Xuefu Zhuyu decoction alleviates deep vein thrombosis through inhibiting the activation of platelets and neutrophils via sirtuin 1/nuclear factor kappa-B pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuefu Zhuyu Decoction (XZD), a renowned traditional Chinese medicine prescription, is widely employed for the management of conditions characterized by qi-stagnation and blood stasis. Although its anti-thrombotic effect on deep vein thrombosis (DVT) patients has been clinically observed, the underlying mechanism remains largely unexplored. AIM OF THE STUDY: Our aim was to investigate the mechanisms by which XZD exerted its effect on DVT. MATERIALS AND METHODS: The ultra performance liquid chromatography (UPLC) technique was employed to evaluate quality of XZD. To examine the effect of XZD on DVT, a DVT rat model with inferior vena cava (IVC) stenosis was established. The 4D-label-free proteomics approach was then utilized to uncover the possible mechanisms of XZD against DVT. Based on proteomics, citrullinated histone H3 (CitH3), along with serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) were observed the inhibitory activity of XZD on neutrophil activation. Subsequently, the marker of platelet activation, specifically glycoprotein IIb (CD41) and glycoprotein IIIa (CD61), were assessed along with the secretion of von Willebrand factor (vWF) to investigate the inhibitory activity of XZD on platelet activation. Finally, we explored the impact of XZD on the sirtuin 1 (SIRT1)/nuclear factor kappa-B (NF-κB) pathway, which was associated with the activation of platelets and neutrophils. RESULTS: Eight distinct components were identified for the quality control of XZD. XZD effectively reduced thrombus weight and length in DVT rats, without affecting the coagulation function or hematological parameters in the systemic circulation. Proteomics analysis revealed that XZD alleviated DVT by inhibiting the activation of platelets and neutrophils. The protein expression of CitH3, along with serum levels of TNF-α and IL-1ß, were reduced in XZD-treated DVT rats. Similarly, protein expressions of CD41 and CD61, along with the release of vWF, were markedly down-regulated in XZD-treated DVT rats. Finally, treatment with XZD resulted in an up-regulation of SIRT1 protein expression and a down-regulation of both acetylated NF-κB/p65 and phosphorylated NF-κB/p65 protein expressions in endothelium. CONCLUSIONS: XZD alleviates DVT by inhibiting the activation of platelets and neutrophils at the injured endothelium via the regulation of SIRT1/NF-κB pathway.

Temporary Upregulation of Nrf2 by Naringenin Alleviates Oxidative Damage in the Retina and ARPE-19 Cells.

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO3-induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO3-treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway.

Aqueous extract of Paeoniae Radix Rubra prevents deep vein thrombosis by ameliorating inflammation through inhibiting GSK3ß activity.

BACKGROUND: Deep vein thrombosis (DVT) is a kind of blood stasis syndrome. Paeoniae Radix Rubra (PRR) has long been widely used for eliminating blood stasis in China, but its effect on DVT has not yet been reported. PURPOSE: The present study aimed to assess the potential inhibitory effect of the aqueous extract of PRR (i.e.,PRR dispensing granule, PRRDG) on DVT and explore the underlying mechanism. STUDY DESIGN/METHODS: The chemical profile of PRRDG was analyzed by high-performance liquid chromatography. Sprague-Dawley rats were intragastrically treated with PRRDG (0.625, 1.25 and 1.875 g crude drug/kg/d) once daily for 7 consecutive days. On the sixth day, a model of inferior vena cava (IVC) stenosis-induced DVT was established. All rats were sacrificed on the seventh day. Serum was collected for enzyme-linked immunosorbent assay. Thrombus-containing IVC was weighed and further processed for histopathologic examination, immunohistochemical analysis and western blotting. LiCl and LY294002 were adopted to block and increase the activity of glycogen synthase kinase 3ß (GSK3ß), respectively. RESULTS: The chemical profile analysis showed that paeoniflorin, benzoylpaeoniflorin, albiflorin, gallic acid and catechin were the main constituents of PRRDG. LiCl decreased thrombus weight, reduced the number of inflammatory cells in thrombus and vein wall, down-regulated phosphorylated NF-κB p65 (p-p65) protein expression. Similarly, PRRDG decreased thrombus weight and tissue factor (TF) protein expression. PRRDG reduced the protein expression levels of P-selectin, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in venous endothelium, serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the number of inflammatory cells in thrombus and vein wall. Moreover, PRRDG down-regulated p-p65 protein expression and up-regulated phosphorylated GSK3ß (p-GSK3ß) protein expression. LY294002 abrogated the inhibitory effects of PRRDG on thrombus weight, TF protein expression, TNF-α and IL-1ß serum levels, inflammatory cells influxes, and p-p65 protein expression. CONCLUSION: PRRDG prevents DVT by ameliorating inflammation through inhibiting GSK3ß activity.

Spatholobi Caulis dispensing granule reduces deep vein thrombus burden through antiinflammation via SIRT1 and Nrf2.

BACKGROUND: Deep vein thrombosis (DVT) is a kind of blood stasis syndrome. Spatholobi Caulis (SC) has been widely used for the treatment of blood stasis syndrome in China, but the underlying mechanism remains poorly understood. PURPOSE: The aim of present study was to investigate the anti-DVT mechanism of Spatholobi Caulis dispensing granule (SCDG). STUDY DESIGN/METHODS: A rat model of inferior vena cava (IVC) stenosis-induced DVT and a cell model of oxygen-glucose deprivation (OGD) were performed. Rats were orally administered with SCDG solution once daily for seven consecutive days. IVC stenosis-induced DVT was operated on the sixth day. Thrombi were harvested and weighed on the seventh day. Pathological changes were observed by hematoxylin-eosin (HE) staining. Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß of serum were analyzed by enzyme-linked immunosorbent assay. C-reactive protein (CRP) was measured with turbidimetric immunoassay. Protein expressions in thrombosed IVCs and/or OGD-stimulated EA. hy926 cells were evaluated by western blot and/or immunofluorescence analyses. RESULTS: SCDG dramatically decreased thrombus weight. SCDG decreased tissue factor (TF) protein expression, inflammatory cells influxes in thrombosed vein wall and serum levels of inflammatory cytokines and CRP. Further, SCDG up-regulated Sirtuin 1 (SIRT1) protein expression and down-regulated acetylated-NF-κB p65 (Ace-p65) protein expression. Moreover, SCDG up-regulated nuclear factor-erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions, and down-regulated phosphorylated-NF-κB p65 (p-p65) protein expression. In the OGD cell model, SCDG medicated serum decreased the protein expression of TF. SCDG medicated serum enhanced SIRT1 protein expression and reduced Ace-p65 nuclear protein expression. SCDG medicated serum promoted protein expressions of nuclear Nrf2 and total HO-1, and inhibited translocation of p65. Furthermore, inhibiting SIRT1 and Nrf2 reversed the protective effect of SCDG medicated serum on OGD-induced EA. hy926 cells. CONCLUSION: SCDG may prevent DVT through antiinflammation via SIRT1 and Nrf2.

Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation.

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

LFCseq: a nonparametric approach for differential expression analysis of RNA-seq data.

BACKGROUND: With the advances in high-throughput DNA sequencing technologies, RNA-seq has rapidly emerged as a powerful tool for the quantitative analysis of gene expression and transcript variant discovery. In comparative experiments, differential expression analysis is commonly performed on RNA-seq data to identify genes/features that are differentially expressed between biological conditions. Most existing statistical methods for differential expression analysis are parametric and assume either Poisson distribution or negative binomial distribution on gene read counts. However, violation of distributional assumptions or a poor estimation of parameters often leads to unreliable results. RESULTS: In this paper, we introduce a new nonparametric approach called LFCseq that uses log fold changes as a differential expression test statistic. To test each gene for differential expression, LFCseq estimates a null probability distribution of count changes from a selected set of genes with similar expression strength. In contrast, the nonparametric NOISeq approach relies on a null distribution estimated from all genes within an experimental condition regardless of their expression levels. CONCLUSION: Through extensive simulation study and RNA-seq real data analysis, we demonstrate that the proposed approach could well rank the differentially expressed genes ahead of non-differentially expressed genes, thereby achieving a much improved overall performance for differential expression analysis.