Molybdenum Disulfide Nanoparticles Resist ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Molybdenum Disulfide Nanoparticles Resist Oxidative Stressmediated Impairment of Autophagic Flux and Mitigate Endothelial Cell Senescence and Angiogenic Dysfunctions SunKui Ke, Youlin Lai, Tong Zhou, Lihuang Li, Yange Wang, Lei Ren, and Shefang Ye ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00714 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Molybdenum Disulfide Nanoparticles Resist Oxidative Stress-mediated Impairment of Autophagic Flux and Mitigate Endothelial Cell Senescence and Angiogenic Dysfunctions

Sunkui Ke,†, # Youlin Lai,§, # Tong Zhou,‡ Lihuang Li,‡ Yange Wang,‡ Lei Ren,‡ Shefang Ye,*, ‡

†

Department of Thoracic Surgery, Zhongshan Hospital of Xiamen University, Xiamen,

361004, P. R. China ‡

Department of Biomaterials, College of Materials, Xiamen University, Xiamen

361005, P. R. China §

Department of Obstetrics, Xiamen Maternity and Care Hospital, Xiamen 361000, P.

R. China

Corresponding author: Shefang Ye, PhD, associate professor Department of Biomaterials, College of Materials, Xiamen University, 422 Siming South Road, Xiamen 361005, China Tel: +86-592-2183058; Fax: +86-592-2183058. E-mail: [email protected] #

These authors contributed equally to this work.

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The impairment of autophagy involves oxidative stress-induced cellular senescence, leading to endothelial dysfunctions and the onset of cardiovascular diseases. As molybdenum disulfide nanoparticles (MoS2 NPs), a representative transition metal dichacogenide materials, has great potential as a multifunctional therapeutic agent against various disorders, the present study aimed to investigate whether MoS2 NPs prevents hydrogen peroxide (H2O2)-induced endothelial senescence by modulating autophagic process. Our results showed that pretreatment with MoS2 NPs inhibited H2O2-induced endothelial senescence and improved endothelial functions. Exposure of H2O2 increased p62 level and blocked the fusion of autophagosomes with lysosomes, indicating of impaired autophagic flux in senescent endothelial cells. However, MoS2 NPs pretreatment efficiently suppressed cellular senescence through triggering autophagy and resisting impaired autophagic flux. Furthermore, the genetic inhibition of autophagy by siRNA against Beclin 1 or ATG-5 directly abrogated the protective action of MoS2 NPs on endothelial cells against H2O2-induced senescence.Thus, these results suggested that MoS2 NPs rescue endothelial cells from H2O2-induced senescence by improving autophagic flux, and provide valuable information for the rational design of MoS2-based nanomaterials for therapeutic use in senescence-related diseases. KEY WORDS: MoS2 nanoparticles, senescence, autophagic flux, endothelial dysfunction


Page 2 of 51

Page 3 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION

Aging is characterized by a gradual decline in biological processes due to the increased accumulation of senescent cells, which featured as cell cycle arrest, declined metabolism, and impaired functions.1,2 Vascular endothelium, critically situated at the interface between blood and the vessel, involved in the regulation of vascular homeostasis.3 However, the occurrence of endothelial senescence hampered the endothelial barrier integrity and nitric oxide (NO) bioavailability, resulted in impairment of angiogenesis, and triggered the increased expression of prothrombotic and proinflammatory mediators,4,5 thus contributing to the pathogenesis of cardiovascular disease, such as chemic heart failure, coronary artery disease, and myocardial infarction. It is plausible that delaying cell senescence can enhance viability and function of vascular endothelial cells. Recent studies highlights a critical role of reactive oxygen species (ROS)-induced cellular damage in promoting senescence.6 Therefore, interventions for alleviating oxidative stress may aid in the design of novel therapeutic approaches to delay or prevent the onset of cardiovascular diseases. Autophagy is the highly conserved cellular process of self-catabolic degradation, by which cytoplasmic components including proteins and organelles become engulfed by autophagosomes.7 The resulting autophagosomes ultimately fuse with lysosomes to generate autolysosome, in which degradation occured.8 Recently, autophagy is an emerging field in studies of longevity,9 and growing evidence has indicated that an age-related loss in autophagic activity has been believed to determine the level of



Page 4 of 51

cellular senescence.10 Furthermore, a significant decline in autophagosome formation and maturation, as well as impaired autophagosome-lysosome fusion were observed with age in vitro and in vivo.11,12 Consistent with this hypothesis, a decrease in autophagic activity been suggested to be closely correlated with the senescent endothelial cells, which may contributes to the development of cardiovascular diseases.13 Thus, therapeutic targeting of autophagy represents a potential strategy for treating or preventing vascular diseases. Motivated by nanotechnology, several typical inorganic nanoparticles (NPs), including gold NPs14 and fullerenol,15 have been investigated as efficient inhibitors for cellular senescence. The mechanisms responsible for the inhibitory effects of NPs on senescence are mainly attributed to their antioxidant properties.14,15 However, the pathogenesis of senescence-associated diseases involves several other complicated factors, such as increased ROS accumulation, reduced activity of autophagy, impaired autophagic flux, which may cooperatively exacerbate endothelial cell dysfunctions. Thus, exploring new nanomaterials with multifunctional performance toward senescence is of great importance. Molybdenum

disulfide

nanoparticles

(MoS2 NPs)

as

transition

metal

dichalcogenides matrials has been widely explored for potential applications, including emitting transistors, sensors, optoelectronics, filtration nanodevice, and hydrogen storage media.16 MoS2 has also showed bright prospects in biomedical applications. For example, MoS2 can serve as novel nanoconstructs for multimodal biomedical imaging,17 near-infrared light triggered photodynamic therapy (PDT),18


Page 5 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DNA/protein biosensors,19,20 and genetic engineering21 due to outstanding thermal/mechanical stability, and exceptional electronical and optical properties. Particularly, MoS2 NPs exhibits high biological stability, favorable biocompatibility and low cytotoxicity,22-24 and have been recently used as an efficient antimicrobial and antifungal agent,25 free radical scavengers,22,26,27 β-amyloid peptide aggregation inhibitors.27 Inspired by its multifunctional performance toward various disorders, we thus hypothesized that MoS2 NPs might prevent senescence and dysfunctions of endothelial cells mediated by ROS. As it is generally believed that autophagy is a essential protective mechanism against cellular senescence, the present study aimed to to investigate the potential roles of autophagy and autophagic flux in the inhibitory effect of MoS2 NPs on endothelial senescence. Herein, we demonstrated that as-prepared MoS2 NPs exhibit remarkable capability to inhibit senescence-associated features and resist ROS-mediated impairment of autophagic flux. Furthermore, genetic inhibition of autophagy by siRNA against Beclin or ATG-5 abrogated the protective action of MoS2 NPs on endothelial cells against H2O2-induced senescence.

EXPERIMENTAL SECTION Preparation of MoS2 NPs and Particle Suspensions. MoS2 NPs were synthesized

using a pulsed laser ablation (PLA) method in polyvinylpyrrolidone (PVP) solution as described.27 Briefly, MoS2 powder (99.99% pure; Sigma, St. Louis, MO, USA) was formed into a disc-shaped pellet with a diameter of 15 mm, and the pellet was settled at the bottom of a quartz cell, into which 5 mL of PVP solution was added. Then, a



Nd:YAG pulsed laser (532 nm) operating at 10 Hz with a pusle energy 100 mJ was used for ablation for 10 min under continuously stirring. Finally, the resulting MoS2 NPs were filtered and precipitated by centrifugation at 20000 rpm for 12 h. The morphologies of as-prepared MoS2 NPs were characterized by TEM (TEM-2100, JEOL Ltd., Japan) (Figure 1a). MoS2 NPs exhibited round shape with an average diameter of approximately 100 nm. Figure 1b shows that the multi-layered structure of individual MoS2 NPs is quite clear. The element mappings of samples were examined by field emission scanning electron microscopy S-4800 FESEM (Hitachi, Japan), and results confirmed the coexistence of Mo and S elements in MoS2 NPs (Figure 1c). DLS data were obtained using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK), showing that the average diameter of PVP-MoS2 NPs was 107 nm (Figure 1d), 117 nm (Figure S1a) in culture medium before and after ultrasonic treatment at 6 h, respectively, indicating that the PVP-MoS2 NPs exhibit favourable colloidal stability. As shown in Figure 1e, PVP-functionalized MoS2 NPs exhibited three characteristic peaks at 1630, 1420, and 1280 cm-1, which correspond to C=O, C-H2, and C-N stretching, respectively, by Fourier transform infrared spectroscopy (FT-IR; iN10 MX IR, Nicolet). Futhermore, thermogravimetric analysis (TGA; STA 409 PC, Netzsch,Germany) indicated that 16.3% of PVP was bound on the surface of MoS2 NPs (Figure S1b). These results verified that PVP was successfully anchored to the MoS2 NPs surface. PVP-MoS2 NPs were dispersed in sterile water by sonication, and a series of working solutions was freshly made by diluting the stock solution (1 mg/mL) with cell culture medium supplemented with


Page 6 of 51

Page 7 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10% FBS and mixed thoroughly before use. Cell Cultures and Senescence Associated β-galactosidase (SA-β β -gal) Activity. Human aortic endothelial cells (HAECs) (Lonza, Walkersville, MD, USA) were maintained in endothelial basal medium-2, supplemented with EGM-2 SingleQuots (Lonza, Walkersville, MD, USA) according to the manufacturer’s recommendation. Experiments were carried out using 4-6 passages of HAECs. HAECs at 80% confluency in 12-well plates were preincubated with 50 µg/mL MoS2 NPs for 6 h. After treatment, the medium was removed, and the cells were thoroughly washed thrice with PBS, and then exposed to 400 µM H2O2 up to 5 days. Cytochemical staining for SA-β-gal was detected with SA-β-gal staining kit (Cell Signaling Technology, MA, USA) following the manufacturer’s protocol. Cultures were then examined under phase-contrast microscopy. Cells were scored as cytosolic blue staining, and the percentage of SA-β-gal-positive cells was counted. To evaluate the autophagic flux on the progress of senescence induced by H2O2, various reagents including chloroquine and bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA) were added into the culture medium after H2O2 treatment. Bromodeoxyuridine (BrdU) Incorporation and Propidium Iodide (PI) Cell Cycle Analysis. Cell proliferation was assessed by BrdU incorporation into cellular DNA at 72 h after exposed to H2O2 with or without MoS2 NPs pretreatment. Briefly, HAECs were incubated with 10 µM BrdU (Sigma Aldrich, St Louis, MO, USA) for 30 min. After washing, cells were probed with anti-BrdU antibody (Cell Signaling Technology, Danvers, MA, USA) followed by incubating with an Alexa Fluor



488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Then, cell nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR, USA). BrdU-positive cells were visualized and photographed using a fluorescent microscope (BX51; Olympus, Tokyo, Japan). To gate different phases of cell cycle, HAECs were gathered by trypsinization, washed with PBS, and fixed in 70 % ethanol at -20 °C for 30 min. After washing, cells were stained with 0.5 mL PI/RNase Staining Solution at 37 °C for 1 h in the dark. Cell cycle analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Enzyme-linked Immunosorbent Assay (ELISA). Samples of HAECs culture supernatants were gathered at 24 h following treatment with 400 µM H2O2, and ELISA assay was applied to determine the levels of interleukin (IL)-6 and plasminogen activator inhibitor (PAI)-1 following the manufacturer's protocol (R&D Systems, Minneapolis, MN). Measurements were performed using a Benchmark microplate reader (Bio-Rad Laboratories, CA, USA). Values were normalized to 10,000 cells. Tube Formation and Cell Migration Assay. HAECs were plated on to a Matrigel matrix-coated 48-well plates (BD Biosciences; San Jose, CA) at a density of 5 × 104 cells/well. To enhance the visibility of tube and network formation in Matrigel, cells were labeled with 1 µM Calcein acetoxymethyl ester (AM) (Life Technology, Invitrogen, USA), and incubated for 30 min at 37 °C. After washing, formation of tube structures was examined with a fluorescence microscope. Tube number (a connection between two junctions) and network number (> 2 tubes branching from a


Page 8 of 51

Page 9 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


junction) were counted from each well. Cell migration assay was carried out using BD Falcon TM Cell Culture Inserts (BD Bioscience, Bedford, MA; pore size, 8 µm) in 24-well culture plates, and chamber slides were precoated with 20 µg/ml fibronectin. After treatment, HAECs were resuspended in serum-free medium for 12 h, and plated into the upper chambers, then lower chambers were added with serum-free media or media supplemented with 50 ng/ml VEGF. After 24 h, cells that migrated through the filter were fixed, stained with 0.5% crystal violet, then observed under a microscope. Protein Extraction and Western Blot Analysis. Cells were scraped, harvested and lysed in a RIPA buffer with protease inhibitors (Cocktail; Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were resolved by SDS-PAGE, and electroblotted to a polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were incubated were then incubated with antibodies specific for p21, p16, p53, p-p53, Cathepsin B (Santa Cruz Biotechnology Inc., California, USA), LC3-I/II, Becline 1, ATG-5, GAPDH (Cell Signaling Technology Inc., Danvers, MA). The blots were revealed using horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc., California, USA). Immunoreactive bands were developed using an enhanced chemiluminescence (NEL103001EA; Perkin Elmer, Waltham, MA, USA). The analysis of intensity of chemiluminescence signal was performed using ImageJ software (NIH, Bethesda, MD, USA). The results are presented as arbitrary units. Immunofluorescence. For transient GFP-LC3 transfection, cells at a density of 4.5 × 105 cells/well were plated onto coverslips in 6-well plates as described previously,28



Page 10 of 51

and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. At 24 h posttransfection, HACEs were incubated in complete medium containing 50 nM Lysotracker red DND-99 (Molecular Probes, Eugene, OR) for 1 h. After washing with PBS twice, cells were fixed and counterstained with DAPI, and then observed under an Fluoview FV1000 Confocal Microscope

(Olympus,

Tokyo,

Japan).

The

number

of

LC3-II-positive

autophagosomes/cell in at least 50 GFP-positive cells for each group were counted as discribed.

29

To determine the percentage colocalization of GFP-LC3-II and

LysoTracker Red, images were analyzed with ImageJ software (NIH, Bethesda, MD, USA) and the ratios of green or red cells to merged cells were calculated using the colocalization plug-in according to the method proposed by Mareninova et al.

30

For

immuostaining for γ-H2AX, HACEs cells were incubated with γ-H2AX (Cell Signaling Technology, Danvers, MA, USA) for 1.5 h according to the manufacturers’ protocols. After stained with Hoechst 33342, cells were observed under a fluorescence microscope. Acridine Orange Staining. HACEs were seeded onto on glass coverslips and pretreated with MoS2 NPs (50 µg/mL) for 6 h prior to exposure to 400 µM H2O2 for 6 h. The cells were then stained with acridine orange at 1 µg/ml(Sigma, St. Louis, MO, USA) at 37°C for 15 min, and washed with PBS, and then immediately observed under a microscope.


Page 11 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Determination of Mitochondrial ROS Production, Mitochondrial Membrane Potential (∆Ψm) and Total Cellular ATP level. The production of mitochondrial superoxide radicals in HAECs were measured with a MitoSOX Red fluorescent probe (Invitrogen, Carlsbad, CA) as described previously.28 ∆Ψm was monitored by a mitochondrial-specific dual fluorescence probe, JC-1(Molecular Probes, Eugene, OR) according to the manufacturer's instructions (Molecular Probes, Eugene, OR). The ATP Assay Kit (Beyotime, China) was applied to measure intracellular ATP levels according to the manufacturer’s protocol, and the BCA Protein Assay Kit was used to evaluate the concentration of protein. ATP concentrations were expressed as µmol/g of protein. Transfection of Small Interfering RNA (siRNA). Sequences for siRNA directed against Beclin 1, ATG-5 and control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HAECs were maintained at 37 °C in a humidified atmosphere of 5% CO2, and transfections were performed with cells that reached approximately 80% confluence using Lipofectamine 2000 following the manufacturer’s guidelines (Invitrogen, Carlsbad, CA). For all siRNA experiments, the siRNAs were transfected at a final concentration of 20 nM. After incubation for additional 48 h, immunoblotting assay was performed to assess the knockdown efficiency of Beclin 1 and ATG-5 expression. Statistical Analysis. The results are presented as means ± standard deviation (SD) of three independent experiments. The differences of measurement data was analyzed



by the t-test (two-sided) or one-way ANOVA. A p value less than 0.05 was regarded as statistically significant.

Results H2O2 Treatment Induced Senescence with Dysfunctions in HAECs. H2O2-induced endothelial senescence has been commonly used as a model of oxidative stress-induced senescence.31 In this study, a H2O2-induced senescence in HAECs was established and the progress of cellular senescence was monitored for 5 d (Figure 2a). As shown, exposure of HAECs to H2O2 for 3 days induced an flattened, and enlarged cell phenotypes, exhibiting strong positive staining for SA-β-gal (Figure 2b), a distinguishing biochemical feature of cellular senescence. Cessation of cell proliferation, representing another feature of cellular senescence, was also observed in H2O2-treated cells, as revealed by BrdU incorporation assay (Figure 2b). In addition, the p16 and p21 expression, cyclin-dependent kinase inhibitors known to be responsible for cell cycle arrest,32 and the levels of IL-6 and PAI-1,33 the senescence-associated secretory phenotype of cells, were also elevated in H2O2-treated cells (Figure 2c-e). Pretreatment with MoS2 NPs Inhibits H2O2-induced Senescence. The inhibitory effects of MoS2 NPs on endothelial senescences were evaluated in H2O2-induced senescence model. To initially screen the sensitivity of HAECs to MoS2 NPs, cell viability was determined 24 h after exposed to increasing concentrations of MoS2 NPs by the MTT assay. As shown in Figure S3, MoS2 NPs did not dramatically affect


Page 12 of 51

Page 13 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HAECs cell viability up to 100 µg/mL. To allow for comparison with previous studies, 50 µg/mL of MoS2 NPs was applied in subsequent experiments. As shown in Figure 3a,b the increase in the percentage of SA-β-Gal-positive cells was abolished by pretreatment of HACEs with MoS2 NPs for 6 h as compared to cells treated with H2O2. The anti-senescence effects of MoS2 NPs was also shown in H2O2 treated-HUVECs cells (Figure S4), indicating different senescent endothelial cells might share the same response to MoS2 NPs. To investigate whether MoS2 NPs could protect against endothelial senescence via cell cycle regulation, flow cytometric analysis of PI-stained cells was carried out. As shown in Figure 3a,c, H2O2 treatment caused a significant increase in G2/M phase arrest, while a decrease in G0/G1 phase cells, representing cell cycle arrest. However, pretreatment with MoS2 NPs significantly attenuated this effect. DNA damage often closely correlated with G2/M cell cycle arrest in premature senescence.34 As expected, H2O2 treatment led to a dramatic increase in γ−H2AX phosphorylation in the nucleus of HAECs, and this trend was significantly repressed by MoS2 NPs (Figure 3d). To corroborate the above results, we further examined the levels of proteins expression associated with cell cycle arrest and senescence. As shown in Figure 3e,f, immunoblot analysis indicated that cells exposed to H2O2 exhibited an increase in the expression of p16, p21, accompanied by upregulation of p53 and phosphorylated p53 (Ser15), whereas MoS2 NPs significantly abolished the upregulation of these proteins expression in senescent HAECs. The influence of MoS2 NPs on the toxicity of H2O2 may vary depending on the exposure order. Therefore, we further compared the results obtained from different



Page 14 of 51

exposure processes, which were illustrated in Figure S5. Our results provided evidence that H2O2-mediated upregulation of p16, p21 was greatly decreased by pretreatment with MoS2 NPs, while there was no significant difference observed via the other two manners, i. e., pretreatment with H2O2 or adding simultaneously with MoS2 NPs and H2O2 (Figure S5). The interaction between MoS2 NPs and HAECs cells was analyzed by TEM. As shown in Figure S6a, MoS2 NPs were readily identified within the cells, distributed in the cytoplasm and not found to be tightly attached to the cell surface. The amount of MoS2 NPs uptaken by cells was further verified by the side scatter (SSC) intensity assayed by flow cytometry (Figure S6b), and a time-dependent internalization after treatment of cells with MoS2 NPs at 50 µg/mL was observed. These findings suggested that the protective effects of MoS2 NPs might be attributed to their ability to enter the cells. Pretreatment Dysfunction.

To

with further

MoS2

NPs

investigate

Inhibits whether

H2O2-induced MoS2

NPs

Endothelial

would

prevent

senescence-associated endothelial dysfunctions in H2O2-treated HAECs, in vitro Matrigel angiogenesis assays were performed to examine angiogenic capacities. As shown in Figure 4a-c, there were less developed tubule networks on matrigel formed in senescent HAECs than untreated control, accompanied by a dramatic reduction in cell migration measured by transwell assay. As expected, pretreatment with MoS2 NPs prevented H2O2-mediated inhibition of endothelial tubule formation, cell migration in HAECs (Figure 4a-c). MoS2 NPs alone had no effect on angiogenic


Page 15 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


functions. As shown in Figure S7, H2O2-mediated cell senescence and angiogenic dysfunctions significantly prevented with the increasing concentrations of MoS2 NPs (25-50 µg/mL), and dose-effect relationships were observed. These results indicate that MoS2 NPs protects cells against oxidative stress-induced premature senescence and endothelial dysfunctions. To reveal the potential influence of surface properties of MoS2 NPs on H2O2-induced endothelial senescence and dysfunctions, HAECs were pretreated cells with either uncoated or PVP, and PEG coated MoS2 NPs (Figure S2 and S8). As shown in Figure S8, similiar to PVP-coated MoS2 NPs, pretreatment with PEG-coated MoS2 NPs could also obviously inhibited H2O2-induced endothelial senescence and improved endothelial functions, while uncoated MoS2 NPs was shown to be less effective. MoS2 NPs Induced Autophagy and Increased Autophagic Flux in HAECs. Next, we tracked changes in the formation of autophagosome using GFP-tagged LC3 As shown in Figure 5a, MoS2 NPs treatment significantly increased the punctate distribution of LC3-II in HAECs in a dose-dependent manner. The number of autophagic cells in control was about 10%, whereas in the presence of MoS2 NPs, this number increased up to 58 % (Figure 5b). To confirm the effect of MoS2 NPs on autophagy, we measured the conversion of cytosolic LC3-I into the lipidated LC3-II, which is tightly correlated with autophagosome formation.7 Western blotting indicated that MoS2 NPs dose-dependently increased the protein expression of LC3 II, indicative of the onset of autophagy in HAECs (Figure 5c,d). The protein p62 as a



marker of the autophagic flux is specifically degraded during autophagy.8 Consistently, HAECs treated with MoS2 NPs showed decreased p62 expressions in a dose-dependent manner compared with those in control cells (Figure 5c,e). In addition, there was no dramatic changes in p62 mRNA levels observed (Figures S9), suggesting that the changes in p62 protein levels may be attributed to autolysosomal degradation. We also measured other autophagy-related protein involved in autophagosome formation. Similarly, MoS2 NPs induced an increased in expression levels of ATG-5 and Beclin-1 in dose-dependent manner via immunoblotting analysis (Figure 5c,d). These findings suggested that MoS2 NPs is capable of inducing autophagy in HAECs. MoS2 NPs Resisted H2O2-impaired Autophagic Flux in Senescent HAECs. Accumulation of p62 protein levels has been considered as a marker of autophagic flux.9 Consistent with previous observation,35 the results showed that p62 protein in H2O2-treated HAECs was increased from day 3 to day 5 (Figure 6a,b). In addition, the autophagic flux in H2O2-treated HAECs was also assessed by analyzing p62 levels pretreatment with or without chloroquine (a lysosome acidification inhibitor). As shown in Figure 6c,d, in contrast with the control cells, chloroquine treatment did not cause an increased p62 level in H2O2-treated cells, suggesting that there is already-impairment of autophagic flux in senescent HAECs. We next examined the effect of MoS2 NPs on H2O2-impaired autophagic machinery. As seen in Figure 6e-g, although both H2O2 and MoS2 NPs treatment significantly triggered the conversion of LC3-I to LC3-II, H2O2 treatment resulted in an accumulation of p62 level, which reflected a block in autophagic flux. However,


Page 16 of 51

Page 17 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pretreatment with MoS2 NPs led to a further increase in the conversion of LC3-I to LC3-II, and a decrease in the p62 protein level compared with H2O2-treated HAECs, indicating that MoS2 NPs could resist autophagic flux (Figure 6e-g). We further investigated whether MoS2 NPs treatment affect autophagic flux in senescent HAECs by evaluating LC3-II levels with or without bafilomycin A1, an inhibitor of autophagosome/lysosome fusion. As compared with cells treated with H2O2 alone, bafilomycin A1 led to an increase in the conversion of LC3-I to LC3-II in MoS2 NPs-treated cells (Figure 6h,i). To study the fusion of autophagosome with lysosome, we next investigated autophagy flux by analyzing the colocalization between GFP-LC3 and Lysotracker Red fluorescence. As shown in Figure 7a,b, the numbers of colocalized yellow foci indicative of autophagosomes/lysosomes fusion were significantly increased in the MoS2 NPs-treated group compared with H2O2-treated group. MoS2 NPs Prevented Senescence-associated Lysosomal and Mitochondrial dysfunction. As a defect in autophagosome/lysosome fusion can be attributed to lysosomal dysfunctions,35 we sought to address whether the effect of MoS2 NPs on cellular senescence is associated with lysosomal and mitochondrial dysfunctions. With respect to lysosomal function, we first examined lysosome membrane permeability (LMP) by staining with lysomotropic dye acridine orange (AO). AO accumulates in acidic vesicles such as lysosome wiht red fluorescence, but emits green fluorescence in nucleus or cytoplasm.36 The changes in fluorescence can be applied to directly monitor LMP. As shown in Figure 8a, lysosomes in control cells



appeared as red fluorescence puncta with high intensity. By contrast, a decrease in fluorescence intensity of red puncta was visualized following H2O2 treatment (Figure 8a). As expected, MoS2 NPs treatment significantly attenuated the H2O2-induced decrease in fluorescence intensity in HAECs (Figure 8a,b). As a consequence of LMP, lysosomal proteases such as cathepsins and other hydrolytic enzymes were translocated from the lysosomal lumen to the cytosol.37 Results showed that the abundance of cathepsin B was reduced in senescent cells (Figure 8c,d). Pretreatment with MoS2 NPs, however, reverted the content of cathepsin B in lysosomes. Moreover, our data also confirmed that mitochondrial dysfunctions occurred in H2O2-treated cells, revealed by decreased ∆Ψm, reduced cellular ATP content, and increased mitochondrial ROS (Figure 8e-h). Accordingly, MoS2 NPs substantially prevented the H2O2-indued mitochondrial dysfunctions (Figure 8e-h). MoS2 NPs Prevents H2O2-induced Endothelial Cell Senescence by Promoting Autophagy. To ascertain the role of autophagy in the observed inhibition of H2O2-mediated endothelial senescence by MoS2 NPs, HAECs were transfected with siRNA for Beclin1 or ATG-5, two known core autophagy proteins.38,39 Immunoblotting analysis confirmed that the expression of either Beclin1 or ATG-5 was abrogated in cells transfected with Beclin- or ATG-5-specific siRNA (Figure 9a,b). As shown in Figure 9c,d, the depletion of Beclin or ATG-5 by siRNA treatment effectively blocked the conversion of LC3-I to LC3-II via western blotting analysis following MoS2 NPs treatment, suggesting that autophagy was inhibited in HAECs. The SA-β-Gal staining showed that the inhibitory effect of MoS2 NPs on


Page 18 of 51

Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H2O2-induced endothelial senescence was abolished in Beclin1 siRNA-transfected cells (Figure 9e). Likewise, similar results were obtained in ATG-5 siRNA-transfected cells by assessment of SA-β-gal staining (Figure S10). In addition, the decreased expression of expression of p21 and p16 by MoS2 NPs treatment was similarly prevented in Beclin siRNA-transfected cells (Figure 9g). These data provided mechanistic insights into MoS2 NPs-mediated autophagic regulation in the protection against H2O2-induced endothelial senescence.

DISCUSSION Our results clearly demonstrate that MoS2 NPs protects cells from senescence induced by oxidative stress, and the mechanism underlying the anti-senescence properties mainly involves stimulation of autophagy and enhancement of autophagic flux. Several lines of evidence are presented to support the hypothesis. First, MoS2 NPs attenuated the senescence-associated phenotype of HAECs under oxidative stress, accompanied by an increase in BrdU-positive cells and a reduction in G2/M arrest. Second, MoS2 NPs efficiently suppressed cellular senescence through a mechanism relevant to inducing autophagy and improving autophagic flux, as well as reversing lysosomal and mitochondrial dysfunction. Third, genetic inhibition of autophagy abrogated the protective effect of MoS2 NPs against oxidative stress-induced endothelial senescence. As endothelial senescence is believed to promote the development of many cardiovascular diseases such as atherosclerosis,40 our current findings will hopefully contribute to the development of new treatment strategies for



cardiovascular diseases. Autophagy serve as an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional cellular organelles and proteins in all living cells.41 Abnormalities in autophagy has been observed in aging and age-related diseases,8-10 and accumulating evidence has suggested that the rate of autophagosome formation and maturation as wells as the efficiency of autophagosome/lysosome fusion decline with age.11 As autophagy impairment has been shown to be crucial for senescence under oxidative stress, the restoration of autophagy activity could be a promising therapeutic strategy in treatment of senescence-related disorders.42 Consistent with previous reports,33,43 our findings showed that although an increase in the number of autophagosomes was shown in H2O2-induced senescent endothelial cells, the degradation of p62 and the autophagosome/lysosome fusion decreased, indicating that impaired autophagic flux with lysosomal dysfunction occurred in these cells (Figure 6 and 8). MoS2 NPs has gained the extensive attention due to unique physicochemical properties and multifunctional biological performance, such as antioxidant ability toward various disorders,25-27 thus prompting us to evaluate whether MoS2 NPs protect cells from senescence under oxidative stress through modulating autophagy activity. Based on the well-defined oxidative stress-induced senescence model, we found that pretreatment with MoS2 NPs could significantly resist impaired autophagic flux in senescent HAECs cells. By comparing with uncaoted MoS2 NPs, we showed that both PVP and PEG coated MoS2 NPs were more efficient to attenuate


Page 20 of 51

Page 21 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H2O2-induced endothelial senescence and dysfunctions (Figure S8). Similar cytoprotective autophagy by graphene oxide (GO) and silver NPs was also observed previously.44,45 Therefore,we hypothesized that pretreatment with MoS2 NPs allowed the efficient uptake of NPs by cells (Figure S6), which was responsible for the induction of autophagic effect. Furthermore, the genetic knockout of autophagy by transfection of siRNA specific to Beclin or ATG-5 demonstrated that autophagy is essential for the anti-senescent effect of MoS2 NPs on vascular endothelial cells. These findings supported the notion that autophagy induction is a promising strategy to counteract cellular senescence,46 and clarified the efficiency of functionalized MoS2 NPs-mediated autophagy activation, therefore presenting a reasonable target for anti-aging intervention. To further unravel the mechanism responsible for the anti-aging effect of MoS2 NPs, we particularly evaluated the status of autophagic flux in senescent cells in the presence of MoS2 NPs. The results regarding p62 degradation suggested that autophagic flux was blocked in senescent HAECs cells (Figure 6). Additionally, we also visulized that the colocalization of GFP-tagged LC3 (an autophagosome marker) with LysoTracker Red staining was prevented in H2O2-treated HAECs, demonstrating an impaired autophagosome-lysosome fusion in endothelial senescence. However, pretreatment with MoS2 NPs enhanced the fusion of autophagosome with lysosome, resulting in delaying senescence in H2O2-treated HAECs. Therefore, we attempted to speculate that enhanced autophagy and resistance to impaired autophagic flux are key to MoS2 NPs-mediated senescence prevention in vascular endothelial cells.



It has been suggested that oxidative stress is a fundamental cause in the aging process in endothelial cells, which initiate a programmed cascade of molecular and cellular events, consequently leading to lysosomal and mitochondrial dysfunction.47,48 In this H2O2-induced endothelial senescence model, our data showed that MoS2 NPs reduced cellular mitochondrial ROS level, maintained mitochondrial functions, and increased ATP levels, demonstrating that antioxidant effect of MoS2 NP is at least partially responsible for rescuing endothelial cell from oxidative stress-induced senescence. Similar findings have been reported for MoS2 NPs,27 which has been shown to counteract β-amyloid peptide (Aβ)-induced oxidative stress and associated cell toxicity by reducing the cellular ROS accumulation. Recently, it was proposed that the protective antioxidant/detoxification defense mechanisms mediated by MoS2 NPs may also result from activation of Nrf2/ARE antioxidant signaling leading to induction of phase II antioxidant enzyme.22 Therefore, the enhanced autophagy, resistance to impaired autophagic flux, and antioxidant effects coordinately play an essential role in MoS2 NPs-mediated attenuation of oxidative stress-induced endothelial senescence. In summary, using a H2O2-induced senescence model, we provide the first evidence that the antisenescence effects of MoS2 NP may involve autophagy activation, improvement of autophagic flux, and antioxidant activity, therefore suggesting that MoS2 NPs as multifunctional inhibitors targets multiple pathways to cooperatively prevent oxidative stress-induced endothelial senescence. These results provide significant insights into the role and mechanism of MoS2 NPs in vascular


Page 22 of 51

Page 23 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


senescence by regulating autophagic flux. Therefore, this study opens a new prospective in the design and construction of potent MoS2 NPs-based therapeutic strategies for endothelial senescence-related diseases.

Supporting Information Fig. S1. DLS and TGA analysis of PVP-MoS2 NPs; Fig. S2. SEM images, DLS analysis, and FT-IR spectra of uncoated, and PEG-modified MoS2 NPs; Fig. S3. Cell viability of HAECs cells by MTT after incubating with PVP-coated MoS2 NPs; Fig. S4. MoS2 NPs prevents H2O2-induced senescence in HUVECs. Fig. S5. Effects of PVP-coated MoS2 NPs on H2O2-mediated senescence in HAECs; Fig. S6. Uptake of MoS2 NPs by HAECs cells. Fig. S7. Dose-dependent effects of MoS2 NPs on H2O2-mediated senescence and angiogenic dysfunctions; Fig. S8. The effects MoS2 NPs with or without functionalization on H2O2-mediated senescence and angiogenic dysfunctions; Fig. S9. The effects MoS2 NPs on p62 mRNA levels assayed by quantitative PCR; Fig. S10. ATG5-mediated attenuation of endothelial senescence by MoS2 NPs. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: +86 592 2189650. Fax: +91 592 2189650.



Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the National Nature Science Foundation of China (31271071, 30901175, 81171448, U1505228).

REFERENCES 1.

Blagosklonny M. V. Cell senescence and hypermitogenic arrest. EMBO. Rep. 2003, 4, 358−362. DOI: 10.1038/sj.embor.embor806

2.

Takahashi, A.; Ohtani N.; Yamakoshi, K.; Iida, S.; Tahara, H.; Nakayama, K.; Nakayama, K. I.; Ide, T.; Saya, H.; Hara, E. Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol. 2006, 8, 1291−1297. DOI: 10.1038/ncb1491

3.

Lin, J. R.; Shen, W. L.; Yan, C.; Gao, P. J. Downregulation of dynamin-related protein 1 contributes to impaired autophagic flux and angiogenic function in senescent endothelial cells. Arterioscler Thromb Vasc Biol. 2015, 35,1413−1422. DOI: 10.1161/ATVBAHA.115.305706

4.

Mistriotis, P.; Andreadis, S.T. Vascular aging: Molecular mechanisms and potential treatments for vascular rejuvenation. Ageing Res. Rev. 2017, 37, 94−116 DOI: 10.1016/j.arr.2017.05.006

5.

Paneni, F.; Diaz Cañestro, C.; Libby, P.; Lüscher, T. F.; Camici, G. G. The Aging


Page 24 of 51

Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cardiovascular System: Understanding It at the Cellular and Clinical Levels. J. Am. Coll. Cardiol. 2017, 69, 1952−1967. DOI: 10.1016/j.jacc.2017.01.064 6.

Marazita, M. C.; Dugour, A.; Marquioni-Ramella, M. D.; Figueroa, J. M.; Suburo, A. M. Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression in retinal pigment epithelial cells: Implications for Age-related Macular

Degeneration.

Redox

Biol.

2016,

7,

78 − 87.

DOI:

10.1016/j.redox.2015.11.011 7.

Choi, A.M; Ryter, S. W.; Levine, B. Autophagy in human health and disease. N. Engl. J. Med. 2013, 368, 651−662. DOI: 10.1056/NEJMra1205406

8.

Lionaki, E.; Markaki, M.; Tavernarakis, N. Autophagy and ageing: insights from invertebrate model organisms. Ageing Res. Rev. 2013, 12, 413−428. DOI: 10.1016/j.arr.2012.05.001

9.

Alberti, A.; Michelet, X.; Djeddi, A.; Legouis R. The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans. Autophagy, 2010, 6, 622−633. DOI: 10.4161/auto.6.5.12252

10. Pyo, J. O.; Yoo, S. M.; Jung, Y. K. The interplay between autophagy and aging. Diabetes Metab. J. 2013, 37, 333−339. DOI: 10.4093/dmj.2013.37.5.333 11. Bergamini, E. Autophagy: a cell repair mechanismthat retards ageing and age-associated diseases and can be intensified pharmacologically. Mol. Aspects Med. 2006, 27, 403−410. DOI: 10.1016/j.mam.2006.08.001 12. Rubinsztein, D. C.; Mariño, G.; Kroemer, G. Autophagy and aging. Cell. 2011, 146, 682-695. DOI: 10.1016/j.cell.2011.07.030



Page 26 of 51

13. LaRocca, T. J.; Henson, G. D.; Thorburn, A.; Sindler, A. L.; Pierce, G. L.; Seals, D. R. Translational evidence that impaired autophagy contributes to arterial ageing. J Physiol. 2012, 590, 3305−3316. DOI: 10.1113/jphysiol.2012.229690 14. Chae, S. Y.; Park, S. Y.; Park, J. O.; Lee, K. J.; Park, G. Gardenia jasminoides extract-capped gold nanoparticles reverse hydrogen peroxide-induced premature senescence. J. Photochem. Photobiol. B. 2016, 164, 204 − 211. DOI: 10.1016/j.jphotobiol.2016.09.033 15. Zhuge, C. C.; Xu, J. Y.; Zhang, J.; Li, W.; Li, P.; Li, Z.; Chen, L.; Liu, X.; Shang, P.; Xu, H.; Lu, Y.; Wang, F.; Lu, L.; Xu, G. T. Fullerenol protects retinal pigment epithelial cells from oxidative stress-induced premature senescence via activating SIRT1.

Invest.

Ophthalmol.

Vis.

Sci.

2014,

55,4628 − 4638.

DOI:

10.1167/iovs.13-13732 16. Adini, A. R.; Redlich, M.; Tenne, R. Medical Applications of Inorganic Fullerene-Like Nanoparticles. J. Mater. Chem. 2011, 21, 15121−15131. DOI: 10.1039/c1jm11799h 17. Liu, T.; Shi, S.; Liang, C.; Shen, S.; Cheng, L.; Wang, C.; Song, X.; Goel, S.; Barnhart, T. E.; Cai, W.; Liu, Z. Iron oxide decorated MoS2 nanosheets with double PEGylation for chelator-free radiolabeling and multimodal imaging guided photothermal therapy. ACS Nano. 2015, 9, 950 − 960. DOI: 10.1021/nn506757x 18. Liu, T.; Wang, C.; Cui, W.; Gong, H.; Liang, C.; Shi, X.; Li, Z.; Sun, B.; Liu, Z. Combined photothermal and photodynamic therapy delivered by PEGylated


Page 27 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MoS2 nanosheets. Nanoscale. 2014, 6, 11219−11225. DOI: 10.1039/c4nr03753g 19. Wang, L.; Wang, Y.; Wong, J. I.; Palacios, T.; Kong, J.; Yang, H. Y. Functionalized MoS(2) nanosheet-based field-effect biosensor for label-free sensitive detection of cancer marker proteins in solution. Small. 2014,10, 1101− 1105. DOI: 10.1002/smll.201302081 20. Zhu, C.; Zeng, Z.; Li, H.; Li, F.; Fan, C.; Zhang, H. Single-layer MoS2-based nanoprobes for homogeneous detection of biomolecules. J. Am. Chem. Soc. 2013, 135, 5998−6001. DOI: 10.1021/ja4019572 21. Kou, Z.; Wang, X.; Yuan, R.; Chen, H.; Zhi, Q.; Gao, L.; Wang, B.; Guo, Z.; Xue, X.; Cao, W.; Guo, L. A promising gene delivery system developed from PEGylated MoS2 nanosheets for gene therapy. Nanoscale Res. Lett. 2014, 9, 587. DOI: 10.1186/1556-276X-9-587 22. Pardo, M.; Shustermeiseles, T.; Levinzaidman, S.; Rudich, A.; Rudich, Y. Low Cytotoxicity of Inorganic Nanotubes and Fullerene-Like Nanostructures in Human Bronchial Epithelial Cells: Relation to Inflammatory Gene Induction and Antioxidant Response. Environ. Sci. Technol. 2014, 48, 3457 − 3466. DOI: 10.1021/es500065z 23. Wu, H.; Yang, R.; Song, B.; Han, Q.; Li, J.; Zhang, Y.; Fang, Y.;Tenne, R.; Wang, C. Biocompatible Inorganic Fullerene-Like Molybdenum Disulfide Nanoparticles Produced by Pulsed Laser Ablation in Water. ACS Nano. 2011, 5, 1276−1281. DOI: 10.1021/nn102941b 24. Hao, J.; Song, G.; Liu, T.; Yi, X.; Yang, K.; Cheng, L.; Liu, Z. In Vivo Long-Term



Page 28 of 51

Biodistribution, Excretion, and Toxicology of PEGylated Transition-Metal Dichalcogenides MS2 (M = Mo, W, Ti) Nanosheets. Adv. Sci. (Weinh). 2016, 4, 1600160. DOI: 10.1002/advs.201600160 25. Yang, X.; Li, J.; Liang, T.; Ma, C.; Zhang, Y.; Chen, H.; Hanagata, N.; Su, H.; Xu, M. Antibacterial activity of two-dimensional MoS2 sheets. Nanoscale. 2014, 6, 10126−10133. DOI: 10.1039/c4nr01965b 26. Ji, DK.; Zhang, Y.; Zang, Y.; Li, J.; Chen, GR.; He, XP.; Tian, H. Targeted Intracellular Production of Reactive Oxygen Species by a 2D Molybdenum Disulfide

Glycosheet.

Adv.

Mater.

2016,

28,

9356 − 9363.

DOI:

10.1002/adma.201602748 27. Han, Q.; Cai, S.; Yang, L.; Wang, X.; Qi, C.; Yang, R.; Wang, C. Molybdenum Disulfide Nanoparticles as Multifunctional Inhibitors against Alzheimer's Disease. ACS

Appl.

Mater.

Interfaces.

2017,

9,

21116 − 21123.

DOI:

10.1021/acsami.7b03816 28. Ye, S.; Yang, P.; Cheng, K.; Zhou, T.; Wang, Y.; Hou, Z.; Jiang, Y.; Ren, L. Drp1-dependent mitochondrial fission mediates toxicity of positively charged graphene in microglia. ACS Biomater. Sci. Eng. 2016, 2, 722–733. DOI: 10.1021/acsbiomaterials.5b00465 29. Zhang, S. F.; Wang, X. Y.; Fu, Z. Q.; Peng, Q. H.; Zhang, J. Y.; Ye, F.; Fu, Y. F.; Zhou, C. Y.; Lu, W. G.; Cheng, X. D.; Xie, X. TXNDC17 promotes paclitaxel resistance via inducing autophagy in ovarian cancer. Autophagy. 2015, 11, 225–238. DOI: 10.1080/15548627.2014.998931


Page 29 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30. Mareninova, O. A.; Hermann, K.; French, S. W.; O'Konski, M. S.; Pandol, S. J.; Webster, P.; Erickson, A. H.; Katunuma, N.; Gorelick, F. S.; Gukovsky, I.; Gukovskaya, A. S. Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis. J. Clin. Invest. 2009, 119, 3340−3355. DOI: 10.1172/JCI38674 31. Wang, Z.; Wei, D.; Xiao, H. Methods of cellular senescence induction using oxidative

stress.

Methods

Mol.

Biol.

2013,1048,

135 − 144.

DOI:

10.1007/978-1-62703-556-9_11 32. He, L.; Chen, Y.; Feng, J.; Sun, W.; Li, S.; Ou, M.; Tang, L. Cellular senescence regulated by SWI/SNF complex subunits through p53/p21 and p16/pRB pathway. Int. J. Biochem. Cell Biol. 2017, 90, 29−37. DOI: 10.1016/j.biocel.2017.07.007 33. Oubaha, M.; Miloudi, K.; Dejda, A.; Guber, V.; Mawambo, G.; Germain, M. A.; Bourdel, G.; Popovic, N.; Rezende, F. A.; Kaufman, R. J.; Mallette, F. A.; Sapieha, P. Senescence-associated secretory phenotype contributes to pathological angiogenesis in retinopathy. Sci. Transl. Med. 2016, 8, 362ra144. DOI: 10.1126/scitranslmed.aaf9440 34. Anwar, T.; Khosla, S.; Ramakrishna, G. Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status. Cell Cycle. 2016, 15, 1883−1897. DOI: 10.1080/15384101.2016.1189041 35. Tai, H.; Wang, Z.; Gong, H.; Han, X.; Zhou, J.; Wang, X.; Wei, X.; Ding, Y.; Huang, N.; Qin, J.; Zhang, J.; Wang, S.; Gao, F.; Chrzanowska-Lightowlers, Z. M.; Xiang, R.; Xiao, H. Autophagy impairment with lysosomal and



mitochondrial stress-induced

dysfunction senescence.

is

an

important

Autophagy.

Page 30 of 51

characteristic

2017,

13,

of

oxidative

99 − 113.

DOI:

10.1080/15548627.2016.1247143 36. Pierzyńska-Mach, A.; Janowski, P. A.; Dobrucki, J. W. Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles. Cytometry A. 2014, 85, 729 − 737. DOI: 10.1002/cyto.a.22495 37. Yu, L.; McPhee, C. K.; Zheng, L.; Mardones, G. A.; Rong, Y.; Peng, J.; Mi, N.; Zhao, Y.; Liu, Z.; Wan, F.; Hailey, D. W.; Oorschot, V.; Klumperman, J.; Baehrecke, E. H.; Lenardo, M. J. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 2010, 465, 942 − 946. DOI: 10.1038/nature09076 38. Wesselborg, S.; Stork, B. Autophagy signal transduction by ATG proteins: from hierarchies to networks. Cell Mol. Life Sci. 2015, 72, 4721 − 4757. DOI: 10.1007/s00018-015-2034-8 39. Fu, L. L.; Cheng, Y.; Liu, B. Beclin-1: autophagic regulator and therapeutic target in cancer. Int. J. Biochem. Cell Biol. 2013, 45, 921 − 924. DOI: 10.1016/j.biocel.2013.02.007 40. Donato, A. J.; Morgan, R. G.; Walker, A. E.; Lesniewski, L. A. Cellular and molecular biology of aging endothelial cells. J Mol Cell Cardiol. 2015, 89, 122–135. DOI: 10.1016/j.yjmcc.2015.01.021 41. Li, Y. Y.; Zhou, Y. L.; Wang, H. Y.; Perrett, S.; Zhao, Y. L.; Tang, Z. Y.; Nie, G.


Page 31 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J. Chirality of Glutathione Surface Coating Affects the Cytotoxicity of Quantum Dots.

Angew.

Chem.

Int.

Ed.

2011,

50,

5860–5864

DOI:

10.1002/anie.201008206 42. Rajawat, Y. S.; Hilioti, Z.; Bossis, I. Aging: Central Role for Autophagy and the Lysosomal Degradative System. Ageing Res. Rev. 2009, 8, 199−213. DOI: 10.1016/j.arr.2009.05.001 43. Han. X.; Tai, H.; Wang, X.; Wang, Z.; Zhou, J.; Wei, X.; Ding, Y.; Gong, H.; Mo, C.; Zhang, J.; Qin, J.; Ma, Y.; Huang, N.; Xiang, R.; Xiao, H. AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation. Aging Cell. 2016, 15, 416−427. DOI: 10.1111/acel.12446 44. Liu, Y.; Wang, X.; Wang, J.; Nie, Y.; Du, H.; Dai, H.; Wang, J.; Wang, M.; Chen, S.; Hei, T. K.; Deng, Z.; Wu, L.; Xu, A. Graphene Oxide Attenuates the Cytotoxicity and Mutagenicity of PCB 52 via Activation of Genuine Autophagy. Environ Sci Technol. 2016, 50, 3154–3164. DOI: 10.1021/acs.est.5b03895 45. Lin, J.; Huang, Z. H.; Wu, H.; Zhou, W.; Jin, P. P.; Wei, P. F.; Zhang, Y. J.; Zheng, F.; Zhang, J. Q.; Xu, J.; Hu, Y.; Wang, Y. H.; Li, Y. J.; Gu, N.; Wen, L. P. Inhibition of autophagy enhances the anticancer activity of silver nanoparticles. Autophagy. 2014, 10, 2006–2020. DOI: 10.4161/auto.36293 46. Saraswat, K.; Rizvi, S. I. Novel strategies for anti-aging drug discovery. Expert. Opin. Drug Discov. 2017, 12, 955−966. DOI: 10.1080/17460441.2017.1349750 47. Regina, C.; Panatta, E.; Candi, E.; Melino, G.; Amelio, I.; Balistreri, C. R.;



Annicchiarico-Petruzzelli, M.; Di Daniele, N.; Ruvolo, G. Vascular ageing and endothelial cell senescence: Molecular mechanisms of physiology and diseases. Mech. Ageing Dev. 2016,159:14−21. DOI: 10.1016/j.mad.2016.05.003 48. El Assar, M.; Angulo, J.; Rodríguez-Mañas, L. Oxidative stress and vascular inflammation in aging. Free Radic. Biol. Med. 2013, 65, 380 − 401. DOI: 10.1016/j.freeradbiomed.2013.07.003


Page 32 of 51

Page 33 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures and legends Figure 1. Physicochemical properties of MoS2 NPs. (a) Typical TEM image, and (b) high-magnification TEM image of MoS2 NPs. (c) Elemental mapping images (Mo, S) of MoS2 NPs. (d) Size distribution of MoS2 NPs in cell culture medium measured by DLS. (e) FT-IR spectra of PVP-functionalized MoS2 NPs.

Figure 2. Stress-induced senescence cell model was established in HAECs by H2O2 treatment. Cells were treated with or without 400 µM H2O2 for 45 min, and transferred to a 24-well plate in complete medium for the indicated days, then SA-β-gal-positive cells were detected by using a SA-β-gal staining kit. (a) The SA-β-gal staining and BrdU-positive cells were calculated and the results were presented as percentage of stained cells. (b) Representative images of SA-β-gal staining and BrdU immunostaining of cells. (c,d) Western blotting and quantitative analysis of p21 and p16 protein levels in H2O2-induced senescent HAECs. (e) Expression level of IL-6, PAI-1 quantified by ELISA assay in H2O2-induced senescent HAECs. Data represent the mean ± SD of three separate experiments. *P

Molybdenum Disulfide Nanoparticles Resist ... - ACS Publications

Recommend Documents