein levels, respectively. The CK, LDH, SOD, CAT and GSH-Px activities and MDA level were determined using commercial kits. ROS levels were determined by DCFH-DA assay. Mitochondrial membrane potential was measured by JC-1 assay. Dual-luciferase reporter assay was used to detect the interaction between miR-140-5p and VEGFA. BVZ could inhibit HCM proliferation and induce apoptosis. miR-140-5p was upregulated in response to BVZ treatment and miR-140-5p restraint could alleviate HCM damage caused by BVZ treatment. In contrast, VEGFA and 14-3-3γ expressions were down-regulated by BVZ, and miR-140-5p could inhibit the expression of 14-3-3γ by directly targeting VEGFA. Moreover, VEGFA suppression enhanced HCM injury stimulated by BVZ and partially reversed the functional role of the miR-140-5p inhibitor in BVZ-treated cells. Taken together, miR-140-5p promoted BVZ-treated cardiomyocyte toxicity by targeting the VEGFA/14-3-3γ signal pathway. Collectively, miR-140-5p mediated the BVZ-induced cytotoxicity to cardiomyocytes by targeting the VEGFA/14-3-3γ signal pathway, indicating that miR-140-5p may be a novel target for treating BVZ-induced cardiotoxicity.

The saponin D39 blocks dissociation of non-muscular myosin heavy chain IIA from TNF receptor 2, suppressing tissue factor expression and venous thrombosis.

BACKGROUND AND PURPOSE: Non-muscular myosin heavy chain IIA (NMMHC IIA) plays a key role in tissue factor expression and venous thrombosis. Natural products might inhibit thrombosis through effects on NMMHC IIA. Here, we have shown that a natural saponin, D39, from Liriope muscari exerted anti-thrombotic activity in vivo, by targeting NMMHC IIA. EXPERIMENTAL APPROACH: Expression and activity of tissue factor in endothelial cells were analysed in vitro by Western blot and simplified chromogenic assays. Interactions between D39 and NMMHC IIA were assessed by serial affinity chromatography and molecular docking analysis. D39-dependent interactions between NMMHC IIA and TNF receptor 2 (TNFR2) were measured by immunofluorescence, co-immunoprecipitation and proximity ligation assays. Anti-thrombotic activity of D39 in vivo was evaluated with a model of inferior vena cava ligation injury in mice. KEY RESULTS: D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior vena cava-ligated mice dose-dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3ß-NF-κB signalling pathways. CONCLUSIONS AND IMPLICATIONS: D39 inhibited tissue factor expression and thrombus formation by modulating the Akt/GSK3ß and NF-κB signalling pathways through NMMHC IIA. We identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.

An integrated shotgun proteomics and bioinformatics approach for analysis of brain proteins from MCAO model using serial affinity chromatograph with four active ingredients from Shengmai preparations as ligands.

Identification and validation of disease-relevant target proteins for natural products is an essential component of modern pharmaceutical research. In the present study, an integrated shotgun proteomics and bioinformatics approach was established to profile the interaction of active small molecules derived from ShengMai preparations (SMXZF) with hundreds of endogenously expressed proteins from middle cerebral artery occlusion (MCAO) model. Affinity-based proteomic strategies for isolation and identification of targets for the bioactive components is a classic, but still powerful approach. The proteins bound by SMXZF of the brain tissue proteins from MCAO model via serial affinity chromatograph were analyzed by nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) and all MS/MS spectra were then automatically searched by the SEQUEST program. A total of 154 proteins had been identified, with the molecular weight ranging from 21,369.6 to 332,393.21 and the pI from 4.32 to 10.88. Bioinformatic analysis was also implemented to better understand the identified proteins. In the gene ontology (GO) annotation, all the identified proteins were classified into 39, 18 and 12 groups according to biological process, cellular component and molecular function, respectively. KEGG pathways analysis of the identified proteins was conducted with 46 corresponding pathways found. In addition, the gene network was also constructed to analyze the relationship of these genes each other. Further validation of some targets were performed in MCAO model by Western blotting. The results indeed supported the notion that proteins MAPK/ERK1/2, CaMKII and VIM were involved in the disease development of MCAO and played an essential role in the protective effect of SMXZF. This study highlights the effectiveness and reliability of this integrated shotgun proteomics and bioinformatics approach, which is a promising paradigm for target identification and elucidating the mechanism of natural products in future research.

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4.

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3'-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)-a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway-by directly binding their 3'-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1ß were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.

A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways.

The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san (SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT-13: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide (H2O2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways . The PC12 cells were exposed to H2O2 (400 µmol·L(-1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase (LDH), the intracellular content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 µg·mL(-1). SMXZF also inhibited H2O2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H2O2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H2O2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H2O2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

An integrated pathway interaction network for the combination of four effective compounds from ShengMai preparations in the treatment of cardio-cerebral ischemic diseases.

AIM: SMXZF (a combination of ginsenoside Rb1, ginsenoside Rg1, schizandrin and DT-13) derived from Chinese traditional medicine formula ShengMai preparations) is capable of alleviating cerebral ischemia-reperfusion injury in mice. In this study we used network pharmacology approach to explore the mechanisms of SMXZF in the treatment of cardio-cerebral ischemic diseases. METHODS: Based upon the chemical predictors, such as chemical structure, pharmacological information and systems biology functional data analysis, a target-pathway interaction network was constructed to identify potential pathways and targets of SMXZF in the treatment of cardio-cerebral ischemia. Furthermore, the most related pathways were verified in TNF-α-treated human vascular endothelial EA.hy926 cells and H2O2-treated rat PC12 cells. RESULTS: Three signaling pathways including the NF-κB pathway, oxidative stress pathway and cytokine network pathway were demonstrated to be the main signaling pathways. The results from the gene ontology analysis were in accordance with these signaling pathways. The target proteins were found to be associated with other diseases such as vision, renal and metabolic diseases, although they exerted therapeutic actions on cardio-cerebral ischemic diseases. Furthermore, SMXZF not only dose-dependently inhibited the phosphorylation of NF-κB, p50, p65 and IKKα/ß in TNF-α-treated EA.hy926 cells, but also regulated the Nrf2/HO-1 pathway in H2O2-treated PC12 cells. CONCLUSION: NF-κB signaling pathway, oxidative stress pathway and cytokine network pathway are mainly responsible for the therapeutic actions of SMXZF against cardio-cerebral ischemic diseases.

[Study on action mechanism of Danhong injection based on computational system biology approach].

Danhong injection is a compound preparation of traditional Chinese medicine Salvia miltiorrhiza and Carthamus tinctorius, and has been widely applied in treating coronary heart diseases and ischemic encephalopathy in clinic. Despite the complexity of its chemical compounds and the diversity of targets, especially in system biology, there have not a report for its action mechanism as a whole regulatory biological network. In this study, protein data of S. miltiorrhiza and C. tinctorius were searched in TCMGeneDIT database and agilent literature search (ALS) system to establish the multi-component protein network of S. miltiorrhiza, C. tinctorius and Danhong injection. Besides, the protein interaction network was built based on the protein-protein interaction in Genecards, BIND, BioGRID, IntAct, MINT and other databases. According to the findings, 10 compounds of S. miltiorrhiza and 14 compounds of C. tinctorius were correlated with proteins. The 24 common compounds had interactions with 81 proteins, and formed a protein interaction network with 60 none-isolated nodes. The Cluster ONE module was applied to make an enrichment analysis on the protein interaction network and extract one sub-network with significant difference P <0.05. The sub-network contains 23 key proteins, which involved five signaling pathways, namely Nod-like receptor signaling pathway, epithelial cell signaling in helicobacter pylori infection, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and neurotrophin signaling pathway through KEGG signaling pathway mapping. In this study, the computational system biology approach was adopted to preliminarily explain the molecular mechanism of main compounds of Danhong injection in preventing and treating diseases and provide reference for systematic studies on traditional Chinese medicine compounds.

Meta-analysis of digoxin use and risk of mortality in patients with atrial fibrillation.

There is an ongoing debate on the safety of digoxin use in patients with atrial fibrillation (AF). To address this issue, the investigators assembled a synthesis of the available evidence on the relation between digoxin and all-cause mortality in patients with AF. PubMed and the Embase database were systematically searched to identify all eligible studies examining the association between digoxin use and the mortality risk in AF. Overall hazard ratios and 95% confidence intervals were calculated using the random-effects model. Eleven observational studies were identified that met the inclusion criteria, 5 of which additionally used propensity score matching for statistical adjustment. In total, 318,191 patients were followed up for a mean of 2.8 years. Overall, digoxin use was associated with a 21% increased risk for mortality (hazard ratio 1.21, 95% confidence interval 1.12 to 1.30). Sensitivity analyses found the results to be robust. In the propensity score-matched AF patients, digoxin use was associated with a 17% greater risk for mortality (hazard ratio 1.17, 95% confidence interval 1.13 to 1.22). When the AF cohort was grouped into patients with and without heart failure, the use of digoxin was associated with an increase in mortality in patients with and those without heart failure, and no significant heterogeneity was seen between the groups (p >0.10). In conclusion, the results suggest that digoxin use was associated with a greater risk for mortality in patients with AF, regardless of concomitant heart failure. A well-powered randomized trial is necessary to reveal the true effect of digoxin.

Network pharmacology-based prediction and verification of the molecular targets and pathways for schisandrin against cerebrovascular disease.

AIM: To illuminate the molecular targets for schisandrin against cerebrovascular disease based on the combined methods of network pharmacology prediction and experimental verification. METHOD: A protein database was established through constructing the drug-protein network from literature mining data. The protein-protein network was built through an in-depth exploration of the relationships between the proteins. The computational platform was implemented to predict and extract the sensitive sub-network with significant P-values from the protein-protein network. Then the key targets and pathways were identified from the sensitive sub-network. The most related targets and pathways were also confirmed in hydrogen peroxide (H2O2)-induced PC12 cells by Western blotting. RESULTS: Twelve differentially expressed proteins (gene names: NFKB1, RELA, TNFSF10, MAPK1, CHUK, CASP8, PIGS2, MAPK14, CREB1, IFNG, APP, and BCL2) were confirmed as the central nodes of the interaction network (45 nodes, 93 edges). The NF-κB signaling pathway was suggested as the most related pathway of schisandrin for cerebrovascular disease. Furthermore, schisandrin was found to suppress the expression and phosphorylation of IKKα, as well as p50 and p65 induced by H2O2 in PC12 cells by Western blotting. CONCLUSION: The computational platform that integrates literature mining data, protein-protein interactions, sensitive sub-network, and pathway results in identification of the NF-κB signaling pathway as the key targets and pathways for schisandrin.