| Gene name: | SIRT3 |
| Aging type: | Prevent |
| Aging characteristic: |
| Tissue type: | Nucleus pulposus |
| Cell name: | NP Cells |
| Gene ID: | 23410 |
| Category: | protein coding |
| Phenotype: | Intervertebral disc degeneration |
| Experimental category: | L |
| PMID: | 33565085 |
| Experiment: | SA-β-gal activity assay//Western blot |
| Description: | The proportion of SA‐β‐Gal‐positive cells and p16INK4α expression in the LV‐shSIRT3 + H2O2 group was significantly higher than those in the LV‐Ctrl and LV‐Ctrl + H2O2 groups, whereas overexpression of SIRT3 distinctly decreased the percentage of SA‐β‐Gal‐positive cells and p16INK4α expression. In addition, whether at the protein or messenger RNA (mRNA) level, p16INK4α and SASP (such as IL‐1β,IL‐6, MMP3, and MMP9) among groups of NPC exhibited a very similar trend, which was consistent with the results of SA‐β‐Gal staining. |
| Regulatory pathway: | AMPK-PGC‐1α |
| R-AG-Pathway: | Activation |
| Pathway experiment: | ROS Assay//Immunofluorescence//Western blot//SA-β-gal activity assay//qRT-PCR |
| Pathway description: | Furthermore, activation of AMPK in NPC noticeably reduced high the iNOS expression and ROS production caused by downregulation of SIRT3; conversely, the inhibition of AMPK in NPC counteracted the decline in iNOS expression and ROS production after SIRT3 overexpression. Additionally, western blot analysis showed that the expression of iNOS and the ratios of Ac‐SOD2/SOD2 in each group were similar to the results of iNOS immunofluorescence staining. In Figure S4F, we found that the SOD2 activity in the LV‐shSIRT3 + AICAR + H2O2 group was significantly higher than that in the LV‐shSIRT3 + H2O2 group, whereas, in the LV‐SIRT3 + Compound C + H2O2 group, its activity was obviously lower than that in the LV‐SIRT3 + H2O2 group. Subsequently, we observed that the activation of AMPK activity in NPC significantly reduced the number of SA‐β‐Gal‐positive cells induced by LV‐shSIRT3; in contrast, the inhibition of AMPK activity in NPC evidently increased the number of SA‐β‐Gal‐positive cells reduced by LV‐SIRT3. In addition, the results of immunofluorescence staining also illustrated that the activation of AMPK declined the expression of p16INK4α, which is highly expressed in NPC by silencing SIRT3; in contrast, the inhibition of AMPK activity in NPC obviously increased the expression of p16INK4α after LV‐SIRT3 intervention. Moreover, RT‐PCR and western blot results demonstrated that the expression changes of p16INK4α and SASP (including IL‐1β,IL‐6, MMP3, and MMP9) among the different groups showed consistency at the mRNA and protein levels. |
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