| External factors: | Antcin M |
| Aging type: | Prevent |
| Aging characteristic: |
| Category: | Chemical compounds |
| Phenotype: | Aging |
| Experiment: | SA-β-gal activity assay//Western blot//Flow cytometry//MTT assay |
| Description: | We examined the protective effects of antcins on HG-induced HNDF senescence, cells were co-incubated with HG and antcins (ANA, ANH and ANM) for 72 h, senescence was measured by SA-β-gal assay. Treatment with ANM showed significant protection against HG-induced HNDF senescence as evidenced by reduction in a number of SA-β-gal positive cells from 9.59-fold to 1.51-fold, whereas ANA and ANH showed moderate inhibition as SA-β-gal positive cells were reduced to 7.46-fold and 8.13-fold, respectively. In addition, results from immunoblotting analysis confirmed that HG-induced upregulation of senescenceassociated proteins such as p16INK4A and p21CIP1 were significantly downregulated by ANM, whereas ANA and ANH showed a moderate inhibition, which is concomitant with the result of SA-β-gal assay. A two-fold increase in cell proliferation was observed in the ANM treatment group.Our results demonstrated that treatment with HG caused cell-cycle arrest in the G1-S transition phase, as the proportion of cells in the G0/G1 phase was significantly increased to 71.2% compared to 46.5% in the NG group.Treatment with ANM eliminated the effect of HG and reduced the cell population in the G0/G1 phase to 49.5%,which is similar to the control (NG) group. |
| Target gene: | NRF2 |
| R-EF-Target gene: | Activation |
| Official symbol(s): | NRF2 |
| Target gene experiment: | Western blot//RT-PCR//SA-β-gal activity assay//Knockdown//Luciferase reporter assay//Immunofluorescence |
| Target gene description: | To determine whether ANM augments Nrf2 transcriptional activity, we used ARE-harboring luciferase reporter assay.a remarkable increase in luciferase activity was observed in cells that were cotreated with HG and ANM or NAC which showed 8.5-fold and 8.2-fold increase, respectively. Transcriptional activation of Nrf2 is dependent upon the rate of nuclear export followed by disassociation from cytoplasmic Keap1. Results from immunofluorescence analyses showed that Nrf2 expression in the nucleus was barely observed in the control (NG) and the HG treatment groups, whereas elevated Nrf2 expression in the nucleus was observed in the ANM or NAC treatment groups . |
| Regulatory pathway: | SIRT1 |
| R-EF-Pathway: | Upregulation |
| Official symbol(s): | SIRT1 |
| Pathway experiment: | Western blot//RT-PCR//SA-β-gal activity assay//Knockdown//Luciferase reporter assay |
| Pathway description: | RT-PCR analysis indicated that SIRT-1, SIRT-3 and SIRT-6 levels were significantly increased in the ANM treatment group compared to the control group.In order to ascertain whether the protective effect of ANM was SIRT-1 dependent, the effect of ANM in SIRT1 silenced HNDFs was investigated under HG conditions.In control siRNA transfected cells, treatment with ANM significantly inhibited HG-induced senescence as assessed by SA-β-gal activity. However, in SIRT-1 siRNA transfected cells, SA-β-gal activity remained partially elevated despite the presence of ANM or RES . |
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