Senescence in human mesenchymal stem cells (MSCs) not only contributes to organism aging and the development of a variety of diseases but also severely impairs their therapeutic properties as a promising cell therapy. Studies searching for efficient biomarkers that represent cellular senescence have attracted much attention; however, no single marker currently provides an accurate cell-free representation of cellular senescence. Here, we studied characteristics of MSC-derived microvesicles (MSC-MVs) that may reflect the senescence in their parental MSCs. We found that senescent late passage (LP) MSCs secreted higher levels of MSC-MVs with smaller size than did early passage (EP) MSCs, and the level of CD105+ MSC-MVs decreased with senescence in the parental MSCs. Also, a substantially weaker ability to promote osteogenesis in MSCs was observed in LP than EP MSC-MVs. Comparative analysis of RNA sequencing showed the same trend of decreasing number of highly-expressed miRNAs with increasing number of passages in both MSCs and MSC-MVs. Most of the highly-expressed genes in LP MSCs and the corresponding MSC-MVs were involved in the regulation of senescence-related diseases, such as Alzheimer's disease. Furthermore, based on the miRNA profiling, transcription factors (TF) and genes regulatory networks of MSC senescence, and the datasets from GEO database, we confirmed that expression of miR-146a-5p in MSC-MVs resembled the senescent state of their parental MSCs. Our findings provide evidence that MSC-MVs are a key factor in the senescence-associated secretory phenotype of MSCs and demonstrate that their integrated characteristics can dynamically reflect the senescence state of MSCs representing a potential biomarker for monitoring MSC senescence.
BackgroundNeuron loss, glial activation and vascular degeneration are common sequelae of ischemia-reperfusion (I/R) injury in ocular diseases. The present study was conducted to explore the ability of curcumin to inhibit retinal I/R injury, and to investigate underlying mechanisms of the drug effects.Methodology/Principal FindingsDifferent dosages of curcumin were administered. I/R injury was induced by elevating the intraocular pressure for 60 min followed by reperfusion. Cell bodies, brn3a stained cells and TUNEL positive apoptotic cells in the ganglion cell layer (GCL) were quantitated, and the number of degenerate capillaries was assessed. The activation of glial cells was measured by the expression level of GFAP. Signaling pathways including IKK-IκBα, JAK-STAT1/3, ERK/MAPK and the expression levels of β-tubulin III and MCP-1 were measured by western blot analysis. Pre-treatment using 0.01%–0.25% curcumin in diets significantly inhibited I/R-induced cell loss in GCL. 0.05% curcumin pre-treatment inhibited I/R-induced degeneration of retinal capillaries, TUNEL-positive apoptotic cell death in the GCL, brn3a stained cell loss, the I/R-induced up-regulation of MCP-1, IKKα, p-IκBα and p-STAT3 (Tyr), and down-regulation of β-tubulin III. This dose showed no effect on injury-induced GFAP overexpression. Moreover, 0.05% curcumin administered 2 days after the injury also showed a vaso-protective effect.Conclusions/SignificanceCurcumin protects retinal neurons and microvessels against I/R injury. The beneficial effects of curcumin on neurovascular degeneration may occur through its inhibitory effects on injury-induced activation of NF-κB and STAT3, and on over-expression of MCP-1. Curcumin may therefore serve as a promising candidate for retinal ischemic diseases.
Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling has been found in several types of human cancer, including hepatocellular carcinoma (HCC). NVP-BEZ235 is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against HCC in preclinical models. Autophagy is a cellular lysosomal degradation pathway essential for the regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mTOR signaling and often counteracts the efficacy of certain cancer therapeutic agents. In this study, we explored the role of autophagy in apoptosis induced by NVP-BEZ235 in two HCC cell lines, Hep3B and PLC/PRF/5, and identified the mechanism of combinatorial treatment. NVP-BEZ235 was effective in inhibiting the growth of the two HCC cell lines possibly though induction of apoptosis. NVP-BEZ235 also potently increased the expression of LC3-II and decreased the expression of p62, indicating induction of autophagy. When NVP-BEZ235 was used in combination with Atg5 siRNA or the autophagy inhibitor 3-methyladenine (3-MA), enhancement of the inhibitory effects on the growth of HCC cells was detected. In addition, enhanced induction of apoptosis was observed in cells exposed to the combination of NVP-BEZ235 and Atg5 siRNA or 3-MA. Thus, induction of autophagy by NVP-BEZ235 may be a survival mechanism that counteracts its anticancer effects. Based on these data, we suggest a strategy to enhance the anticancer efficacy of BEZ235 by blockade of autophagy. Thus, our study provides a rationale for the clinical development of combinations of NVP-BEZ235 and autophagy inhibitors for the treatment of HCC and other malignancies.
Oxidized low-density lipoprotein (ox-LDL)-induced endothelial damage contributes to the initiation and pathogenesis of atherosclerosis. Salidroside can alleviate atherosclerosis and attenuate endothelial cell injury induced by ox-LDL. However, the mechanisms involved in this process are not fully understood. Therefore, the purpose of the present study was to investigate the role of the adenosine monophosphate-activated protein kinase (AMPK)/sirtuin (SIRT)1 pathway in the protection of salidroside against ox-LDL-induced human umbilical vein endothelial cells (HUVECs) injuries. The results revealed that salidroside reverses ox-LDL-induced HUVECs injury as demonstrated by the upregulation of cell viability and downregulation of LDH release. In addition, salidroside increased the expression of the SIRT1 protein in ox-LDL-treated HUVECs. Next, it was demonstrated that SIRT1 knockdown induced by transfection with small interfering (si)RNA targeting SIRT1 (siSRT1) abolished the protection of salidroside against ox-LDL-induced HUVECs injuries. This was illustrated by a decrease in cell viability and an increase in LDH release, caspase-3 activity and apoptosis rate. Furthermore, salidroside mitigated ox-LDL-induced reactive oxygen species production, upregulated malondialdehyde content and NADPH oxidase 2 expression and decreased superoxide dismutase and glutathione peroxidase activities, while these effects were also reversed by siSIRT1 transfection. In addition, it was demonstrated that salidroside suppressed ox-LDL-induced mitochondrial dysfunction as demonstrated by the increase in mitochondrial membrane potential and decreases in cytochrome c expression, and Bax/Bcl-2 reductions. However, these effects were eliminated by SIRT1 knockdown. Finally, it was demonstrated that salidroside significantly upregulated the phosphorylated-AMPK expression in ox-LDL-treated HUVECs and AMPK knockdown induced by transfection with AMPK siRNA (siAMPK) leads to elimination of the salidroside-induced increase in cell viability and the decrease in LDH release. Notably, siAMPK transfection further decreased the expression of SIRT1. In conclusion, these results suggested that salidroside protects HUVECs against ox-LDL injury through inhibiting oxidative stress and improving mitochondrial dysfunction, which were dependent on activating the AMPK/SIRT1 pathway.
Purpose Bacterial biofilms on the surface of prostheses are becoming a rising concern in managing prosthetic joint infections. The inherent resistant features of biofilms render traditional antimicrobial therapy unproductive and revision surgery outcomes uncertain. This situation has prompted the exploration of novel antimicrobial strategies. The synergy of ultrasound microbubbles and vancomycin has been proposed as an efficient alternative for biofilm eradication. The purpose of this study was to evaluate the anti-biofilm effect of stimulated phase-shift acoustic nanodroplets (NDs) combined with vancomycin. Materials and methods We fabricated lipid phase-shift NDs with a core of liquid perfluoropentane. A new phase change mode for NDs incorporating an initial unfocused low-intensity pulsed ultrasound for 5 minutes and a subsequent incubation at 37°C into a 24-hour duration was developed. Methicillin-resistant Staphylococcus aureus (MRSA) biofilms were incubated with vancomycin and NDs under the hybrid stimulation. Biofilm morphology following treatment was determined using confocal laser scanning microscopy and scanning electron microscopy. Resazurin assay was used to quantify bactericidal efficacy against MRSA biofilm bacteria. Results NDs treated sequentially with ultrasound and heating at 37°C achieved gradual and substantial ND vaporization and cavitation in a successive process. NDs after stimulation were capable of generating stronger destruction on biofilm structure which was best characterized by residual circular arc margins and more dead bacteria. Furthermore, NDs combined with vancomycin contributed to significantly decreasing the metabolic activity of bacteria in MRSA biofilms ( P <0.05). Conclusion Phase-shift acoustic NDs could exert a significant bactericidal effect against MRSA biofilms through a new stimulation mode. Acoustic NDs present advantages over microbubbles for biofilm damage. This anti-biofilm strategy could be used either alone or as an enhancer of traditional antibiotics in the control of prosthetic joint infections.
Hypoxia resulting from apnea in patients with sleep apnea is an important factor in heart disease. We designed the present study to determine whether dexmedetomidine (DEX) has a direct protective effect against hypoxia-reoxygenation-induced left ventricular dysfunction without systemic hemodynamic and humoral effects. Isolated rat hearts were exposed to 60-min hypoxia followed by 30-min reoxygenation with 0, 10, or 100 nM DEX prehypoxia administration (n = 7 each group). In a second experiment (n = 7), 100 nM DEX was administered posthypoxia. In a third experiment (n = 7 each group), an alpha 2 antagonist, yohimbine was given with and without 100 nM DEX prehypoxia administration. DEX prehypoxia, but not posthypoxia, administration significantly improved the recovery of left ventricular developed pressure after reoxygenation (0, 10, 100 nM DEX prehypoxia or 100 nM DEX posthypoxia values were 53 +/- 6, 64 +/- 9, 78 +/- 13, or 62 +/- 12 mm Hg [mean +/- sd]) and reversed by yohimbine, 58 +/- 8 mm Hg, respectively. We conclude that DEX exerts the direct protective effect on the left ventricular dysfunction caused by hypoxia-reoxygenation through mainly alpha 2-adrenergic stimulation before and during the hypoxic period.
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