Fig 1: Hyperglycaemic conditions increase senescence which is modulated via SIRT1 activity. The role of SIRT1 in hyperglycaemic induced senescence was examined. (a) Positive cellular senescence represented via blue β-galactosidase activity seen in representative micrographs. Senescence increased significantly day 4 in osteogenic conditions, with a further significant increase in the hyperglycaemic conditions. SRT1720 activation of SIRT1 significantly decreased this trend, whereas Sirtinol induced inhibition of SIRT1 significantly increased senescence in all treatments (n = 4 and 5FoV). (b) p16 mRNA expression was increased in osteogenic conditions and further increased in hyperglycaemic conditions compared to control untreated cells, whereas SIRT1 activation significantly decreased p16 expression, and Sirtinol significantly increased levels of p16 transcript (n = 4). (c) p21 mRNA abundance was also observed. Levels of p21 transcript significantly increased in hyperglycaemic conditions, further enhanced via SIRT1 inhibition, with a significant reduction detected in SRT1720 treated cells (n = 4). (d) p53 mRNA expression was significantly increased during hyperglycaemic treatment and further enhanced with SIRT1 inhibition. SIRT1 activation significantly reduced p53 mRNA production. (n = 4) (e) Chromatin immunoprecipitation was performed with an anti-acetyl-lysine antibody. Acetylation of the p53 promotor region increased during hyperglycaemic treatment, which was reversed with the addition of SIRT1 activator SRT1720 (n = 3). *P < 0.05, **P < 0.005, ***P < 0.001. Scale Bars = 100 μm.
Fig 2: SRT2104 significantly improved the PE-like symptoms in SIRT1+/− mice. (A) Schematic diagram of the treatment of SIRT1+/− mice from early pregnancy to late pregnancy. (B) Representative appearance of fetuses and placentas from vehicle and SRT2104 groups. (C–G) The embryo-resorption rate (C), the weight of the placenta (D), and the weight of the live fetus (E) in vehicle (n = 8) and SRT2104 (n = 7) groups. (F) The SBP at basic condition, early PG, and mid PG, late PG from vehicle (n = 8) and SRT2104 (n = 9) groups. (G) ΔBP (late PG—basic condition) in vehicle (n = 8) and SRT2104 (n = 9) groups. (H) The concentration of urinary protein in vehicle (n = 4) and SRT2104 (n = 7) group at late PG. (I) Masson staining and PAS staining of mice kidney tissues from vehicle (n = 4) and SRT2104 (n = 4) groups. (J) Masson staining of mice placental tissues in vehicle (n = 4) and SRT2104 (n = 4) groups. (K) The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student’s test (and nonparametric tests). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.
Fig 3: Activation of SIRT1 relives RUNX2 and Osteocalcin activation. (a) RUNX2 mRNA significantly decreased via SRT1720 activation and significantly increased via Sirtinol treatment (n = 4–9). (b) Acetylation of RUNX2 promoter region was determined via chromatin immunoprecipitation. RUNX2 promotor acetylation was significantly reduced with SRT1720 activation (n = 3). (c) RUNX2 protein was significantly decreased via SRT1720 activation and significantly increased via Sirtinol treatment (n = 3). (d) OCN activity measured via fluorescence increased in hyperglycaemic conditions compared to osteogenic, with SIRT1 activator attenuating this process and SIRT1 inhibitor Sirtinol increasing OCN fluorescence (n = 4 and 5FoV), OCN mRNA followed a similar pattern, with a decrease in OCN transcripts in SRT1720 activated cells (n = 4–9). *P < 0.05, **P < 0.005, ***P < 0.001. Scale Bars = 10 μm.
Fig 4: Suggested mechanism of SIRT1 within vascular calcification. High glucose, phosphate and calcium lead to a reduction in SIRT1 expression and a simultaneous increase in osteogenic markers, RUNX2, OCN and ALP alongside the induction of a senescent phenotype, exacerbating the calcification of vascular SMCs.
Fig 5: SIRT1+/− mice demonstrated pre-eclampsia-like symptoms. (A) Schematic diagram of SIRT1+/− mice construction. (B) Genotype of SIRT1+/− mice using PCR and agarose gel electrophoresis. (C) Immuofluorescence for SIRT1 in mice placental tissues from SIRT1flox/flox (n = 3) and SIRT1+/− (n = 7) mice. (D) Representative appearance of fetuses and placentas from SIRT1flox/flox and SIRT1+/− groups. (E–G) The embryo-resorption rate (E), the weight of the placenta (F), and the weight of the live fetus (G) in SIRT1flox/flox (n = 19) and SIRT1+/− (n = 31) mice. (H) The gene ratio of fetuses at E18.5 and P28 (E18.5: the 18.5th day of gestation, P28: the 28th day in postnatal age). (I) The systolic blood pressure (SBP) at basic condition, early PG (pregnancy), mid PG, late PG from SIRT1flox/flox (n = 20), and SIRT1+/− (n = 33) groups. (J) ΔBP (late PG—basic condition) in SIRT1flox/flox (n = 20) and SIRT1+/− (n = 33) groups. (K) The correlation of ΔBP and SIRT1 gene values in fetuses. (L) Masson staining and PAS staining of mice kidney tissues from SIRT1flox/flox (n = 3) and SIRT1+/− (n = 7) groups. (M) The concentration of urinary protein in SIRT1flox/flox (n = 6) and SIRT1+/− (n = 13) groups at late PG. (N) Masson staining of mice placental tissues in SIRT1flox/flox (n = 3) and SIRT1+/− (n = 7) groups. (O) The labyrinth/junctional zone ratio of each group. Error bars, mean ± SD. The data were analyzed by one-way ANOVA (and nonparametric or mixed) and unpaired Student’s test (and nonparametric tests). ** p < 0.01; *** p < 0.001; **** p < 0.0001, ns: no significance.
Supplier Page from Abcam for Human SIRT1 ELISA Kit