Fig 2: Aspirin alleviates Aβ-induced senescence and mitochondrial dysfunctions via upregulation of SIRT pathway. (a–g) HMC3 cells were preincubated with aspirin for 4 h followed by treatment with 10 μM Aβ for 72 h. (a) Protein levels of SIRT1, PAI-1, and p21 were detected by western blotting. (b) The quantification of relative protein levels in (a). (c) The representative SA-β-gal staining images. (d) Quantification of the percentage of SA-β-gal cells. Scar bar, 50 μm. (e) The cells were stained with JC-1 dye and the fluorescence intensity was quantified using BioTek reader. (f) HMC3 cells were incubated with DCFH-DA and Hoechst; then, the fluorescence intensity was detected by BioTek reader. (g) The fluorescent latex beads were added to the medium and incubated at 37°C for 3 h. The fluorescence intensity was quantified using BioTek reader. The data are presented as mean ± SEM, n ≥ 3 independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, analyzed by one-way ANOVA followed by Bonferroni's test.

Fig 3: Aβ-induced gene expression of senescence in human microglial cells. (a–h) Human microglial cells were treated with 10 μM Aβ for different times. (a–d) Relative genes expression levels of PAI-1, p21, CCND1, and CCNA1 were measured by qPCR. (e) Protein levels of p53, PAI-1, and p21 were detected by western blotting. (f–h) The quantification of relative protein levels in (e). (i–n) Human microglial cells were stimulated with Aβ (1-10 μM) for 72 h. (i) Western blotting was used to investigate protein levels of p53, PAI-1, and p21. (j–l) The quantification of relative protein levels in (i). (m) The representative SA-β-gal staining images. (n) Quantification of the percentage of SA-β-gal cells. Scar bar, 100 μm. (o–q) Senescence-associated proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 were detected by qPCR. The data are presented as mean ± SEM, n ≥ 3 independent experiments; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, analyzed by one-way ANOVA followed by Bonferroni's test.

Fig 4: (A) Chrysanthemum coronarium L. (CC) extract reduces the senescent phenotype in H2O2 (100 μmol/L)-stimulated HUVECs, as shown by β-gal staining. The magnification was set to 40×. Scale bar = 50 μm. (B) Quantitative analysis of positive SA-β-gal stained cells. Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). (C) CC reduced the expression of p21, p53, and PAI-1 induced by H2O2. (D–F) The relative ratios from comparison to β-actin in duplicate blots are shown in a graph chart. Control cells (CB) received the vehicle alone. The T Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). **, p < 0.01, ***, p < 0.001, versus CB; #, p < 0.05, ###, p < 0.001, vs. H2O2.

Fig 5: PAI-1 appears to play a role in the PI3K/Akt signaling process regulated by CAV-1. (A) The heat map of the 25 genes correlated to CAV-1. (B) Venn diagram of the overlapping genes among significantly co-expressed genes, significantly DEGs and EMT-associated genes of CAV-1. (C) Spearman correlation between CAV-1 and 48 Overlapping genes. (D) Volcano plot of DEGs. Blue and red dots indicate the significantly down-regulated and up-regulated DEGs, respectively. DEGs, differentially expressed genes. (E) The positive correlation between CAV-1 expression and SERPINE1(PAI-1) expression. (F) The positive correlation between PI3K expression and SERPINE1(PAI-1) expression.