npcs senescence  (Beyotime)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited NPCs proliferation. (A) Measurement of lactic acid content in NPCs using a lactic acid detection kit (n = 3). (B) The effect of different concentrations or treatment times of lactic acid on the proliferation of NPCs as determined by CCK-8 assay (n = 3). (C-D) The effect of different concentrations of lactic acid on the proliferation of NPCs as determined by EdU staining. Proliferating NPCs appeare red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (E-F) The effect of different concentrations of lactic acid on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (G-H) The effect of different concentrations of lactic acid on DNA double stranded as determined by γ-H2AX; DNA double-stranded-damaged cells appear red, and nuclei were counterstained with DAPI (blue) (scale bar = 50 μm. n = 3). (I-J) The effect of different concentrations of lactic acid on the NPCs senescence as determined by SA-β-gal assay; senescent cells appear blue (scale bar = 200 μm. n = 3). (K-L) Western blotting analysis showing the effect of different lactic acid concentrations on collagen II, MMP9 and MMP13 expression and quantification of the results. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: High concentrations of lactic acid inhibited the proliferation of NPCs via the Akt/p21/p27/cyclin D1 pathway. (A-D) Western blotting analysis showing the effect of different lactic acid concentrations on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (E-H) Western blotting analysis showing the effect of lactic acid and/or SC79 on p21, p27, and cyclin D1 expression and quantification of the results (n = 3). (I-J) The effect of lactic acid and/or SC79 on the proliferation of NPCs as measured by EdU staining. Proliferating NPCs appear red, and nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (K-L) The effect of lactic acid and/or SC79 on the cell cycle of NPCs; the box positions represent negative controls (n = 3). (M-N) The effect of lactic acid and/or SC79 on the senescence of NPCs as determined by SA-β-gal assay. The senescent cells appeared blue (scale bar = 200 μm. n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: Western Blot, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Lactic acid regulates the function of NPCs by binding to specific sites of Akt. (A) Detection of Akt kinase activity in NPCs after treatment with different concentrations of lactic acid (n = 3). (B) Domain structure of Akt. (C-D) Molecular docking diagram of lactic acid and Akt. (E) Western blotting analysis showed Akt expression after knocking down Akt with shRNA and transfecting AktWT, AktK39A, AktL52A and AktK39A,L52A into NPCs respectively. (F) Detection of Akt kinase activity in NPCs of different groups (n = 3). (G-H) The effect of site mutation of Akt on the proliferation of NPCs (red) as determined by EdU staining. Nuclei were counterstained with Hoechst 33,258 (blue) (scale bar = 100 μm. n = 3). (I-J) The effect of lactic acid (10 mM) on the senescence of Akt site-mutated NPCs (blue) as determined by SA-β-gal assay (scale bar = 200 μm. n = 3). (K-L) The effect of lactic acid (10 mM) on ROS production of Akt site-mutated NPCs as determined by a DCFH-DA probe. The FL1 subset was set according to the ROS intensity of the negative control (n = 3). (M-P) Western blotting analysis showing the effect of lactic acid (10 mM) on collagen II, MMP9 and MMP13 expression in Akt site-mutated NPCs and quantification of the results (n = 3). (Q-T) MST analysis of lactic acid in the interaction of NT-647-NHS-labeled Akt. Kd represents the dissociation constant. La represents lactic acid; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns represents no statistical significance

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing, shRNA, Mutagenesis, Staining, Negative Control, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt

doi: 10.1007/s00018-023-05094-y

Figure Lengend Snippet: Schematic diagram of the signaling pathways involved in the promotion of senescence and oxidative stress in NPCs by and corresponding IVDD by lactic acid

Article Snippet: A SA-β-gal Staining kit (C0602, Beyotime) was used to assess the effect of lactic acid on NPCs senescence.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: Characterization of adipocytes and Adipo-sEVs. (a) Oil red O staining of adipocytes differentiated from HS-5. (b) Morphology of Adipo-sEVs under TEM. Scale bar, 200 nm. (c) Western blotting analysis indicated that Adipo-sEVs expressed sEV markers, such as CD9, CD63, and TSG101, and were negative for GM130. (d) Particle size distribution of sEVs measured by NTA. (e) IF analysis of DiO-labelled Adipo-sEV internalization by NPCs and EPCs. Scale bar, 30 μ m. (f) Levels of NAMPT and CD63 in HS5-sEVs and Adipo-sEVs. (g) Densitometric quantification of the relative band intensity in (f). n = 3 per group. ( ∗ P < 0.05 compared with HS5-sEVs).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: Staining, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: Adipo-sEVs can ameliorate the senescent phenotypes of NPCs and age-related dysfunction in vitro. (a) Representative micrographs of NPCs stained with SA- β -gal in different treatment groups. SA- β -gal-positive cells are shown in blue. Scale bar, 100 μ m. (b) Percentage of SA- β -gal-positive cells. n = 3 per group. (c) IF staining for Ki-67 (red). DAPI was used to stain the nuclei. Scale bar, 50 μ m. (d) Percentage of Ki-67-positive cells. n = 3 per group. (e) The expression of P16 was assessed by western blotting. (f) Densitometric quantification of the relative band intensity in (e). n = 3 per group. (g) The expression of Collagen II, Aggrecan, MMP-3, and ADAMTS-4 was assessed by western blotting. (h) Densitometric quantification of the relative band intensity in (g). n = 3 per group. (i) Quantification of mRNA expression for SASP (TNF- α , IL-6, and IL-8). n = 3 per group. ( ∗ P < 0.05 compared with IL-1 β (-) and Adipo-sEV (-) group; # P < 0.05 compared with IL-1 β (+) and Adipo-sEV (-) group).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: In Vitro, Staining, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: Adipo-sEVs deliver NAMPT into senescent NPCs and enhance NAD + and Sirt1 activity. (a) Adipo-sEVs increased NAMPT and Sirt1 levels in senescent NPCs, but not Sirt3 and Sirt5 levels, as detected by western blotting. (b) Densitometric quantification of the relative band intensity in (a). n = 3 per group. (c) IF staining for Sirt1 (red). DAPI was used to stain the nuclei. Scale bar, 50 μ m. (d) NAD + levels in NPCs treated with IL-1 β and those cotreated with IL-1 β and Adipo-sEVs. ( ∗ P < 0.05 compared with the control group; # P < 0.05 compared with the IL-1 β group).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: Activity Assay, Western Blot, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: sEVs secreted from NAMPT-knockdown adipocytes exhibited attenuated function on rejuvenating senescent NPCs. (a) Representative micrographs of EPCs stained with SA- β -gal in different treatment groups. SA- β -gal-positive cells are shown in blue. Scale bar, 100 μ m. (b) Percentage of SA- β -gal-positive cells. n = 3 per group. (c) IF staining for Ki-67 (red). DAPI was used to stain the nuclei. Scale bar, 50 μ m. (d) Percentage of Ki-67-positive cells. n = 3 per group. (e) The expression of P16 was assessed by western blotting. (f) Densitometric quantification of the relative band intensity in (e). n = 3 per group. (g) The expression of Collagen II, Aggrecan, MMP-3, and ADAMTS-4 was assessed by western blotting. (h) Densitometric quantification of the relative band intensity in (g). n = 3 per group. (i) Quantification of mRNA expression for SASP (TNF- α , IL-6, and IL-8). n = 3 per group. (j) NAMPT and Sirt1 expression were assessed by western blotting. (k) Densitometric quantification of the relative band intensity in (j). n = 3 per group. (l) NAD + levels in different treatment groups. ( ∗ P < 0.05 compared with the IL-1 β group; # P < 0.05 compared with the IL-1 β + CTRL-sEV group).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: Staining, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: Inhibition of Sirt1 attenuates the therapeutic effects of Adipo-sEVs on senescent NPCs. (a) Representative micrographs of NPCs stained with SA- β -gal in different treatment groups. SA- β -gal-positive cells are shown in blue. Scale bar, 100 μ m. (b) Percentage of SA- β -gal-positive cells. n = 3 per group. (c) IF staining for Ki-67 (red). DAPI was used to stain the nuclei. Scale bar, 50 μ m. (d) Percentage of Ki-67-positive cells. n = 3 per group. (e) The expression of P16 was assessed by western blotting. (f) Densitometric quantification of the relative band intensity in (e). n = 3 per group. (g) The expression of Collagen II, Aggrecan, MMP-3, and ADAMTS-4 was assessed by western blotting. (h) Densitometric quantification of the relative band intensity in (g). n = 3 per group. (i) Quantification of mRNA expression for SASP (TNF- α , IL-6, and IL-8). n = 3 per group. ( ∗ P < 0.05 compared with IL-1 β group; # P < 0.05 compared with the IL-1 β +sEV group).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: Inhibition, Staining, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Small Extracellular Vesicles Derived from Adipocytes Attenuate Intervertebral Disc Degeneration in Rats by Rejuvenating Senescent Nucleus Pulposus Cells and Endplate Cells by Delivering Exogenous NAMPT

doi: 10.1155/2021/9955448

Figure Lengend Snippet: Intradiscal injection of Adipo-sEVs attenuated the development of IVDD in rats by rejuvenating senescent NPCs and EPCs. (a) MRIs of the indicated groups were obtained eight weeks after needle puncture. (b) Pfirrmann MRI grade scores. (c) H&E staining of IVDs in the indicated groups eight weeks after needle puncture. Scale bar, 1 mm. (d) IF staining for P16 (green) four weeks after needle puncture. DAPI was used to stain the nuclei. Scale bar, 1 mm. (e) Quantification of mRNA expression for Sirt1 in NP and EP. n = 3 per group. ( ∗ P < 0.05 compared with the control group; # P < 0.05 compared with the IVDD group; % P < 0.05 compared with the IVDD+CTRL-sEV group).

Article Snippet: We treated the senescent NPCs with Adipo-sEVs and 10 nM of nicotinamide (a Sirt1 inhibitor; Beyotime) for seven days and found that Adipo-sEVs failed to reduce the number of senescent cells, restore the Ki-67 expression, or downregulate the level of P16 (Figures – ).

Techniques: Injection, Staining, Expressing

Journal: Orthopaedic Surgery

Article Title: Inhibition of the P53 / P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells

doi: 10.1111/os.12886

Figure Lengend Snippet: Identification of senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) and uptake of SNPC‐Exo by NPC. (A) Characteristics of SNPC‐Exo under transmission electron microscope. (B) Western blot analyses of exosomal protein markers CD63 and Tsg101 and negative protein calnexin. CL, NPC lysate; EXO, senescent NPC‐derived exosomes. (C) Uptake of SNPC‐Exo by NPC. SNPC‐Exo were stained with CM‐Dil (B) and NPC were stained with CM‐Dio (C). Uptake was observed by fluorescence microscope.

Article Snippet: Senescent NPC were cultured in DMEM‐F12 containing 10% exosome‐free fetal bovine serum.

Techniques: Derivative Assay, Transmission Assay, Microscopy, Western Blot, Staining, Fluorescence

Journal: Orthopaedic Surgery

Article Title: Inhibition of the P53 / P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells

doi: 10.1111/os.12886

Figure Lengend Snippet: Senescent nucleus pulposus cell (NPC)‐derived exosomes (SNPC‐Exo) accelerated senescence and inhibited the proliferation of NPC. (A, B) Western blot analysis of P16, P21, and P53 in SNPC‐Exo‐treated NPC. (C) Relative fold change of absorbance between EXO group and control group at 450 nm. (D, E) Relative percentage of SA‐β‐gal positive cells between EXO group and control group. (F, G) Colony‐forming units assay of NPC. (H, I) The cell cycle phases of NPC were determined by flow cytometry analyses. CON, NPC cultured alone; EXO, NPC treated with SNPC‐Exo; NS, no significance. All data is shown as mean ± SD. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Senescent NPC were cultured in DMEM‐F12 containing 10% exosome‐free fetal bovine serum.

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Cell Culture

Journal: Orthopaedic Surgery

Article Title: Inhibition of the P53 / P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells

doi: 10.1111/os.12886

Figure Lengend Snippet: P21 and P53 regulated the proliferation and senescence of nucleus pulposus cell (NPC) senescent NPC‐derived exosomes (SNPC‐Exo) treatment. (A, B) The expression of P21 and P53 in NPC was inhibited by siRNA transfection. (C, D) Relative percentage of SA‐β‐gal positive cells among groups after corresponding treatments. (E) Relative fold change of absorbance among groups after Corresponding treatments at 450 nm. (F) The cell cycle phases of NPC were determined by flow cytometry analyses. CON, NPC cultured alone; EXO, NPC treated with SNPC‐Exo; EXO+siP21, NPC treated with SNPC‐Exo and P21 siRNA; EXO+siP53, NPC treated with SNPC‐Exo and P53 siRNA; NC, NPC treated with siRNA negative control; NS, no significance; siRNA, NPC treated with P21 or P53; siRNAEXO+NC, NPC treated with SNPC‐Exo and siRNA negative control. All data are shown as mean ± SD. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Senescent NPC were cultured in DMEM‐F12 containing 10% exosome‐free fetal bovine serum.

Techniques: Derivative Assay, Expressing, Transfection, Flow Cytometry, Cell Culture, Negative Control

Journal: Aging (Albany NY)

Article Title: Kinsenoside ameliorates intervertebral disc degeneration through the activation of AKT-ERK1/2-Nrf2 signaling pathway

doi: 10.18632/aging.102302

Figure Lengend Snippet: Kin suppresses NPC apoptosis and senescence under oxidative stress. ( A ) The NPCs were treated with different concentrations of Kin (0, 5, 10, 25 or 50 μg/ml) alone for 24 h, or Kin (0, 5, 10 or 25 μg/ml) for 2 h before receiving TBHP (100 μM) for 24 h. The cell viability was assessed by CCK-8. ( B ) The western blotting of C-caspase3 in the NPCs treated with different concentrations of TBHP (0, 20, 50 or 100 μM) for 24 h or TBHP (100 μM) for indicated time points (0, 6, 12 or 24 h). ( C ) The western blotting of p16 in the NPCs treated with different concentrations of TBHP (0, 20, 50 or 70 μM) for 48 h or TBHP (70 μM) for indicated time points (0, 12, 24 or 48 h). ( D , E ) The western blotting and quantitative protein levels of C-caspase3 and p16 in the NPCs as treated above. ( F ) The apoptosis rate in NPCs, treated with different concentrations of Kin (0, 5, 10 or 25 μg/ml) for 2 h before receiving TBHP (100 μM) for 24h, was analyzed by flow cytometry using Annexin V-APC/PI. ( G ) The cell proliferation was determined by quantification of EdU-positive cells in the NPCs treated with different concentrations of Kin (0, 5, 10 or 25 μg/ml) for 2 h before receiving TBHP (70 μM) for 24 h; scale bar: 100 μm. All data are expressed as mean ± SD of at least three independent experiments.

Article Snippet: The NPC senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining kit (Beyotime) according to the protocols.

Techniques: CCK-8 Assay, Western Blot, Flow Cytometry