Weixia Cai

SIRT1 Is a Regulator in High Glucose-Induced Inflammatory Response in RAW264.7 Cells

Sepsis is defined as a systemic inflammatory response syndrome that disorders the functions of host immune system, including the imbalance between pro- and anti-inflammatory responses mediated by immune macrophages. Sepsis could also induce acute hyperglycemia. Studies have shown that the silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, mediates NF-κb deacetylation and inhibits its function. Therefore, SIRT1 is likely to play an important role in high glucose-mediated inflammatory signalings. Here we demonstrate that high glucose significantly downregulates both the mRNA and protein levels of SIRT1 and upregulates the mRNA level and the release of two pro-inflammatory cytokines, IL-1β and TNF-α, in RAW264.7 macrophages. Interestingly, the reduced level of SIRT1 by high glucose is remarkably upregulated by SIRT1 activator SRT1720, while the level and the release of IL-1β and TNF-α significantly decrease with the use of SRT1720. However, when the function of SIRT1 is inhibited by EX527 or its expression is suppressed by RNAi, the upregulated level and release of IL-1β and TNF-α by high glucose are further increased. Taken together, these findings collectively suggest that SIRT1 is an important regulator in many high glucose-related inflammatory diseases such as sepsis.

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization.

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.

MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA

Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor‐1 (PAI‐1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR‐181c and miR‐10a were differentially‐expressed and targeted uPA and PAI‐1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR‐181c knockdown or miR‐10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR‐181c–uPA and miR‐10a–PAI‐1 regulatory pathways have an integral role in HS pathogenesis.

Inhibition of Notch signaling leads to increased intracellular ROS by up-regulating Nox4 expression in primary HUVECs

The essential roles of Notch pathway in angiogenesis have been reported for years. However, how Notch pathway plays its role in regulating endothelial cells remains largely unknown. In this study we found that blockade of Notch signaling with a γ-secretase inhibitor increased reactive oxygen species (ROS) in primary human umbilical vein endothelial cells (HUVECs) under both normaxic and ischemia/reperfusion (I/R) conditions. Abruption of ROS generation with ROS scavengers or specific inhibitors of ROS production in HUVECs abolished Notch blockade-induced HUVEC proliferation, migration and adhesion, suggesting that the regulation of Notch pathway on endothelial cell behavior is at least partially dependent on its down-regulation of ROS level. We further showed that the enhanced generation of ROS after blocking Notch signal was accompanied by augmented expression of Nox4, which led to increased phosphorylation of VEGFR2 and ERK in HUVECs. In summary, our results have shown that Notch signaling regulates ROS generation by suppressing Nox4, and further modulates endothelial cell proliferation, migration and adhesion.

Efficacy and safety of BRAF inhibition alone versus combined BRAF and MEK inhibition in melanoma: a meta-analysis of randomized controlled trials

Recent clinical studies have shown that combination therapy of BRAF and MEK inhibition provides more survival benefit than BRAF inhibition monotherapy. However, the adverse events due to BRAF and MEK inhibitors impact the physical comfort and social life of patients. Thus, in this study we have undertaken a meta-analysis of randomized controlled trials to compare the efficacy and adverse events risk between monotherapy and combination therapy. We identified the relevant studies by searching PubMed, EMBASE and Google scholar databases, between the year January 2000 and May 2016. Based on the heterogeneity, the fixed- or random-effects models were employed to analyze the efficacy and the incidence rate of adverse events. In addition, the subgroup analyses were conducted to overcome the effects of heterogeneity. Finally, our study included five RCTs, involving 1730 patients for this meta-analysis. The fixed-effects model demonstrated that combination therapy of BRAF and MEK inhibition provided more survival benefit in terms of ORR, PFS and OS (P < 0.00001). But, the combination therapy also significantly increased the incidences of pyrexia, chills, vomiting, chorioretinopathy, retinal detachment, hypertension, night sweats, increased aspartate aminotransferase and creatine kinase levels (P < 0.05) as compared to monotherapy. But, based on the significantly better survival outcomes, the combined BRAF and MEK inhibition will obviously be the mainstay therapy for the BRAF V600-mutant melanoma. However, a set of adverse events should be paid attention when physicians consider combination therapy.

SIRT1 regulates inflammation response of macrophages in sepsis mediated by long noncoding RNA

Molecular mechanisms for macrophage immune responses modulated by SIRT1 during sepsis remain unclear. Here, we show that SIRT1 expression is down-regulated in macrophages from mouse sepsis model or LPS stimulation. SIRT1 expression in macrophages correlates with low levels of a long noncoding RNA (lncRNA)-NONMMUT003701 [named as lncRNA-CCL2]. SIRT1 inhibits lncRNA-CCL2 expression via sustaining a repressive chromatin state in the lncRNA-CCL2 locus. The inflammation cytokines expression is downregulated by knockdown of lncRNA-CCL2. Such inhibition can be reversed partly by decreased SIRT1 activity. Thus, this work uncovers previously unidentified mechanisms in which SIRT1 associates with lncRNA and lncRNA regulates macrophage inflammatory response.

Murine Sertoli cells promote the development of tolerogenic dendritic cells: a pivotal role of galectin-1.

Sertoli cells (SCs) possess inherent immunosuppressive properties and are major contributors to the immunoprivileged status of mammalian testis. SCs have been reported to inhibit the activation of B cells, T cells and natural killer cells but not dendritic cells (DCs). Herein, we present evidence that co‐culture with SCs results in a persistent state of DC immaturity characterized by down‐regulation of the surface molecules I‐A/E, CD80, CD83, CD86, CCR7 and CD11c, as well as reduced production of pro‐inflammatory cytokines. SC‐conditioned DCs (SC‐DCs) displayed low immunogenicity and enhanced immunoregulatory functions, including the inhibition of T‐cell proliferation and the promotion of Foxp3+ regulatory T‐cell development. Mechanistically, the activation of p38, extracellular signal‐regulated kinase 1/2, and signal transducer and activator of transcription 3 was suppressed in SC‐DCs. More importantly, we demonstrate that galectin‐1 secreted by SCs plays a pivotal role in the differentiation of functionally tolerogenic SC‐DCs. These findings further support the role of SCs in maintaining the immunoprivileged environment of the testis and provide a novel approach to derive tolerogenic DCs, which may lead to alternative therapeutic strategies for the treatment of immunopathogenic diseases.

Insulin protects against damage to pulmonary endothelial tight junctions after thermal injury: Relationship with zonula occludens-1, F-actin, and AKT activity

Intensive insulin therapy during critical illness protects the endothelium and thereby prevents organ failure. This study tested the hypothesis that insulin directly affects the attenuation of burn injury‐induced damage to pulmonary endothelial tight junction and investigated the underlying mechanisms. Sprague Dawley rats with severe burn injury were randomized to treatment with insulin dissolved in normal saline (maintenance of blood glucose at a level between 5.0 and 7.0 mmol/L) or normal saline alone (in vivo treatment). Pulmonary damage was evaluated. Rat pulmonary microvascular endothelial cells were treated with 20% burn serum or 20% burn serum + insulin (in vitro treatment). Selected cultures were pretreated with phosphatidylinositol 3‐kinase/protein kinase B (AKT) inhibitor (LY294002). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine phalloidin and were examined. Cell extracts were obtained to assess zonula occludens‐1, occludin, and phosphorylated AKT levels by immunoblotting. Treatment with insulin attenuated the pulmonary edema, hemorrhage, and inflammatory cell infiltration of rats with severe burn injury. Burn serum significantly enhanced monolayer permeability to albumin, whereas treatment with insulin (10−7 mol/L) limited this effect. Meanwhile, insulin (10−7 mol/L) reduced burn serum‐induced F‐actin stress fiber formation and decreased zonula occludens‐1 expression. LY294002 decreased cytoplasmic AKT phosphorylation and inhibited the protection effects of insulin. Through the phosphatidylinositol 3‐kinase/AKT pathway, insulin independent of glucose toxicity can attenuate increased pulmonary endothelial permeability induced by burn injury. The effect is attributed to the attenuation of the architectural disruption of protein components of the endothelial tight junction. This result is useful in inhibiting multiple organ failure after burn injury.

Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development

SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J−/− mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J−/− macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J−/− mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.

Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K/Akt signaling pathway

Introduction: Adipose tissue‐derived stem cells (ADSCs) have been shown to enhance wound healing via their paracrine function. Exosomes, as one of the most important paracrine factors, play an essential role in this process. However, the concrete mechanisms that underlie this effect are poorly understood. In this study, we aim to explore the potential roles and molecular mechanisms of exosomes derived from ADSCs in cutaneous wound healing. Methods: Normal human skin fibroblasts and ADSCs were isolated from patient skin and adipose tissues. ADSCs were characterized by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Exosomes were purified from human ADSCs by differential ultracentrifugation and identified by electron microscopy, nanoparticle tracking, fluorescence confocal microscopy and western blotting. Fibroblasts were treated with different concentrations of exosomes, and the synthesis of collagen was analyzed by western blotting; the levels of growth factors were analyzed by real‐time quantitative PCR (RT‐PCR) and ELISA; and the proliferation and migration abilities of fibroblasts were analyzed by real‐time cell analysis, CCK‐8 assays and scratch assays. A mouse model with a full‐thickness incision wound was used to evaluate the effect of ADSC‐derived exosomes on wound healing. The level of p‐Akt/Akt was analyzed by western blotting. Ly294002, a phosphatidylinositol 3‐kinases (PI3K) inhibitor, was used to identify the underlying mechanisms by which ADSC‐derived exosomes promote wound healing. Results: ADSC‐derived exosomes were taken up by the fibroblasts, which showed significant, dose‐dependent increases in cell proliferation and migration compared to the behavior of cells without exosome treatment. More importantly, both the mRNA and protein levels of type I collagen (Col 1), type III collagen (Col 3), MMP1, bFGF, and TGF‐&bgr;1 were increased in fibroblasts after stimulation with exosomes. Furthermore, exosomes significantly accelerated wound healing in vivo and increased the level of p‐Akt/Akt in vitro. However, Ly294002 alleviated these exosome‐induced changes, suggesting that exosomes from ADSCs could promote and optimize collagen deposition in vitro and in vivo and further promote wound healing via the PI3K/Akt signaling pathway. Conclusions: This study demonstrates that ADSC‐derived exosomes can promote fibroblast proliferation and migration and optimize collagen deposition via the PI3K/Akt signaling pathway to further accelerate wound healing. Our results suggest that ADSCs likely facilitate wound healing via the release of exosomes, and the PI3K/Akt pathway may play a role in this process. Our data also suggest that the clinical application of ADSC‐derived exosomes may shed new light on the use of cell‐free therapy to accelerate full‐thickness skin wound healing and attenuate scar formation. Graphical abstract Schematic diagram shows how the wound healing effect of ADSC‐Exos is mediated by the activation of the PI3K/Akt signaling pathways. Figure. No Caption available. HighlightsADSC‐Exos are internalized into HDFs and regulate their biological behaviors and functions.ADSC‐Exos play an important role in accelerating wound healing via activating PI3K/Akt signaling pathway.ADSC‐Exos may serve as a cell‐free therapy for the potential clinical treatment of wound healing.