Capsaicin Receptor TRPV1 Delays Aortic Aging in Atherosclerotic Mice by Inhibiting the ISG15 ‐p53 Pathway

Figures

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  FIGURE 1. Screening and validation of DEGs in C57BL/6 and Apoe −/− mice. (A) Heat map showing the 21 most DEGs in the aorta between the control and Apoe −/− groups. (B, C) KEGG and GO analysis of DEGs. (D) PPI analysis of the 21 DEGs in relation to genes associated with AS and aging. (E) Real‐time PCR detection revealed that TRPV1 expression in the aortas of Apoe −/− mice on a high‐fat diet was significantly downregulated at the mRNA level. (F) Western blot detection revealed that TRPV1 expression in the aortas of Apoe −/− mice on a high‐fat diet was significantly downregulated. (G) Immunofluorescence double‐labelling detection revealed that TRPV1 expression was significantly reduced in vascular endothelial and smooth muscle cells in the aortic sinus of Apoe −/−mice on a high‐fat diet. C57BL/6 mice fed a normal diet were used as the control group and Apoe −/− mice fed a high‐fat diet were used as the Apoe −/− group (AS mice). **p < 0.01. Scale bar: 20 μm.

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  FIGURE 2. TRPV1 deficiency promotes aortic aging in AS mice. (A) β‐gal staining of the aortic sinus showed that the area of β‐gal positive staining significantly increased after TRPV1 deletion. EVG staining also showed thinner elastic fibres and reduced collagen fibres. (B) In Apoe −/− mice on a high‐fat diet with TRPV1 deletion, the expression of all SASP proteins except CCL5 was significantly increased in aortic tissue. (C) Expression of aging‐related proteins was significantly increased in TRPV1‐deficient AS mice. *p < 0.05, **p < 0.01. Scale bar: 10 μm or 20 μm.

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  FIGURE 3. RNA sequencing screening of target genes affected by TRPV1 in vascular aging. (A) Volcano plot showing the top 20 DEGs in AS (Apoe −/− high‐fat diet) and TRPV1‐deficient AS (Apoe −/− Trpv1 −/−) mice on a high‐fat diet. (B, C) KEGG and GO analysis of the DEGs. (D) PPI analysis of the 20 DEGs and genes related to AS and aging. (E) Real‐time PCR validation of the expression of the 20 DEGs at the mRNA level, with results consistent with RNA sequencing. *p < 0.05, **p < 0.01.

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  FIGURE 4. TRPV1 deficiency leads to increased ISG15 expression in the aorta in response to a high‐fat diet. (A) PPI analysis reveals the regulatory relationship between ISG15 and validated DEGs associated with aging‐related proteins. (B) Western blot analysis shows that ISG15 expression in the aortas of Apoe −/− mice fed a high‐fat diet is significantly increased following TRPV1 deficiency. (C) Immunofluorescence double labelling demonstrates that TRPV1 deficiency significantly increases ISG15 expression in the aortic endothelial cells of AS mice. (D) In vitro culture of EA.hy926 cells with the TRPV1 antagonist capsazepine and the TRPV1 agonist capsaicin revealed that capsazepine significantly promoted ISG15 expression, while capsaicin significantly inhibited it. (E) In vitro culture of A7r5 cells with the TRPV1 antagonist capsazepine and the TRPV1 agonist capsaicin showed that capsazepine significantly promoted ISG15 expression, whereas capsaicin failed to inhibit it. **p < 0.01. Scale bar: 50 μm.

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  FIGURE 5. ISG15 participates in the regulation of vascular endothelial cell senescence. (A) β‐gal staining shows that ISG15 overexpression promotes senescence in normal cells, while ISG15 interference in damaged cells inhibits senescence. (B) A CCK8 assay shows that ISG15 overexpression inhibits cell proliferation, while ISG15 interference in damaged cells promotes cell proliferation. (C) The angiogenesis assay shows that ISG15 overexpression inhibits angiogenesis, while ISG15 interference in damaged cells promotes angiogenesis. (D) Real‐time PCR detection of SASP secretion showed that ISG15 overexpression promoted SASP secretion, while ISG15 interference in damaged cells inhibited SASP secretion. (E) In vascular endothelial cells, ISG15 overexpression promoted the secretion of age‐related proteins, while ISG15 interference in damaged cells inhibited the secretion of age‐related proteins. *p < 0.05, **p < 0.01. Scale bar: 50 or 100 μm.

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  FIGURE 6. Interference with ISG15 expression inhibits vascular aging in TRPV1‐deficient mice. (A) Real‐time PCR validation of ISG15 expression at the mRNA level in Apoe −/− Trpv1 −/− mice, showing significant downregulation of ISG15 expression after interference. (B) Western blot validation of ISG15 expression at the protein level in Apoe −/− Trpv1 −/− mice after ISG15 interference, showing significant downregulation of ISG15 expression. (C) β‐gal staining showed that the area of aged cells in the aortic sinus of mice was significantly reduced following ISG15 interference. (D) Immunofluorescence double‐labelling staining showed that p16 expression was significantly downregulated in both vascular endothelial and smooth muscle cells of the aortic sinus after ISG15 interference. (E) Real‐time PCR detection of SASP expression in mouse aortic tissue showed that the expression of all other SASP proteins was significantly downregulated after ISG15 interference, except for CCL5, which remained unchanged. (F) Western blot detection of aging‐related protein expression in mouse aortic tissue showed that, after ISG15 interference, the expression of all aging‐related proteins was significantly reduced.…

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  FIGURE 7. TRPV1 targets ISG15 to regulate vascular endothelial cell senescence. (A) β‐gal staining shows that the TRPV1 antagonist capsazepine exacerbates ox‐LDL‐induced cell senescence. When ISG15 is interfered with, however, the senescence‐promoting effect of capsazepine is significantly weakened. (B) Capsazepine significantly enhances the ox‐LDL‐inhibited cell proliferation effect, while interfering with ISG15 significantly reduces this effect of capsazepine. (C) In EA.hy926 cells with interfered ISG15, the inhibitory effect of capsazepine on angiogenesis was significantly reduced. (D) In EA.hy926 cells with interfered ISG15, the promoting effect of capsazepine on SASP (excluding CCL5) expression was significantly weakened. (E) Capsazepine significantly increased the expression of p21 and p53, but had no effect on the expression of p16 and γH2A.X. After ISG15 was interfered with, capsazepine only increased the expression of p16 and γH2A.X, and had no effect on the expression of p21 and p53. *p < 0.05, **p < 0.01. Scale bar: 50 or 100 μm.

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  FIGURE 8. ISG15 promotes vascular endothelial cell senescence by activating p53. (A) Overexpression of ISG15 in EA.hy926 cells promotes the phosphorylation of p53 and p21, while inhibiting the phosphorylation of Rb. After interfering with p53, the phosphorylation of p53 and p21 is weakened, while the phosphorylation of Rb is enhanced. (B) Interfering with p53 significantly inhibited ISG15's promotion of cellular senescence. (C) The inhibitory effect of ISG15 on cell proliferation was significantly weakened after p53 was interfered with. (D) Interfering with p53 significantly inhibited the inhibitory effect of ISG15 on angiogenesis. (E) Interfering with p53 significantly inhibited ISG15's promotion of SASP secretion in vascular endothelial cells. *p < 0.05, **p < 0.01. Scale bar: 50 or 100 μm.