Representative TEM and SEM images of (A, C) MWCNTs-COOH and (B, D) MWCNTs-PEG.

<p><b>(E) FT-IR spectra of MWCNTs-COOH and MWCNTs-PEG.</b></p

MWCNTs-COOH and MWCNTs-PEG induced ROS generation in RAW 264.7 cells.

<p>Cells were treated with or without 75 µg/mL of MWCNT samples for the indicated times, and the ROS levels were measured by (A) DCF staining and (B) HE staining using a flow cytometer or (C) fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. Hydrogen peroxide (H₂O₂) (1 mM) was used as the positive control. *<i>p</i><0.05 compared to control sample, #<i>p</i><0.05 compared to MWCNTs-PEG. Data represent similar results from three independent experiments.</p

MWCNTs-COOH and MWCNTs-PEG induced a mitochondria-associated death pathway in RAW 264.7 cells.

<p>(A, B) MWCNTs-COOH and MWCNTs-PEG induced the loss of Δ<i>Ψ</i>m in RAW 264.7 cells. Cells treated with or without 75 µg/mL of MWCNT samples for the indicated times were stained with the mitochondria-selective JC-1 dye, and then analyzed by flow cytometry and fluorescence microscope. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *<i>p</i><0.05 compared to control sample, #<i>p</i><0.05 compared to MWCNTs-PEG. (C) Western blot analysis of the effects of MWCNTs-COOH and MWCNTs-PEG on translocation of cyto c, and expression of Bax and Bcl-2 in RAW 264.7 cells. Cells were treated with or without 75 µg/mL of MWCNT samples for 24 h. Data represent similar results from three independent experiments.</p

Effects of MWCNTs-COOH and MWCNTs-PEG on NADPH oxidase activity.

<p>RAW 264.7 cells were treated with or without 75 µg/mL of either (A) MWCNTs-COOH or (B) MWCNTs-PEG for up to 12 h, and NADPH oxidase was measured. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *<i>p</i><0.05 compared to control sample. (C) MWCNTs-COOH and MWCNTs-PEG stimulated membrane translocation of p47^phox and p67^phox. After treatment of RAW 264.7 cells with or without MWCNT samples for 12 h, cell lysates were isolated from the membrane fraction. Western blot analysis was used to detect p47^phox and p67^phox. Blots are representative of at least three independent experiments. (D) Immunolocalization of p47^phox after exposure of RAW 264.7 cells to 75 µg/mL of MWCNT samples for 12 h. Images are representative of three independent experiments.</p

Cytotoxic effects of MWCNTs-COOH and MWCNTs-PEG on macrophages.

<p>(A) RAW 264.7 cells and (B) primary rat peritoneal macrophages were incubated with or without indicated concentrations of <i>f</i>-MWCNTs samples for 24 h. At the end of the incubation period, the WST-1 assay was performed to evaluate the cytotoxicity. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *<i>p</i><0.05 compared to control sample, #<i>p</i><0.05 compared to MWCNTs-PEG.</p

Effects of MWCNTs-COOH and MWCNTs-PEG on NF-κB activation.

<p>RAW264.7 cells were treated with 75 µg/mL of either MWCNTs-COOH or MWCNTs-PEG for the indicated times, then EMSA assay and western blot analysis were used to determine (A) NF-κB DNA-binding activity and (B) levels of cytoplasmic IκBα and nuclear p65, respectively. (C) Translocation of p65 from cytosol to nuclei. RAW264.7 cells were treated with or without 75 µg/mL of MWCNT samples for 6 h, and p65 was then immunofluorescently stained (green). (D) Pretreatment of RAW264.7 cells with ROS scavenger (NAC, DPI) inhibited NF-κB activation. Data represent similar results from three independent experiments.</p

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

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), representative transition metal dichacogenide materials, have 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

MWCNTs-COOH and MWCNTs-PEG induced the activation of caspase-3 and −9 in RAW 264.7 cells.

<p>Cells were treated with or without either MWCNTs-COOH or MWCNTs-PEG for 24 h. The activation of caspase-3, −8, −9 and PARP was measured by (A) caspase activity or (B) western blot analysis. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *<i>p</i><0.05 compared to control sample, #<i>p</i><0.05 compared to MWCNTs-PEG. Representative images of the activation of caspase-3, −8, −9, and PARP by western blot analysis are shown.</p

Characterization of MWCNTs-COOH and MWCNTs-PEG.

*<p>Average diameters and lengths of MWCNTs-COOH and MWCNTs-PEG were determined by TEM images (n = 20).</p

Cellular uptake and distribution of MWCNTs-COOH and MWCNTs-PEG.

<p>(A) Quantitative analysis of cellular uptake of MWCNTs-COOH and MWCNTs-PEG by RAW 264.7 cells as shown by flow cytometry using the light SSC parameter. Cells were treated with or without <i>f</i>-MWCNT samples for the indicated time and assayed for SSC intensity by flow cytometry. Data are representative of three independent experiments and are expressed as the mean ± SD of at least three experiments. *<i>p</i><0.05 compared to control sample, #<i>p</i><0.05 compared to MWCNTs-PEG. (B) Intracellular distribution of MWCNTs-COOH and MWCNTs-PEG in RAW 264.7 cells viewed under a fluorescence microscope. Cells were incubated with or without FITC-labeled <i>f</i>-MWCNTs samples for 12 h, and then processed for CLSM examination.</p