Enalapril alleviates cellular senescence.

(A, D) SA-β-Gal staining (A) and statistical analysis of SA-β-Gal ratios (D) in IMR90 cells after enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (B, E) Ki67 immunofluorescence experiment (B) and statistical analysis of Ki67 intensity (E) after enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (C) Western blot analysis showing the protein levels of p16 and p21 in IMR90 cells after enalapril treatment. (F) Heatmap showing cell cycle-related gene (green) upregulation and SASP gene (purple) downregulation after enalapril treatment. (G) RNA expression of SASP factors in IMR90 cells after enalapril treatment, with blue bars representing the control group and red bars representing the enalapril-treated group. (H) Growth curves of IMR90 cells after enalapril treatment. The x-axis represents the passage time, while the y-axis represents population doubling (PD). A two-tailed t test was employed; ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Phosphorylated Smad1/5/9 mediate the anti-senescence effect of enalapril.

(A) Western blot results for the detection of pSmad5, pSmad1/5/9 and other proteins after enalapril treatment. (B) GSEA showing the enrichment of the BMP signaling pathway after enalapril treatment. NES, normalized enrichment score. ES, enrichment score. (C) Profile of pSmad1/5/9 enrichment at the TSS regions in the control group (Ctrl) and the enalapril-treated group (Enalapril). (D) Heatmap of cell cycle (green) and SASP-related genes (purple) in the control group (Ctrl), BMP receptor inhibitor-treated group (LDN193189, LDN), and enalapril and BMP receptor inhibitor-cotreated group (EP+LDN). (E) Western blot analysis showing the protein levels of pSmad1/5/9, p16, and p21 in response to different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. (F) SA-β-Gal staining (left) and corresponding ratio statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (G) Ki67 immunofluorescence experiment (left) and intensity statistical chart (right) of IMR90 cells treated with different combinations of enalapril, a BMP receptor inhibitor (LDN193189, LDN), and BMP4. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t test was employed; ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

pSmad1/5/9 regulate ID-induced inhibition of p16, p21 and SASP.

(A) Changes in the peaks of all transcription factors identified by CUT&Tag following enalapril treatment, with red dots indicating transcription factors with increased peak intensity (Upregulated TFs). (B) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the ID1 region between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. (C) Western blot analysis showing the changes in the protein levels of ID1 and ID2 following enalapril treatment. (D) Changes in the RNA levels of ID1 and ID2 following enalapril treatment. (E) Western blot analysis showing the protein levels of ID1 and ID2 following treatment with a BMP receptor inhibitor (LDN193189, LDN). (F, I) Western blot showing the changes in the protein levels of p16 and p21 following the knockdown of ID1 or ID2 (F) or the inhibition of ID1 (I). (G) RNA expression of SASP factors after ID knockdown, with pink representing ID1 knockdown and orange representing ID2 knockdown. (H) Normalized average RNA expression levels of selected SASP factors and cell cycle arrest factors after enalapril treatment and ID knockdown. Positive values indicate upregulation, while negative values indicate downregulation. The values represent the expression levels relative to those of the Ctrl. (J) Western blot analysis showing pSmad1/5/9 levels following ID1 and ID2 knockdown. (K) SA-β-Gal staining (left) and SA-β-Gal ratio quantification (right) in the control group (Ctrl) and ID-knockdown groups (ID1_KD, ID2_KD) with or without enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (L) Ki67 immunofluorescence intensity in the control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. A two-tailed t test was employed, ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

pSmad1/5/9 increase the expression of antioxidative genes.

(A) Bar plot showing the enriched GO terms and pathways associated with the CUT&Tag-upregulated peaks (left) and the upregulated RNA-seq genes (right) following enalapril treatment. (B) Heatmap showing the changes in the RNA expression of antioxidative genes. (C) Profile plot showing the increase in the pSmad1/5/9 binding signal of antioxidative genes in the TSS region following enalapril treatment. (D) Western blot analysis showing the protein levels of representative antioxidative genes after enalapril treatment. (E) Relative RNA levels of TXN, GPX4, and PRDX5 during cellular senescence. The blue bars represent young cells, and the red bars represent senescent cells. (F, H) Integrative Genomics Viewer (IGV) showing pSmad1/5/9 signals near the PRDX5 (F) and TXN (H) regions between the control group (Ctrl) and the enalapril treatment group (Enalapril). The vertical yellow boxes indicate regions with increased signal intensity. (G) ChIP-qPCR results showing pSmad1/5/9 levels at many peaks following enalapril or combination treatment with enalapril and LDN193189. The y-axis represents the normalized pSmad1/5/9 signals relative to 10% input. pSmad1/5/9 enrichment for HPRT1 and HBB served as a negative control, and pSmad1/5/9 enrichment for ID1 and ID2 served as positive controls, as previously described. A two-tailed t test was employed, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (I) Detection of ROS fluorescence levels via the DCFH-DA probe in young cells, senescent cells (Ctrl), cells treated with enalapril, and cells treated with a combination of enalapril and LDN193189. Scale bars, 200 μm.

Enalapril exerts its anti-aging effects on mouse organs via pSmad1/5/9-antioxidative genes.

(A) A schematic workflow of the mouse feeding and experimental procedures. Created with BioRender.com. (B) Heatmap of RNA levels of SASP factors in various organs following enalapril treatment (EP). (C) Cytokine array analysis of secreted proteins (below) and relative quantitation (above) of SASP factors in control serum (Ctrl) and enalapril-treated serum (Enalapril). (D) Western blot analysis showing the protein levels of pSmad1/5/9 and antioxidative genes in various organs following enalapril treatment. (E) Heatmap of the RNA levels of BMP signaling pathway-related genes and antioxidative genes in the brain, kidney, and liver following enalapril (EP) treatment.

Enalapril mitigates age-related degenerative changes in aged mice.

(A) Changes in the body weights of the mice after enalapril feeding. (B, C) Movement trajectory (B) and spontaneous alternation rate (C) in the Y-maze of the mice after enalapril treatment. (D, E) Changes in the grip strength (D) and rotarod fall time (E) of the mice after enalapril treatment. (F, G) Heatmap of movement trajectories (F) and the number of entries into the central area (G) in the open-field test after enalapril treatment. (H–M) Alterations in the serum levels of aspartate transaminase (AST) (H), creatinine (CREA) (I), lactate dehydrogenase (LDH) (J), triglycerides (TGs) (K), low-density lipoprotein cholesterol (LDL) (L), and high-density lipoprotein cholesterol (HDL) (M) after enalapril treatment in mice. (N) SA-β-Gal staining of the brain, liver, and spleen after enalapril treatment. Scale bars, 20 μm. (O, P) Periodic Acid-Schiff (PAS) staining (O) and Congo red staining (P) of the brain after enalapril treatment. Scale bars, 50 μm. (Q) Sirius red staining of the kidney after enalapril treatment. Scale bars, 50 μm. (R) Oil Red O staining of the spleen, kidney and liver after enalapril treatment. Scale bars, 20 μm. (S) Hematoxylin and eosin (H&E) staining of the liver after enalapril treatment. Scale bars, 50 μm.

Summary schematic.

It showing pSmad1/5/9-mediated combating cellular senescence of enalapril, and improvement of organ function in mice. Specifically, enalapril increases the level of pSmad1/5/9 and Smad4, promoting the expression of downstream genes, such as antioxidative and cell cycle related genes, thereby promoting cell proliferation and reducing ROS. Moreover, enalapril mitigates age-related degenerative changes, including enhanced memory, improved kidney function, increased liver metabolism and other organ functions. Created with BioRender.com.

Primers used in RT-qPCR

Primers used in pSmad1/5/9 ChIP-qPCR

shRNA target sequence used in knock down experiment

Antibodies

The expression of various senescence markers during IMR90 cell senescence.

(A) SA-β-Gal staining (left) and statistical analysis of SA-β-Gal ratios (right) during IMR90 cell senescence. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (B) Western blot analysis showing the protein levels of p16 and p21 in IMR90 cells at different passage. (C) Ki67 immunofluorescence experiment (left) and statistical analysis of Ki67 intensity (right) during IMR90 cell senescence. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (D) RNA expression of SASP factors during IMR90 cell senescence, with blue bars representing the young group and red bars representing the old group. A two-tailed t-test was employed, ns indicates no significant difference, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Alterations in critical regulators of cellular senescence.

(A) Western blot analysis showing the changes in protein levels of key targets in classical senescence signaling pathways following enalapril treatment. (B) Western blot analysis showing the changes in Smad and BMP-related protein levels during IMR90 cell senescence.

pSmad1/5/9 alleviates cellular senescence phenotypes.

(A) Western blot analysis showing the changes in protein levels following BMP4 treatment. (B) SA-β-Gal staining (left) and corresponding ratio analysis chart (right) following BMP4 treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Ki67 immunofluorescence (right) and corresponding ratio analysis chart (left) following BMP4 treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, **p < 0.01, ****p < 0.0001.

Reduction of pSmad1/5/9 correlates with cellular senescence.

(A) Western blot showing the protein levels after the addition of LDN193189. (B) SA-β-gal staining (left) after adding LDN193189 and its corresponding staining proportion statistical chart (right). Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Immunofluorescence staining for Ki67 (left) after adding LDN193189 along with the corresponding intensity statistical chart (right). Scale bars, 80 μm. Enlarged scale bars, 40 μm. (D) Western blot showing the protein levels following BMPR1A knockdown. (E) Relative RNA levels of BMPR1A following BMPR1A knockdown. (F) SA-β-gal staining (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A-knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 200 μm. Enlarged scale bars, 100 μm. (G) Ki67 immunofluorescence (left) and its corresponding staining proportion statistical chart (right) in the control group (Ctrl) and BMPR1A-knockdown groups (BMPR1A_KD) with enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, ns indicates no significant difference, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Knockdown of ID accelerates cellular senescence.

(A) Western blot showing changes in ID protein levels during cellular senescence. (B) SA-β-Gal staining (left) following knockdown of ID1 and ID2, along with corresponding statistical chart (right). Scale bars, 200 μm. Enlarged scale bars, 100 μm. (C) Ki67 immunofluorescence (left) following knockdown of ID1 and ID2, along with corresponding statistical chart (right). Scale bars, 80 μm. Enlarged scale bars, 40 μm. A two-tailed t-test was employed, *p < 0.05, ***p < 0.001. (D) Ki67 immunofluorescence in control group (Ctrl) and ID knockdown groups (ID1_KD, ID2_KD), with or without enalapril treatment. Scale bars, 80 μm. Enlarged scale bars, 40 μm. (E) Heatmap of SASP factor expression in mouse bone marrow cells following ID knockout from previously reported data(Fei et al., 2023).

Effects of enalapril on the expression changes of genes related to organ physiological functions in mice.

(A) In multiple organs, enalapril increases the enrichment of GSEA pathways associated with improved physiological function. (B) Western blot analysis showing changes in Id1 protein levels in various organs of mice.

Effects of reduced pSmad1/5/9 levels on the expression changes of genes in mice.

(A) Cytokine array analysis of secreted proteins (above) and relative quantitation (below) of SASP factors in enalapril-treated serum (Enalapril) and co-treated serum (Enalapril+LDN). (B) Western blot analysis showing the protein levels of pSmad1/5/9 and antioxidative genes in various organs following enalapril and LDN co-treatment.

Effects of reduced pSmad1/5/9 levels on aging-related behaviors in mice.

(A, D) Changes in the rotarod fall time (A) and grip strength (D) of the mice after enalapril and LDN treatment. (B, C) The number of entries into the central area (B) and heatmap of movement trajectories (C) in the open-field test after enalapril and LDN treatment. (E, F) Spontaneous alternation rate (E) and movement trajectory (C) in the Y-maze of the mice after enalapril and LDN treatment.

Enalapril ameliorates pathological phenotypes in aged mice.

(A-C) SA-β-Gal staining quantification chart of the brain (A), liver (B) and spleen (C) after enalapril treatment. (D, E) Periodic Acid-Schiff (PAS) staining (D) and Congo red staining (E) quantification chart of the brain after enalapril treatment. (F) Sirius red staining quantification chart of the kidney after enalapril treatment. (G-I) Oil Red O staining quantification chart of the spleen (G), kidney (H) and liver (I) after enalapril treatment. (J) Hematoxylin and eosin (H&E) staining quantification chart of the liver after enalapril treatment.