External factors
| External factors: |
β-Hydroxybutyrate |
| Category: |
Chemical compounds |
| Experiment: |
SA-β-gal activity assay//qRT-PCR//Flow cytometry |
| Description: |
To test the anti-senescence efficacy of β-HB, primary human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (hASMCs) were exposed to H2O2 with or without β-HB. As expected, H2O2 (150 mM, 3 days) exposure increased numbers of senescence-associated β-galactosidase- (SA β-gal) positive cells with senescence-related morphological transformations: enlarged, flat, and multinucleated appearance of cells. The addition of β-HB (4 mM, 3 days) markedly reduced senescence, while acetoacetate (AcAc, 4 mM, 3 days) had no effects on H2O2-induced senescence.As expected, β-HB also prevented senescence-related morphological transformations.Quantitative real-time PCR (qRT-PCR) of HUVECs samples indicated that β-HB effectively suppressed the expression of IL-6 and IL-1a triggered by H2O2. Quantitation of cell-cycle distribution showed that β-HB dramatically induced G0 with simultaneous reductions of G1 and S/G2 phases . |
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Regulatory relationship
| Target gene:
|
HNRNPA1//OCT4A |
| R-EF-Target gene:
|
Downregulation//Upregulation |
| Official symbol(s): |
HNRNPA1//POU5F1 |
| Target gene experiment: |
SA-β-gal activity assay//Western blot//Pull-down assay//MALDI-TOF mass analysis//qRT-PCR |
| Target gene description: |
We identified hnRNP A1 as a β-HB-binding protein using MALDI-TOF mass analysis. To confirm binding affinity, a pull-down assay was performed using SP-β-HB beads, and hnRNP A1 was validated with a specific antibody. The binding between hnRNP A1 and β-HB-conjugated beads was disturbed when the interaction was challenged with excess β-HB, S-β-HB, β-HB ester, or butyrate acting as binding competitors. Furthermore, silencing hnRNP A1 triggered senescence of HUVECs and accelerated the response to H2O2 .Both β-HB and S-β-HB were able to upregulate Oct4A, Lamin B1, p27, and phospho-eIF2a in thoracic aorta, indicating cell quiescence. Interestingly, β-HB-induced Oct4 protein elevation was only observed in the aorta, brain, and heart. Oct4A mRNA was also increased byβ-HB and S-β-HB injection, especially in the aorta, brain cortex, and heart. |
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Aging network
Annotation:
The green line represents Upregulation.
The purple line represents Downregulation.
The orange line represents Activation.
The yellow line represents Inhibition.
The gray line represents Unclear.
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